HFP2017Advanced Lecture

CourseHuman Fungal Pathogens

Molecular Mechanisms of Host-Pathogen Interactions and

Virulence

May 13th-19th 2017

La Colle sur Loup

France

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Contents

Sponsors 4

General Information 6

Timetable 10

Programme 11

Abstracts 22

Posters 98

Index 243

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Organising Committee

Carol Munro (United Kingdom)Christophe d’Enfert (France)

Anita Sil (USA)

Vice Chairs (HFP2019 organisers):

Julian Naglik Alex Andrianopoulos

International Scientific Advisory Board

Robert Cramer (USA)Cécile Fairhead (France)

Matt Fisher (United Kingdom)Neil Gow (United Kingdom)

Ilse Jacobsen (Germany)Bruce Klein (USA)

Jim Kronstad (Canada)Oliver Kurzai (Germany)

Stuart Levitz (USA)Xiaorong Lin (USA

Robin May (United Kingdom)Suzanne Noble (USA)

Anne Puel (France)Jessica Quintin (France)Ana Traven (Australia)

Ted White (USA)

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Sponsors

The organisers of HFP 2017 would like to thank all their sponsors and supporters for their generous support.

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General Information

Venue

Belambra “Les Terrasses de Saint Paul de Vence”Chemin de Montmeuille

06480 La Colle sur Loup

France

Phone: +33 (0)4 93 32 96 96

Registration

The conference registration desk shall be open on Saturday 12 May from 15.00 – 19.00 and the following mornings, 13 to 18 May, from 8.15 to 8.45 prior to the start of the talks.

Conference Facilities

Meeting room and equipment

The meeting room (Riviera) is located near the main building of the centre. It is equipped with a LCD projector, an overhead projector, and microphones. Two laptop computers will be available (one PC, one Mac). Speakers should plan to be in the meeting room 30 min. prior to their session to upload their presentation onto these computers. It is therefore recommended that speakers bring their PowerPoint presentation on a memory stick. Alternatively, speakers using presentation software other than PowerPoint can bring their own laptop. Please note that equipment for 35 mm slides will not be provided at this meeting.

Posters

Poster boards are located in room Kandinsky. Posters should be posted on Sunday May 14 before the session starts and removed Thursday May 18.

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Posters should have a maximum size of 100 cm x 120 cm (w x h)

Participants should be at their poster on the following days: • Sunday May 14: Posters with the letter A

• Monday May 15: Posters with the letter B

• Wednesday May 17: Posters with the letter C

Materials are provided to attach the posters to the boards.

Accommodation

General

Rooms have been booked for the nights of May 13 - 18 inclusive (6 nights), with departure after breakfast on Friday May 19. All participants will stay in the Club Belambra residence in single accommodation (studio) or small apartments (two separate bedrooms and a shared bathroom).

Extra Nights

Please contact the venue, Club Belambra, directly if you require extra accommodation in addition to the nights included in the conference booking. Extra nights are at your own expense and should be paid by the participants.

Meals

Breakfast will be served buffet style from 7.30 am. Times for lunch and dinner are as shown in the conference programme. Mineral water and coffee are served at each meal. Additional beverages are at participants’ own expense.

Site Services

Telephone calls

There are no telephones in the bedrooms. There are several public phones on the conference site. These operate with calling cards that can be bought at the reception desk. A public phone operating with coins is available in the main building.

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Photocopies and Faxes

Photocopies may be made, and faxes sent and paid for at the Club Belambra reception.

Computer Facilities and Internet Connection

WiFi internet access is available in the lobby. There is an internet café in La Colle sur Loup.

Means of Payment to the Conference Site

Credit cards are accepted by the Club Belambra reception desk (not at the bar) and all currency payments should be in Euro. It is not possible to change foreign currency at the venue.

All additional expenses, such as drinks (except those served during meals), telephone calls, tours, etc. are at participants’ own expense and should be paid upon check-out from the Club Belambra.

Bank Facilities

The nearest bank, the Caisse d’Epargne is in La Colle sur Loup (2 km, 15 min. walk). This bank has an ATM where withdrawals with an international credit card are possible. Exchange of foreign currencies or withdrawal of Euros with an international credit card is possible at Nice airport.

Leisure Activities and Tourism

Weather

Weather in May is normally sunny with temperatures ranging from 15 to 20°C during the day and 8 to 12°C during the night. However, showers are also possible.

On SiteThe venue is set within a 25 acres private pine-tree forest and provides numerous recreational and leisure facilities: bar with terrace, TV room, American pool table, outdoor swimming pool, volleyball and basketball courts, bowls pitch and 3 tennis courts.

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The Surrounding Area

The countryside provides opportunities for hiking, rock-climbing and canyoning. La Colle-sur-Loup is close to the traditional village of Saint-Paul-de-Vence with the Maeght Fundation displaying an impressive collection of modern art. It is also close to Nice with the Matisse museum and the Chagall museum, to Vence with the Chapel of the Rosary imagined and achieved by Henri Matisse, and to Vallauris with the Picasso museum. It is also close to Biot famous for its glass workshop and Grasse famous for its perfume industry. Excursions will be organized during the free afternoon of May 16th.

Social Programme

• Saturday May 13 - Welcome drinks will be served

• Thursday May 18 - Conference Dinner and Aperitif will be served in the evening

• Tuesday May 16 - Excursions on the free afternoon. Four buses will be organized going respectively to Saint-Paul-de-Vence and the Maeght Foundation, Nice, Biot and Antibes, and Monaco.

Participants will be asked to select their preferred excursion at registration as there is limited availability for each excursion (53 persons each). Buses will be organized to return to the site from the different locations.

On the evening of May 16th you will be free to make your own dinner arrangements.

Some Useful Information Insurance

The meeting organizers do not provide insurance and cannot take responsibility for accidents or illnesses that might occur during the week, in the course of travel to and from the venue. It is therefore the responsibility of each participant to check their health insurance requirements.

Shopping Hours

The nearest shops (newspapers, toiletries, etc) are in La Colle sur Loup, about 15 minute walk from the Club Belambra. Major shopping can be done in Nice. Opening times of shops in France in general are: Monday through Saturday: 09.00 to 12.00 and 14.00 to 19.00 but many close on Monday.

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Timetable

Saturday Sunday Monday Tuesday Wednesday Thursday Friday May 13 2017 May 14 2017 May 15 2017 May 16 2017 May 17 2017 May 18 2017 May 19 2017

07.30 - 09.00 Breakfast Breakfast Breakfast Breakfast Breakfast Breakfast

Session 3: Discovering novel

antifungal strategies 08.30 - 10.00

Coffee

Session 4: Interactive

workshop on mycobiome in

health & disease 10.30 - 12.15

12.15 - 13.30 Lunch Lunch Lunch Lunch Lunch

13.30 - 15.45

Workshop 1 15.45 - 16.45

Workshop 2 15.45 - 16.45

Workshop 3 15.45 - 16.45

Workshop 4 15.45 - 17.00

Coffee Coffee Coffee Coffee

Elevator Session 1 17.10-18.00

Elevator Session 2 17.10-18.00

Elevator Session 3 17.10-18.00

Concluding remarks and award

ceremony 17.20-18.00

18.0019.00

Dinner

20.40 Keynote Lecture

21.30 Welcome Reception 20.30-22.30 20.30-22.30 20.30-22.30

Session 5: Host-pathogen

interactions, the host perspective

09:00 - 12:15

Free afternoon and evening

Registration

Drinks, Dinner and Farewell Party

Session 6: Host-pathogen

interactions, the pathogen

perspective 09:00 - 12:15

Departure

Dinner Dinner Dinner

Welcome address20.20

Poster Session A

Poster Session B

Poster Session C

15.00

09.00

Session 1: Pathogenic fungi -

genomics, evolution and epidemiology

09.00 - 12.15

Session 2: Molecular and

cellular biology of fungal pathogens

09.00 - 12.15

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Programme

Saturday May13, 2017

15.00 Registration 19.00 Dinner 20.20 Carol Munro

Introductory Remarks 20.25 Carol Munro

Presentation of FEMS and FEBS 20.30 Donna MacCallum

Presentation of ISHAM 20.40 ISHAM-sponsored Keynote lecture: Alistair Brown (K1)

The complexity of host inputs affects fungal outputs and hence virulence

21.30 Welcome reception

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Sunday May14, 2017

07.30 Breakfast 08.15 Registration

09.00 Session 1: Pathogenic fungi - genomics, evolution and epidemiology A session sponsored by the French Society of Medical Mycology Chair: Richard Bennett

09.00 Richard Bennett, Brown University, USA Introduction

09.20 S1/1 Geraldine Butler, University College Dublin, Ireland Genome diversity of Candida species

09.50 S1/2 Antonis Rokas, Vanderbilt University, USA Genomics and the making of fungal metabolic diversity

10.20 Coffee Break 10.40 S1/3 Li-Jun Ma, University of Massachusetts Amherst, USA

Genome evolution and organism adaptation of a cross-kingdom fungal pathogen Fusarium oxysporum

11.10 S1/4 Guilhem Janbon, Institut Pasteur, France The dynamics of the Cryptococcus neoformans transcriptome

11.40 S1/5 Norman Pavelka, A*Star, Singapore

12.15 Lunch 15.45 Workshop 1: Genomics, evolution, epidemiology,

antifungals Chairs: Guilhem Janbon, Cecile Fairhead

15.45 W1/1 Jane Usher, University of Exeter, UK (P1A) Targeting synthetically lethal partners of antimicrobial resistance alleles in Candida glabrata to minimize emergence of drug resistant infection

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16.00 W1/2 Cecile Fairhead, Université Paris Sud, France (P22C) Mating-type switching in Candida glabrata and related species.

16.15 W1/3 Joachim Morschhäuser, University of Würzburg, Germany (P26C) Evolution of fluconazole-resistant Candida albicans strains exhibiting multiple resistance mechanisms by drug-induced sexual recombination

16.30 W1/4 Mélanie Legrand, Institut Pasteur, France (P39C) Candida albicans: a model organism to study mechanisms involved in eukaryotic genome integrity

16.45 Coffee Break 17.10 Elevator session 1

Chairs: Guilhem Janbon, Cecile Fairhead 17.10 E1/1 Selene Mogavero (P5A)

Candidalysin is the Candida albicans haemolytic factor 17.15 E1/2 Paul Donovan (P11A)

Riboswitch Discovery in Saccharomycotina Species 17.20 E1/3 Suelen dos Santos (P17A)

The establishment of a specific response during paracoccidioidomycosis is dependent of CD11c+ cells

17.25 E1/4 Patricia Otieno-Odhiambo (P23A) Pneumocystis: what are the host innate immune mechanisms controlling infection?

17.30 E1/5 Maria Spyrou (P24A) Orchestration of chitin synthesis in Candida albicans

17.35 E1/6 Lisa Lombardi (P27A) Development of a plasmid based system for CRISPR-Cas9 mediated mutation in Candida parapsilosis

17.40 E1/7 Bethany McCann (P35A) From Bug to Drug: In-house production of bespoke antifungal peptide aptamers

17.45 E1/8 Jiyoti Verma (P36A) Insights into host-pathogen interactions by dual RNAseq.

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17.50 E1/9 Catarina Costa (P41A) Are Candida glabrata drug efflux pumps from the MFS clinically relevant?

17.55 E1/10 Courtney Kousser (P42A) Bacterial secreted factors regulate mucormycete germination

18.00 Close 19.00 Dinner 20.30 Poster Session A

Monday May 15, 2017

07.30 Breakfast 08.15 Registration

09.00 Session 2: Molecular and cell biology of fungal pathogens A session sponsored by the British Mycological Society Chair: Ana Traven

09.00 Ana Traven, Monash University, Australia Introduction (sponsored by IUBMB)

09.20 S2/1 Judith Berman, Tel Aviv University, Israel Clearing the FoG: Subpopulation responses to antifungal drugs

09.50 S2/2 Mike Lorenz, University of Texas McGovern Medical School, USA Metabolic adaptations drive resistance to phagocytosis

10.20 Coffee Break 10.40 S2/3 Hubertus Haas, Innsbruck Medical University, Austria

Iron – a key hub in virulence of Aspergillus fumigatus 11.10 S2/4 Anita Sil, University of California San Francisco, USA

Regulation of cell shape and virulence by temperature in Histoplasma capsulatum

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11.40 S2/5 Liz Ballou, University of Birmingham, UK Endogenous Reactive Oxygen Species in Cryptococcus neoformans morphogenesis and pathogenesis

12.15 Lunch 15:45 Workshop 2: Molecular and cell biology of fungal

pathogens Chairs: Ana Traven, Alex Adrianopoulos

15:45 W2/1 Jorge Amich (P2C) Methionine synthases as drug targets for antifungal drug development

16.00 W2/2 Helene Martin-Yken (P3B) The Dark Side of the Wall: Atomic Force Microscopy Revelations on Drug Resistance and Adhesion

16.15 W2/3 Guanghua Huang (P9A) The Regulatory Subunit of Protein Kinase A (Bcy1) in Candida albicans Plays Critical Roles in Filamentation and White-Opaque Switching but is Not Essential for Cell Growth

16.30 W2/4 Miguel Teixeira (P9B) Unveiling transcriptional regulation in Candida species: using the PathoYeastract database

16.45 Coffee Break 17.10 Elevator Session 2

Chairs: Ana Traven, Alex Adrianopoulos 17.10 E2/1 Zanetta Chang (P8B)

Drug resistance via RNAi-dependent epimutations in Mucor circinelloides

17.15 E2/2 Silvia Novohradská (P5B) Pathogenic yeasts express virulence determinants in a new natural model system to defend against amoeba predation

17.20 E2/3 Enrico Garbe (P15B) Characterization of the Candida albicans ECE1 promoter

17.25 E2/4 Ona Rogiers (P13B) The in vivo efficiency of anidulafungin and tigecycline against polymicrobial catheter-associated peritonitis

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17.30 E2/5 Patricia M. Silva (P16B) Temporal and spatial control of fungal filamentous growth

17.35 E2/6 Larissa VG Longo (P17B) The role of APSES transcription factors in Histoplasma capsulatum virulence and adaptation to stress conditions

17.40 E2/7 Delma Childers (P23B) Fungal Transformers: Tracking a Moving Target

17.45 E2/8 Wouter van Genechten (P38B) Fast in vivo super-resolution microscopy and background reduction in Candida albicans.

17.50 E2/9 Fitz Gerald Silao (P42B) Mitochondrial proline metabolism is key to arginine-induced morphogenesis in Candida albicans

17.55 E2/10 Yacine Dahman (P29B) CRISPR-Cas9-engineered C. glabrata strains for the study of virulence factors in animal models

18.00 Close 19.00 Dinner 20.30 Poster Session B

Tuesday May 16, 2017

07.30 Breakfast 08.30 Session 3: Discovering novel antifungal strategies

A session sponsored by BioRad Chair: Damian Krysan

08.30 S3/1 Damian Krysan, University of Rochester Medical Center, USA Adventures in antifungal drug development

09.00 S3/2 Françoise Dromer, Institut Pasteur, France New treatment strategies for cryptococcosis?

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09.20 S3/3 Karin Thevissen, KU Leuven, Belgium Plant defensins and linear plant defensin-derived peptides increase caspofungin's antibiofilm against Candida biofilms in vitro and in vivo

09.40 S3/4 Carlo Petosa, Université de Grenoble Alpes, France Selective BET bromodomain inhibition as a potential antifungal therapeutic strategy

10.00 Coffee Break 10.20 Session 4: Interactive workshop on the mycobiome in

health and disease A session sponsored by the Microbiology Society

Chair: Tom Dawson 10.20 S4/1 Andy Koh, University of Texas, USA

Commensal anaerobic gut microbiota promote Candida albicans colonization resistance

10.40 S4/2 Tom Dawson, A*Star, Singapore Forbidden Kingdom: role of the mycobiome and fungus-host interactions in human skin disease

11.00 S4/3 Mathias L. Richard, INRA AgroParisTech, France Gut fungal microbiota: The yin and yang of Inflammatory Bowel Disease

11.20 S4/4 Luigina Romani, University of Perugia, Italy The regulation of host resistance to fungi by the microbiota: towards a systems-level understanding of fungal diseases

11.40 Open discussion 12.15 Lunch 13.30 Free afternoon and evening

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Wednesday May 17, 2017

07.30 Breakfast 09.00 Session 5: Host-pathogen interactions, the host

perspective A session sponsored by European Confederation of Medical Mycology Chair: Donna MacCallum

09.00 Donna MacCallum University of Aberdeen, UK Introduction

09.20 S5/1 Sarah Gaffen, University of Pittsburgh, USA IL-17 signal transduction and immunity to Candida albicans

09.50 S5/2 Jessica Quintin, Institut Pasteur, France Innate immune memory and fungal infections

10.20 Coffee Break 10.40 S5/3 Georgios Chamilos, University of Crete, Greece

Novel molecular mechanisms regulating phagosome biogenesis during interplay of airborne filamentous fungi with the mammalian phagocytes

11.10 S5/4 Agostinho Carvalho, University of Minho The Genetic architecture of innate antifungal immunity

11.40 S5/5 Chris Mody, University of Calgary, Canada Fungal effector mechanisms: Direct killing by cytolytic lymphocytes

12.15 Lunch 15.45 Workshop 3: Host-pathogen interactions, the host

perspective Chairs: Ilse Jacobsen, Oliver Kurzai

15.45 W3/1 Salome Leibundgut (P1C) The intraspecies diversity of C. albicans triggers qualitatively and temporally distinct host responses that determine the balance between commensalism and pathogenicity.

16.00 W3/2 Darius Armstrong-James (P25B) Calcineurin orchestrates lateral transfer of Aspergillus fumigatus during macrophage cell death

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16.15 W3/3 Steffen Rupp (P35B) Dermal fibroblasts play a central role in skin model protection against C. albicans invasion

16.30 W3/4 Constantin Urban (P44C) Host factor adenosine modulates inflammatory responses in systemic candidiasis

16.45 Coffee Break 17.10 Elevator Session 3

Chairs: Ilse Jacobsen, Oliver Kurzai 17.10 E3/1 Leenah Alaalm (P1C)

Understanding how the Hsp90 Kinase interactome affects Candida albicans virulence

17.15 E3/2 Barbara Koch (P8C) Uncovering new links between mitochondria and hyphal growth of Candida albicans

17.20 E3/3 Robert Tams (P10C) The Kennedy Pathway: A rheostat for virulence in Candida albicans

17.25 E3/4 Amanda Veri (P33C) Investigating the mechanisms by which the transcription factor Hsf1 regulates Candida albicans morphogenesis

17.30 E3/5 Angeliki Andrianaki (P30C) Iron restriction inside the phagosome of macrophages is an essential host defense mechanism against Mucorales

17.35 E3/6 Intan Dewi (P43C) Sialic acids on the surface of Aspergillus fumigatus conidia impacts pathogen recognition and anti-fungal response by PBMCs

17.40 E3/7 Eric Delarze (P36C) Identification of mediators of antifungal tolerance in Candida albicans

17.45 E3/8 Cláudio Duarte-Oliveira (P40C) The long pentraxin PTX3 regulates the pathogenic potential of Trichosporon asahii via differential induction of IL-10

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17.50 E3/9 Philipp Kämmer (P26C) Fingerprinting of Candida spp. blood infections

17.55 E3/10 Grasielle Jannuzzi (P16C) The absence of TLR3 controls paracoccidioidomycosis with increase of cytotoxic CD8+ T cells

18.00 Close 19.00 Dinner 20.30 Poster Session C

Thursday May 18, 2017

07.30 Breakfast 09.00 Session 6: Host-pathogen interactions, the pathogen

perspective A session sponsored by The Burroughs Wellcome Fund Chair: Rebecca Hall

09.00 Rebecca Hall, University of Birmingham, UK Introduction

09.20 S6/1 Alex Brand, University of Aberdeen, USA Contact-sensing and directional growth regulation in Candida albicans hyphae

09.50 S6/2 Bruce Klein, University of Wisconsin-Madison, USA A novel Blastomyces ligand for Dectin-2 with adjuvant activity for vaccination against fungi

10.20 Coffee Break 10.40 S6/3 Sven Krappmann, Universitätsklinikum Erlangen,

Germany Putting fungal virulence in context

11.10 S6/4 Kirsten Nielsen, University of Minnesota, USA Cell Wars: The battle between Cryptococcus titan cells and the immune system

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11.40 S6/5 Julian Naglik, Kings College London, UK Epithelial activation by Candidalysin

12.15 Lunch 15.45 Workshop 4: Host-pathogen interactions, the pathogen

perspective Chairs: Bruce Klein, Julian Naglik

15.45 W4/1 Slavena Vylkova (P2A) Phagosomal neutralization by Candida albicans induces macrophage pyroptosis

16.00 W4/2 Robert Wheeler (P2B) Candida albicans takes a licking during infection

16.15 W4/3 Elaine Bignell (P19A) Mechanistic basis of epithelial damage in response to A. fumigatus infection

16.30 W4/4 Mary Ann Jabra-Rizk (P19B) Therapeutic Implications of Candida albicans and Staphylococcus aureus Mixed Biofilms

16.45 W4/5 Neeraj Chauhan (P41B) Oxidative stress resistance and modulation of macrophage pro-inflammatory response by Candida albicans MAM33

17.00 Coffee Break 17.20 Concluding Remarks and Award Ceremony

Carol Munro 18.00 Close 19.00 Dinner and Farewell party

Friday May 19 2017 From 07.30

Breakfast and departure

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K1

The complexity of host inputs affects fungal outputs and hence virulence

Alistair J.P. Brown Aberdeen Fungal Group, MRC Centre for Medical Mycology, University of Aberdeen, Aberdeen, UK

Fitness attributes such as stress and nutrient adaptation contribute significantly to the virulence of fungal pathogens. Numerous laboratories have contributed to our understanding of stress and nutrient adaptation in Candida albicans. As a result we have a reasonable understanding of how this pathogen responds to certain specific environmental stresses and nutrients, and how these responses are regulated. To minimize confounding factors, these studies have generally defined a specific response under standardised experimental conditions in vitro. It is becoming apparent, however, that these conditions can differ from host niches, which are both complex (i.e. fungal cells are often subjected to multiple host inputs) and dynamic (i.e. host inputs change over time). The implications of these complex and dynamic environmental landscapes upon fungal behaviours and virulence will be discussed.

Abstracts

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S1/1

Genome diversity of Candida species

Geraldine Butler University College Dublin, Dublin, Ireland

Candida albicans and close relatives translate the codon CUG as serine rather than leucine, associated with changes in the tRNACAG. The group containing these species is therefore often referred to as the “CTG clade”. Recent analysis has shown that the switch to CUG serine occurred at the base of the Metschnikowiaceae/ Debaryomycetaceae. Metschnikowiaceae species include many fully sexual species such as the Hyphopichia group and the Metschnikowia group. Most asexual Candida species belong to the Candida/Lodderomyces clade. We used comparative genomic analysis to expand our study the evolution of mating type in sexual and asexual species within the CUG-Ser clade. We are also exploring within-species diversity using genome sequencing. Analysis of C. orthopsilosis genomes suggests that this species has no single origin, and arose from multiple hybridisation events. More recently, we have characterised diversity among C. tropicalis isolates.

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S1/2

Genomics and the making of fungal metabolic diversity

Antonis Rokas Vanderbilt University, Nashville, TN, USA

A defining characteristic of Fungi is that, in contrast to animals, they are typically embedded in their food and digest it externally in the presence of competitors (think of the blue lines of mold in blue cheese). Thus, different fungi specialize in “eating” different foods (hence their diverse primary metabolism), and because digestion happens externally, fungi have also evolved potent food defense mechanisms (hence their diverse secondary metabolism). The cholesterol-reducing drug lovastatin, the antibiotic penicillin, and the potent carcinogen aflatoxin are just a tiny sample of the pharmacopoeia stored in and manufactured by fungal DNA. Our lab studies elucidate how this diversity is generated and maintained through studies of yeasts in the subphylum Saccharomycotina and of filamentous fungi in the genus Aspergillus. For example, as part of the National Science Foundation Dimensions of Biodiversity program, in collaboration with the Hittinger (University of Wisconsin-Madison) and Kurtzman (US Department of Agriculture) labs, we are decoding the genomes of all known species in the Saccharomycotina subphylum to uncover the genetic basis of yeast metabolic diversity. Similarly, jointly with several different collaborating labs, we are investigating the ecology and evolution of gene regulation of secondary metabolic pathways in Aspergillus. In my talk, I will discuss our latest results, using genomic, evolutionary, and functional experiments to elucidate the evolutionary patterns and processes involved in the making of fungal metabolic diversity.

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S1/3

Genome evolution and organism adaptation of a cross-kingdom fungal pathogen Fusarium oxysporum

Li-Jun Ma University of Massachusetts Amherst, Amherst, USA

As a species complex, Fusarium oxysporum causes destructive and intractable wilt diseases in over 100 diverse plant hosts. At the same time, clinical isolates of F. oxysporum have also been reported to cause life-threatening infections in immune-compromised patients. The Fusarium comparative genomics confirmed that horizontally acquired transposon-rich, pathogenicity chromosomes convey fungal pathogenicity and contribute directly to the pathogen niche adaptation in both plant and human hosts. This discovery established F. oxysporum as an effective model to investigate horizontal transfer in eukaryotes. The pathogenicity chromosomes provide a focal point to investigate the genetic mechanisms that underlie pathogenesis against different hosts. However, these highly dynamic pathogenicity chromosomes are associated with fitness costs. This presentation will discuss diverse selection pressures act on these pathogenicity chromosomes and their potential evolutionary consequences in a genome that results in the adaptation of an organism.

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S1/4

The dynamics of the Cryptococcus neoformans transcriptome

Sara Gonzalez-Hilarion, Damien Paulet, Corinne Maufrais, Frédérique Moyrand, Guilhem Janbon Institut Pasteur, Paris, France

Our recent work suggests that a fascinating, complex pattern of RNA molecules composes the Cryptococcus transcriptome and this fungus is emerging for different aspects as an ideal model to study RNA metabolism in eukaryotes. It is also tempting to hypothesize that this complex RNA metabolism provides a mechanism for this yeast to respond to different environmental cues and to be an efficient pathogen. We have recently re-annotated the genomes of both varieties of C. neoformans and proved that introns represent key players in the regulation of gene expression in this yeast. Any modification of the environment results in a global and specific alteration of the transcriptome. For instance, we identified a large number of regulated alternative splicing events although these events poorly affect the diversity of the proteome. We also observed major alterations of RNA molecules number per cell in response to growth condition changes. These “transcriptome size” modifications need to be considered when identifying genes differentially expressed.

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S1/5

Norman Pavelka A*Star, Singapore

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W1/1

Targeting synthetically lethal partners of antimicrobial resistance alleles in Candida glabrata to minimize emergence of drug resistant infection

Jane Usher, Ken Haynes University of Exeter, Exeter, UK

Drug resistance has emerged as a huge problem in many areas of medicine from cancer to infectious diseases. This is driving the development of novel therapeutic strategies. One that is gaining ground, is multi-target therapy, where combinations of drugs targeting different components of a disease network are deployed. A major impediment to this approach is the characterization of suitable targets for combination therapies. To date most combinatorial therapy targets have been selected based on previous biological knowledge of drug mode of action and/or mechanisms of resistance, severely constraining the number of proteins that can be targeted.

Unbiased genome-wide screens will reveal many more components of the interaction networks of known drug targets, which could then be targeted in combination therapies. To test this principle in the context of antimicrobial resistance we have implemented an unbiased genome-wide screening technology, SGA analysis, facilitating characterization of pair-wise synthetic genetic interactions. We performed an unbiased SGA screen with a C. glabrata PDR1+ gain of function allele. PDR1 encodes a transcriptional regulator and gain of function mutations in this gene, are the principal mediators of fluconazole resistance in C. glabrata. We identified a gcn5 null mutation as one negative synthetic interaction with PDR1+. We showed that deletion of GCN5 and/or chemical inhibition of the protein Gcn5, are synthetically lethal with PDR1+. These data demonstrate that deletion or chemical inactivation of a PDR1+ synthetically lethal gene results in cellular death if wild-type PDR1 mutates to a PDR1+ FLZ resistance conferring allele.

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W1/2

Mating-type switching in Candida glabrata and related species.

Cecile Fairhead, Stéphanie Boisnard, Youfang Li Zhou Université Paris Sud, Orsay, France

Although it seems to have lost the capacity for sexual reproduction, the genome of Candida glabrata contains three MAT-like cassettes, and the HO gene, encoding the endonuclease that initiates the mating-type interconversion event. We have published that S. cerevisiae Ho induces mating-type switching in C. glabrata; that this results in massive cell death, and that Ho cuts the additional loci, HMR and HML. Examination of strains in our collection shows mating-type interconversion events, and rearrangements of MAT-like loci. We now show that it is not the additional cleavage of MAT-like loci that induces cell death, but only cleavage of MAT. We also show that this situation is peculiar to C. glabrata, since the closely-related Nakaseomyces delphensis species can undergo mating-type interconversion by expression of the S. cerevisiae HO gene, without the massive cell death observed in C. glabrata. We will discuss these observations and propose hypotheses.

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W1/3

Evolution of fluconazole-resistant Candida albicans strains exhibiting multiple resistance mechanisms by drug-induced sexual recombination

Christina Popp, Joachim Morschhäuser University of Würzburg, Würzburg, Germany

Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is commonly caused by mutations in the target enzyme Erg11 or by gain-of-function mutations in the transcription factors Mrr1, Tac1, and Upc2, which result in overexpression of multidrug efflux pumps and ergosterol biosynthesis genes. Many fluconazole-resistant isolates are homozygous for the mutated genes and exhibit multiple resistance mechanisms. The loss of heterozygosity is frequently accompanied by MTL homozygosity, which enables the cells to switch to the mating-competent opaque cell form. To investigate if sexual recombination may be involved in the evolution of highly drug-resistant strains, we propagated a set of isogenic strains that were heterozygous for ERG11, MRR1, TAC1 and UPC2 alleles with resistance mutations in the presence of fluconazole. This resulted in the emergence of derivatives with increased resistance that had become homozygous for the mutated allele and the mating type locus. When mixing MTLa/a and MTLα/α cells of these strains in all possible combinations, we could isolate mating products containing the genetic material from both parents. The initial mating products did not exhibit higher drug resistance than their parental strains, but further propagation under selective pressure resulted in the loss of the wild-type alleles and increased fluconazole resistance. Our results demonstrate that fluconazole treatment not only selects for resistance mutations but also promotes genomic alterations that confer mating competence. This allows cells in an originally clonal population to exchange individually acquired resistance mechanisms by sexual recombination and generate highly drug-resistant progeny.

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W1/4

Candida albicans: a model organism to study mechanisms involved in eukaryotic genome integrity

Mélanie Legrand1, Adeline Feri1, Timea Marton1, Raphaël Loll-Krippleber1, Corinne Maufrais2, Christophe d’Enfert1 1Institut Pasteur, INRA, Fungal Biology and Pathogencity Unit, Paris, France, 2Institut Pasteur, center for Informatics in Biology, Paris, France

Genomics studies in Candida albicans have highlighted various degrees of tolerance to genome plasticity. In different instances, genome rearrangements have been correlated with adaptation of the cells to various stress both in vivo and in vitro. In particular, loss-of-heterozygosity (LOH) events, aneuploidies and isochromosome formation have been shown to contribute to increasing the resistance to azole antifungals. Genomics studies have also revealed that some chromosome haplotypes cannot be found in the homozygous state, suggesting the presence of recessive lethal alleles on certain chromosome homologs of C. albicans.

Because DNA double-strand breaks (DSBs) have been shown to be very potent initiators of recombination in yeast and other organisms, we investigated the molecular mechanisms involved in the repair of a targeted, I-SceI enzyme-induced DNA DSB in C. albicans. Our data indicated that most of the I-SceI-induced DNA DSBs are repaired by gene conversion in C. albicans. Interestingly, the precise characterization of the progenies that arose from a targeted DSB on Chromosome 4 (Chr4) or Chr7 led to the identification of recessive lethal and deleterious alleles on Chr4 and Chr7 of the C. albicans SC5314 laboratory strain. In addition, high-resolution analysis of an induced DNA DSB revealed template switching events and, unexpectedly, bidirectional homozygosis in C. albicans. Our data suggest that C. albicans is a valid model to study mechanisms involved in eukaryotic genome integrity.

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Candidalysin is the Candida albicans haemolytic factor

Selene Mogavero1, Sarah Höfs1, Alexa Lauer1 ,2, Bernhard Hube1 ,2 1Hans Knöll Institute, Jena, Germany, 2Friedrich Schiller University, Jena, Germany

Candida albicans has recently been discovered to be able to produce an important virulence factor, the hyphae-associated secreted peptide toxin Candidalysin. This toxin has proven to be crucial for the establishment of a mucosal infection (1). But what other roles might this toxin have? C. albicans has long been known to be haemolytic, but the haemolytic factor has never been clearly identified. Previous work suggested that it probably is a secreted mannoprotein (2). Might the yet-not-clearly-identified haemolytic factor actually be Candidalysin? Using both C. albicans mutants and synthetic peptides in haemolysis assays, we can now clearly attribute the full haemolytic activity of C. albicans to its secreted Candidalysin. We further elucidated the mechanism of the toxin-induced red blood cell (RBC) lysis, and found that pH, cholesterol, divalent cations, and purinergic receptor activation play a central role. We also observed a significant variation of our results depending on blood donor, which opens new questions about the mechanism of RBC lysis by Candidalysin. For example, might there be a differential role of cell-surface antigens for Candidalysin susceptibility? Our results shed some new light on C. albicans virulence, with important clinical implications, especially for patients suffering from candidaemia, condition that has been rising in incidence in the past decades, due to the increase in immunocompromised patients.

1. Moyes DL et al., 2016. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature. 532 (7597): 64-68.

2. Watanabe T et al., 1999. Characterization of a haemolytic factor from Candida albicans. Microbiology. 145 (Pt 3): 689-694.

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Riboswitch Discovery in Saccharomycotina Species

Paul Donovan1, Aisling Coughlan2, Des Higgins2, Geraldine Butler1 1School of Biomedical and Biomolecular Science, Conway Institute , University College Dublin, Belfield, Dublin, Ireland, 2School of Medicine and Medical Science, Conway Institute , University College Dublin, Belfield, Dublin, Ireland

Riboswitches are regulatory elements of mRNA that control gene expression, usually by binding a ligand. Thiamine pyrophosphate (TPP) riboswitches have been described in many bacteria, some plant species, and the filamentous fungi Neurospora crassa and Aspergillus oryzae. Early reports suggested that riboswitches were not present in the Saccharomycotina, including Candida and Saccharomyces species.

We used Infernal with the RFAM covariance model to identify riboswitches in Saccharomycotina species. Riboswitches were identified in thiamine biosynthesis genes (THI4 and THI5), and in a gene of unknown function (DUR31). We confirmed that the thiamine-regulated riboswitch mediates alternative splicing in DUR31 in Candida parapsilosis and Ogataea polymorpha. C. parapsilosis and C. albicans DUR31 deletion strains grow in the presence of pyrithiamine, a toxic thiamine analogue. DUR31 therefore likely encodes a thiamine transporter.

DUR31 has been lost from the genome of Saccharomyces cerevisiae and relatives. However, it is present in most other species in the Saccharomycotina. In S. cerevisiae, thiamine is imported by Thi7 and related proteins. This family is restricted to species closely related to S. cerevisiae. We therefore suggest that Dur31 is a major thiamine transporter in most Saccharomycotina species.

Here, we show that riboswitches are widespread in the Saccharomycotina, including most species in the CTG-Ser clade. Surprisingly, they are lost from DUR31 in some of the best-researched species, including C. albicans and C. tropicalis. We found that all TPP riboswitches are lost from the Saccharomycetaceae.

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The establishment of a specific response during paracoccidioidomycosis is dependent of CD11c+ cells

Suelen dos Santos, Sandro de Almeida Universidade de São Paulo, Sao Paulo, SP, Brazil

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America and the most frequent systemic mycosis in Brazil. The dendritic cells (DCs) are antigen presenting cells able to do the link between innate and adaptive immune response that play a pivotal role in important infections caused by airborne pathogens. During the first 12 hours of PCM infection, lung DCs migrate to lymph nodes and induce a mixed pattern of CD4 T cell cytokines, compatible with a Th1/Th2 response. The aim of this work is to characterize the immunological and cellular profile of mice infected with P. brasiliensis after CD11c+ cells depletion and how the lack of this population interferes in the infection outcome. We evaluate the depletion of CD11c+cells in C57BL/6.CD11c-DTR mice, after 24 hour of administration of diphtheria toxin (DTX). With 4ng/g of animal, we get almost 80% CD11c+ cells reduction in the spleen. After this period, we infected the animals intratracheally with P. brasiliensis. After 7 and 15 days we had a CD4+ cells reduction in lymph nodes, and a small proliferation of this cells obtained from spleen in response of P. brasiliensis antigens, in comparison of not depleted animals. In the absence of CD11c+ cells we saw an increase of IL4, IL-6 and IL-10 and a decrease of Il-17 after 7 days of infection. In conclusion, animals without CD11c+ during the infection had a different pattern of cytokines and a small lymphocyte specific proliferation and altogether these results showed the importance of CD11c+ cells during the establishment of PCM infection.

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Pneumocystis: what are the host innate immune mechanisms controlling infection?

Patricia Otieno-Odhiambo1, Nontobeko Mthembu1, Suraj P. Parihar1 ,2, Frank Brombacher1 ,2, Jay Kolls4, Gordon D. Brown3, J. Claire Hoving1 1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa, 2International Centre for Genetic Engineering and Biotechnology, Cape Town, South Africa, 3Aberdeen Fungal Group, Institute of Medical Sciences,University of Aberdeen, Aberdeen, UK, 4The Richard King Mellon Foundation,Institute of Paediatric Research, University of Pittsburgh,School of Medicine, Pittsburgh, USA

The high prevalence of diseases such as HIV/AIDS on the African continent has rendered patients immunocompromised. This is strongly associated with the prevalence of life threatening opportunistic fungal pathogens. Even so, fungal infections are an often overlooked clinical and public health issue. Pneumocystis jirovecii, causes pneumonia (PCP) in HIV/AIDS patients, with an estimated 400,000 cases and mortality of 150,000 per annum. The introduction of highly active antiretroviral therapy has certainly improved the HIV pandemic outlook but PCP related morbidity and mortality remain high. Innate immunity drives pathogen recognition with significant downstream effects on adaptive responses. Both Toll like receptors (TLRs) and C-type lectin receptors (CLRs) are key pattern recognition receptors (PRRs) involved in antifungal immunity. Evidence suggests that Pneumocystis recognition involves PRRs as well. By using a Pneumocystis mouse model and mice deficient in CLRs including Dectin-1, Clecsf8 (Cle4d, Mcl, Dectin 3) or the downstream signalling molecule PKCδ (protein kinase c delta), we hope to dissect the innate immune response in Pneumocystis infection. Preliminary data using PKCδ-/- mice that has been shown to engage Syk Kinase-coupled C-type lectin receptors, shows higher lung burden compared to the wild type mice suggesting Pkcδ plays a role in the immunity against Pneumocystis infection. With the indicative preliminary results, and continual investigation, we hope to discover new insights into underlying host mechanisms involved in protective immunity to Pneumocystis which may lead to improved treatments for immunocompromised hosts.

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E1/5Orchestration of chitin synthesis in Candida albicans

Maria Spyrou, Megan Lenardon, Alistair Brown, Neil Gow University of Aberdeen, Aberdeen, UK

Septation is a highly regulated process, fundamental for cell viability, taking place during cell division. Understanding the biochemistry and cell biology of fungal septation may lead to the identification of tractable drug targets for future antifungal chemotherapeutic strategies. In the pathogenic fungus C. albicans, a primary septum which is made out of chitin is formed between two dividing cells. In C. albicans, chitin is synthesised by four chitin synthases: Chs1, Chs3, Chs2 and Chs8 and all four enzymes localise to sites of septation. Strains expressing pairs of fluorescently tagged chitin synthases were generated to examine timing and position of recruitment in relation to each other. Based on data obtained by live-cell fluorescence microscopy, a model is proposed demonstrating the spatial and temporal distribution of all four chitin synthases during septation in C. albicans hyphae. We demonstrate for the first time that Chs3 arrives at the septation site first prior to the other three chitin synthases and that Chs1 Chs2 and Chs8 form contractile rings that either disappear or are retained as spots upon completion of septation. Proteomic analyses of GFP pull down assays identified further proteins that may be part of the septation complex. These data are sued to present a new model of cell wall synthesis during septation in this fungus.

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Development of a plasmid based system for CRISPR-Cas9 mediated mutation in Candida parapsilosis

Lisa Lombardi, Siobhán Turner, Fang Zhao, Geraldine Butler School of Biomedical and Biomolecular Science, Conway Institute, University College Dublin, Dublin, Ireland

The RNA-guided CRISPR-Cas9 endonuclease system has revolutionized genome editing in eukaryotes, and its popularity exponentially increases, with almost 1500 papers published in 2016. The CRISPR-Cas9 elements (Cas9 and guide RNA, gRNA) can be used in the fungal pathogen Candida albicans to knockout gene function (Vyas et al., 2015; Mitchell et al., 2016). However, the published methods require either integration of CAS9 into the genome of the host organism, or its transient expression and integration of a selectable marker. Here we describe the first plasmid-based CRISPR-Cas9 system in Candida species. We have developed a method for gene editing in the diploid yeast Candida parapsilosis, which is frequently responsible for infections in infants. The autonomously replicating plasmid confers Nourseothricin resistance, thus avoiding the need for integrating a selectable marker. Both CAS9 and the gRNA are carried on the same plasmid, and ribozymes flank the gRNA to allow its expression from a PolII promoter (Gao et al., 2014). The system successfully mutated ADE2 in a panel of C. parapsilosis strains, including clinical isolates, with an efficiency ranging from 20 to 80%. The plasmid is lost in absence of selection, so that detrimental effects of prolonged Cas9 expression can be avoided. Overall, this system may allow high throughput generation of mutations in relevant clinical isolates, as well as simultaneously target several genes, which would facilitate the characterisation of gene families in C. parapsilosis.

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From Bug to Drug: In-house production of bespoke antifungal peptide aptamers

Bethany McCann, Christopher Grice, Darren Thomson, Margherita Bertuzzi, Elaine Bignell University of Manchester, Manchester, Greater Manchester, UK

Of around 250 Aspergillus species, Aspergillus fumigatus is the most pathogenic to humans causing invasive fungal infections, and killing upwards of 200,000 people annually. However the number of available antifungal drugs is limited and occurrence of antifungal resistance is increasing.

Peptide aptamers (PAs) can be defined as short; 8-20 amino acids in length, combinatorial peptide fragments doubly constrained within inert protein scaffolds which exhibit functionality much like antibodies. High specificity and affinity for target proteins permits PAs the ability to modulate functionality of targets. Such ligand based perturbation of function paves the way for the elucidation of novel drug targets and the design of potential inhibitory agents against virulence-causing mechanisms.

A. fumigatus mutants lacking the pH-dependent transcription factor PacC, or an upstream pH-responsive receptor protein PalH, display highly attenuated virulence resulting in non-invasive phenotypes. Inhibition of PalH mediated pH signalling is therefore an attractive novel antifungal strategy.

In an initial screen for selection of anti-PalH PAs, we used a yeast membrane two hybrid approach expressing the PalH receptor as bait, and a library of thioredoxin constrained peptide aptamers as preys. The approach successfully identified PalH-binding PAs, however in all instances expression in A. fumigatus delivered gain of pH function phenotypes. We describe the development of two critical new tools, of PAs causing loss of pH signalling, using direct selection in A. fumigatus: a randomised library of Peptide Aptamers (PAs) expressed, under regulatable control, in A. fumigatus, additionally a split-fluorescence approach for direct detection of protein-protein interactions, and their subsequent inhibition.

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E1/8Insights into host-pathogen interactions by dual RNAseq.

Jiyoti Verma1, Timothy Tucey1, Paul Harrison2, Traude Beilharz1, Thomas Naderer1, Ana Traven1 1Infection and Immunity Program and the Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Australia, 2Victorian Bioinformatics Consortium, Monash University, Melbourne, Australia

Candida albicans is the most prevalent fungal species causing human infections. Macrophages are the first line of immune defence against fungal infections. We have previously established that Candida co-culture with macrophages causes host cell death in two distinct phases termed Phase I and II. Phase I death lasts for the first 6-8 hours and results from NLRP3/caspase 1 inflammasome-dependent pyroptosis. Phase II death starts later, it is caspase 1-independent, and it kills the majority (60-70%) of the macrophage population under our experimental conditions, showing it is dominant over pyroptosis. In order to understand host and fungal factors that coordinate these distinct macrophage death pathways, we have performed dual RNAseq of Candida infecting murine bone-marrow-derived macrophages at time points corresponding to early and mid Phase I death, and early Phase II death. We observed changes to the transcriptional programs linked to host antimicrobial responses, host cell death pathways and host and pathogen metabolism as the infection progressed through the Phase I and Phase II stages of host cell death. Our data shows that the transcriptional activators Tye7 and Gal4 play a role in enabling C. albicans to adapt after it escapes from macrophages following pyroptosis, thereby controlling the ability of the pathogen to rapidly elicit Phase II death. Collectively, our study addresses how, by adapting to intracellular and then extracellular infection environments and usurping changes in host physiology during infection, C. albicans is able to thrive and trigger macrophage death.

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Are Candida glabrata drug efflux pumps from the MFS clinically relevant?

Catarina Costa1 ,2, Jonathan Ribeiro1 ,2, Isabel M. Miranda3 ,4, Ana Silva-Dias3 ,4, Mafalda Cavalheiro1 ,2, Sofia Costa-de-Oliveira3 ,4, Acácio R. Gonçalves3 ,4, Miguel C. Teixeira1 ,2 1Department of Bioengineering, Instituto Superior Técnico, University of Lisbon, Lisbon, Portugal, 2iBB - Institute for Bioengineering and Biosciences, Biological Sciences Research Group, Lisbon, Portugal, 3Department of Pathology, Division of Microbiology, Faculty of Medicine, University of Porto, Porto, Portugal, 4CINTESIS - Center for Health Technology and Services Research, Faculty of Medicine of the University of Porto, Porto, Portugal

Azole drug resistance in Candida species has long been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters. Recently, drug efflux pumps from the Candida glabrata Drug:H+ Antiporter (DHA) family were found to play a role in this process. CgAqr1 [1] and CgQdr2 [2] confer resistance to imidazole antifungals, CgQdr2 expression being controlled by CgPdr1. CgTpo3 [3], CgTpo1_1 and CgTpo1_2 [4] confer resistance to imidazoles, but also triazole antifungals. These transporters were shown to catalyze azole extrusion across the plasma membrane [1-4]. A link between DHA gene expression and the clinical acquisition of antifungal drug resistance was recently demonstrated [5]. After screening 138 Candida glabrata clinical isolates for azole drug resistance, harvested from patients attending two major Hospitals in Portugal, ten susceptible and ten resistant to clotrimazole were selected for further analysis. The transcript levels of CgAQR1, CgQDR2, CgTPO1_1 and CgTPO3 were found to be significantly upregulated in resistant isolates when compared to the susceptible ones, with a level of correlation similar to that observed for the ABC encoding gene CgCDR2. Altogether, our results suggest that azole resistance is not the result of the action of a single effector, but rather the combined action of several drug efflux pumps from the ABC and MF Superfamilies.

[1] Costa et al, Front Microbiol, 4:170, 2013; [2] Costa et al, AAC, 57:3159, 2013; [3] Costa et al, JAC, 69:1767, 2014; [4] Pais et al, MCP, 15:57, 2016; [5] Costa et al, Front Microbiol, 7:526, 2016.

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E1/10Bacterial secreted factors regulate mucormycete germination

Courtney Kousser, Kerstin Voelz, Rebecca A Hall Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK

Within the human body, microorganisms reside as part of a complex and varied ecosystem, where they rarely exist in isolation. Therefore, bacteria and fungi have co-evolved to develop elaborate and intricate relationships, utilising both physical and chemical communication mechanisms. Mucormycetes are spore-forming fungi belonging to the order Mucorales and are the causative agent of potentially fatal mucormycosis in immunocompromised individuals. Key to the pathogenesis of mucormycetes is the ability to swell and germinate leading to penetration of the surrounding tissues, angioinvasion, vessel thrombosis, and tissue necrosis. Mycomycete spores are found ubiquitously in the environment and in wounds, where they encounter a myriad of bacterial and fungal species including Pseudomonas aeruginosa. Here we show that culture supernatants from P. aeruginosa inhibit the germination of Rhizopus microsporus spores, without affecting spore swelling. This inhibition of germination was not due to the presence of quorum sensing molecules, but was instead due to the iron restriction, as addition of exogenous iron restored germination. We hypothesise that P. aeruginosa secreted iron siderophores, which sequester the available iron inhibiting fungal germination. Therefore, treatment of P. aeruginosa in trauma wounds could result in the release of this inhibition of germination, leaving the patient prone to an underlying fungal infection.

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S2/1Clearing the FoG: Subpopulation responses to antifungal drugs

Judith Berman, Alex Rosenberg, Alon Dahan Tel Aviv University, Ramat Aviv, Israel

...

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Metabolic adaptations drive resistance to phagocytosis

Michael Lorenz University of Texas McGovern Medical School, Houston, TX, USA

Macrophages are a key component of our innate antimicrobial defenses, yet Candida albicans is highly resistant to these cells. This interaction is dynamic and complex, with remarkable changes in gene expression, physiology, and metabolism in the fungal cell that stem from the recognition that the phagosome is a nutrient-poor environment. Potential carbon sources in this organelle include proteins, amino acids, carboxylic acids, and N-acetylglucosamine (GlcNAc) and C. albicans has adapted its metabolism to utilize these compounds far more efficiently than non-pathogenic model fungi. Remarkably, a common side effect of these disparate catabolic pathways is the extrusion of basic compounds, such as ammonia, that neutralize acidic environments, including the phagolysosome; neutral pH is the signal that induces the characteristic hyphal differentiation that drives escape from the phagocyte. Surprisingly, the genetic requirements for neutralization on the different compounds are non-overlapping; that is, mutations of the transcription factors Stp2 and Cwt1, block neutralization on amino acids and carboxylic acids, respectively, but have no phenotypes on the other. Likewise, the GlcNAc transporter Ngt1 is required only during growth on this molecule. Mutants impaired in any one of these phenomena survive phagocytosis less readily and are less cytotoxic to macrophages. Thus, C. albicans has evolved three independent and likely partially redundant mechanisms to remodel the phagolysosome and promote resistance to macrophage killing.

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Iron – a key hub in virulence of Aspergillus fumigatus

Hubertus Haas Medical University Innsbruck, Innsbruck, Austria

The most common airborne fungal pathogen, the mold Aspergillus fumigatus, employs low-affinity and reductive iron assimilation, vacuolar iron deposition, and the siderophore system to ensure supply with and to avoid toxicity of this essential metal. Siderophore functions comprise uptake, intracellular transport and storage of iron. Siderophore production proved to be crucial not only for adaptation to iron starvation conditions but also for germination, asexual and sexual propagation, antioxidative defense, microbial competition as well as virulence in plant and animal hosts. Control of iron homeostasis including siderophore production is mediated by two central transcription factors, the GATA-factor SreA and the bZip-factor HapX, which are interconnected in a negative transcriptional feed-back loop. SreA represses excessive iron uptake, while HapX functions depend on ambient iron availability: during iron starvation HapX represses iron-consuming pathways and activates iron uptake, while during iron excess HapX activates vacuolar iron detoxification. Moreover, the SREBP transcription factor SrbA links iron metabolism, ergosterol biosynthesis, triazole drug resistance and hypoxia adaptation. Deficiency in HapX or SrbA, but not SreA, attenuates virulence of A. fumigatus in murine models of aspergillosis, which underlines the crucial role of adaptation to iron limitation in virulence. Recent studies in animal models demonstrated that fungal siderophore uptake can be exploited to image aspergillosis via positron emission tomography using 68Gallium-labelled siderophores. Moreover, siderophore production proved to be an attractive new biomarker for aspergillosis.

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Regulation of cell shape and virulence by temperature in Histoplasma capsulatum

Sinem Beyhan1 ,2, Mark Voorhies1, Lauren Rodriguez1, Anita Sil1 1University of California San Francisco, San Francisco, CA, USA, 2JCVI, La Jolla, CA, USA

The long-term goal of our research is to determine how environmental signals such as temperature regulate morphology and virulence in the fungal pathogen Histoplasma capsulatum. H. capsulatum grows in a filamentous form in the soil; once inhaled into a mammalian host, these cells switch their growth program to a parasitic yeast form that subverts the innate immune system to cause disease. Temperature is a key signal that regulates this morphogenetic switch. We identified the first transcriptional regulators required for growth in the yeast form in response to host temperature. These factors are orthologous to key developmental regulators in other fungi, and represent critical elements of the temperature-dependent regulatory circuit in H. capsulatum. We have now identified a number of additional signaling proteins that are critical for the appropriate morphologic and/or transcriptional response to temperature. Ultimately we hope to elucidate an integrated pathway of thermosensory and thermoresponsive proteins required for H. capsulatum to thrive either in the soil or in the mammalian host.

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Endogenous Reactive Oxygen Species in Cryptococcus neoformans morphogenesis and pathogenesis

Christina Nikolakopoulou2, Vic Key Tew2, Elizabeth Ballou1 ,2 1Institute of Microbiology and Infection, School of Biosciences, University of Birmigham, Birmingham, UK, 2MCR Centre for Medical Mycology, Aberdeen Fungal Group, University of Aberdeen, Aberdeen, UK

The human fungal pathogen Cryptococcus neoformans causes an estimated 1 million infections each year, and more than 600,000 deaths world wide. Unlike fungal pathogens such as Candida albicans and Aspergillus species, Cryptococcus causes disease through uncontrolled proliferation as a budding yeast. Reactive Oxygen Species (ROS) are typically considered to be damaging agents that must be tightly regulated by the cell. Exogenous ROS, such as those generated by phagocytic cells, promote host pathogen defense, and endogenous ROS generated by mitochondria lead to DNA damage. However, endogenously produced ROS have also been demonstrated to be key signaling molecules regulating morphogenetic transitions and host mutualism in a number of mammalian, plant, and fungal systems. A major source of these endogenous ROS is the NADPH Oxidase complex, comprised of the Rac GTPase, NADPH Oxidase (Nox), and Nox Regulators (NoxR). The Rac/Nox complex is highly conserved throughout eukaryotes. Here, we investigateC. neoformans genes involved in the positive and negative regulation of endogenously produced ROS. We demonstrate a role for these genes in hyphal morphogenesis, and yeast phase stress resistance, cell ploidy, morphogenetic transitions, and pathogenesis.

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Methionine synthases as drug targets for antifungal drug development

Jennifer Scott, Elaine Bignell, Jorge Amich Manchester Fungal Infection Group, University of Manchester, UK

There are only three classes of antifungals, echinocandins, polyenes and azoles, and only the last can be orally administered. The continued and widespread emergence of drug resistance highlights a desperate need to develop new antifungal treatments. We recently demonstrated that methionine synthase (MetH) is essential for Aspergillus fumigatus viability and virulence, which lead us to propose it as a promising drug target. MetH is also essential in Candida albicans and Cryptococcus neoformans, suggesting a broad spectrum of action for possible novel drugs. To validate the target further, we have constructed a metH_tetOFF regulatable strain and investigated the effects of down-regulating gene expression for A. fumigatus growth. This system has the invaluable advantage that expression can be shut down in mycelia, which mimics the action of a drug on an established infection and therefore overcomes the flaw of using null mutants for virulence studies. We have observed that as little as 0.1 µg/ml doxycycline (dox) prevents colony formation, suggesting effective transcriptional repression. Indeed, 30 minutes after addition to mycelia, dox produces a sustained reduction in metH expression, which causes a strong reduction in fungal growth on both solid and liquid media. We are currently testing the use of the tetOFF system in murine models of aspergillosis, which will hopefully allow us to demonstrate, with unprecedented certainty, that methionine synthase is a valid drug target for the development of novel antifungals. The tetOFF system may be an invaluable new tool to validate targets in vivo without the requirement of an actual drug.

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The Dark Side of the Wall: Atomic Force Microscopy Revelations on Drug Resistance and Adhesion.

Helene Martin-Yken1, Cécile Formosa-Dague1, Marion Schiavone1, Etienne Dague2 1LISBP INRA Universite de Toulouse, Toulouse, France, 2LAAS CNRS, Toulouse, France

The cell wall of yeast and fungi plays a crucial role in the way these cells sense, respond and adapt to environmental perturbations. Using recent Atomic Force Microscopy technological developments, the biophysical consequences of different stresses on the major human fungal pathogen, Candida albicans, were imaged and measured. Morphological changes were characterized at the nanoscale, including surface roughness, elasticity and adhesive properties. Exposure to the antifungal Caspofungin was shown to cause a deep cell wall remodeling with major modifications of chitin and beta-glucan content. Remarkably, a low dose of Caspofungin (i.e., 0.5 × MIC) provoked a strong expression of adhesive proteins on the cell surface of C. albicans, a side effect highly relevant considering its wide spread medical use. Moreover, Single Molecule Force Spectroscopy (SMFS) experiments by AFM allowed us to visualize the organization of these adhesins, to map them on the cell surface and to quantify the adhesion forces, including on cells undergoing mophogenetic differentiation. Combined with molecular biology tools, this approach enabled us to unravel the particular contribution of previously uncharacterized proteins (PGA22 and PGA59) to C. albicans adhesion mechanism. In addition, functionalizing the AFM tip with antibodies allows following the appearance of specific proteins, while precisely mapping them at the cell surface, and even measuring the time scale of their progression through the cell wall. The example of Hwp1 appearance on geminating hyphal tubes will be illustrated.

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The Regulatory Subunit of Protein Kinase A (Bcy1) in Candida albicans Plays Critical Roles in Filamentation and White-Opaque Switching but is Not Essential for Cell Growth

Xuefen Ding, Chengjun Cao, Qiushi Zheng, Guanghua Huang Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

The conserved cAMP-dependent protein kinase (PKA) is composed of the regulatory and catalytic subunits and acts as the central component of the cAMP signaling pathway. In the human fungal pathogen Candida albicans, the PKA regulatory subunit Bcy1 plays a critical role in the regulation of cell differentiation and death. It has long been considered that Bcy1 is essential for cell viability in C. albicans. In the current study, surprisingly, we found that Bcy1 is not required for cell growth, and we successfully generated a bcy1/bcy1 null mutant in C. albicans. Deletion of BCY1 leads to multiple cellular morphologies and promotes the development of filaments. Filamentous and smooth colonies are two typical morphological types of the bcy1/bcy1 mutant, which can undergo spontaneous switching between the two types. Cells of filamentous colonies grow better on a number of different culture media and have a higher survival rate than cells of smooth colonies. In addition, deletion of BCY1 significantly increased the frequency of white-to-opaque switching on N-acetylglucosamine-containing medium. The bcy1/bcy1 null mutant generated herein provides the field a new resource to study the biological functions of the cAMP signaling pathway in C. albicans.

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Unveiling transcriptional regulation in Candida species: using the PathoYeastract database

Pedro T. Monteiro3 ,4, Pedro Pais1 ,2, Catarina Costa1 ,2, Isabel Sá-Correia1 ,2, Miguel C. Teixeira1 ,2 1iBB - Institute for Bioengineering and Biosciences, Lisbon, Portugal, 2Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal, 3Department of Computer Science and Engineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal, 4INESC-ID, Lisbon, Portugal

It is essential to understand the structure and functioning of the transcription networks regulating the early response to clinically relevant environmental changes, in Candida species, to be able to understand and circumvent the long term acquisition of virulence and drug resistance-related phenotypes. In that sense, the PathoYeastract (http://pathoyeastract.org) database was developed for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in the pathogenic yeasts Candida albicans and C. glabrata [1]. This freely available information system currently includes 28,000 unique documented regulatory associations between transcription factors (TF) and target genes and 107 DNA binding sites, considering 134 TFs in both species. Being a twin of the YEASTRACT database [2], PathoYeastract makes available bioinformatics tools to predict the TFs involved in the regulation of a gene or genome-wide transcriptional response. Each search can be filtered to selected only specific environmental conditions or experimental evidence. Promoter analysis tools and visualization tools for the representation of TF regulatory networks are also provided. A preliminary comparative genomics setup for the study of cross-species evolution of regulatory networks is further offered, aiming the prediction of gene and genomic regulation based on orthologous regulatory associations described for other yeast species.

[1] Monteiro et al, Nucleic Acids Res, 45:D597, 2017; [2] Teixeira et al, Nucleic Acids Research, 42: D161, 2014.

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Drug resistance via RNAi-dependent epimutations in Mucor circinelloides

Zanetta Chang1, Blake Billmyre1, Soo Chan Lee2, Joseph Heitman1 1Duke University, Durham, North Carolina, USA, 2University of Texas at San Antonio, San Antonio, Texas, USA

The opportunistic fungal infection mucormycosis is notable for high mortality as well as increasing incidence. Treatment is complicated by the fact that Mucor circinelloides, a major cause of mucormycosis, demonstrates high intrinsic resistance to most antifungal agents. However, the mechanisms driving this extensive resistance remain poorly understood. Previous work demonstrated that Mucor is capable of developing transient resistance to the antifungal FK506 through a novel, RNA interference-dependent mechanism known as epimutation. Epimutants silence the drug target gene and can be selected by exposure to FK506; the target gene is re-expressed in these strains following passage without selective pressure. This silencing process involves the generation of small RNAs (sRNA) against the target gene via core RNAi pathway proteins. To further investigate the role of epimutation in Mucor’s intrinsic antifungal resistance, we studied the development of resistance to a second drug, 5-fluoroorotic acid (5-FOA). We have identified epimutants that exhibit resistance to 5-FOA without mutations in either of the target genes, pyrF or pyrG. We conducted sRNA hybridization analysis to document the presence of sRNA against pyrF or pyrG in these epimutants, and that this sRNA is lost after reversion to drug sensitivity. Analysis of sRNA libraries generated from these epimutants demonstrated expression of sRNA against the pyrF and pyrG loci, respectively. From this, we conclude that epimutation is a general mechanism through which Mucor can develop resistance to multiple antifungal agents.

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Pathogenic yeasts express virulence determinants in a new natural model system to defend against amoeba predation

Silvia Novohradská1, Renáta Tóth2, Sascha Brunke1, Attila Gácser2, Jörg Linde1, Falk Hillmann1 1Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena, Germany, 2Department of Microbiology, University of Szeged, Szeged, Hungary

Candida, Aspergillus and Cryptococcus are the leading causative agents of systemic mycoses. Many of their representatives have also been isolated from non-human sources, suggesting that their virulence potential might have been partially shaped in their natural niche as a strategy to counteract environmental predators. Previously we demonstrated that phagocytic interactions of filamentous fungi are conserved between soil amoeba and macrophages. We have now established a new natural amoeba model system to study evolutionary forces that could have led to the generation and maintenance of virulence traits in human pathogenic yeasts. Protostelium fungivorum is an exclusively mycophagous amoeba, widely spread in nature, which we have successfully isolated and completely sequenced its genome. Large-scale feeding experiments revealed a broad prey spectrum within yeasts and filamentous fungi. With an extreme efficiency, the yeasts were recognized, phagocytosed and killed in a range of few minutes. As C. parapsilosis was found to be a preferential prey, we used this pathogen in a dual-transcriptome approach to identify targets of this predatory-prey interaction. Amoeba-responsive genes included those involved in the elevated metal efflux, oxidative stress response, filamentous growth and secreted lipase, suggesting predatory selection pressure on these important virulence determinants. As a killing mechanism, we propose mobilization of internal copper resources leading to an impaired oxidative stress response and intoxication inside of the acidic phagolysosome. Constructed deletion mutants for the most promising targets will further reveal if traits that have originated to counteract with natural predators could also have supported the resistance against innate immune cells.

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Characterization of the Candida albicans ECE1 promoter

Enrico Garbe1 ,2, Ronny Martin1 ,3, Oliver Kurzai1 ,3, Slavena Vylkova2 1Septomics Research Center, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany, 2Research Group Host Fungal Interfaces, Friedrich Schiller University, Jena, Germany, 3Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany

ECE1 is one of the most abundant genes in C. albicans hyphae. It encodes a cytolytic peptide causing damage to host cells membranes. Like other core filamentation response genes as ALS3 and HWP1, ECE1 has a very long 5’ intergenic region of 3197 bp. In this study we examined which parts of this intergenic region are required for ECE1 regulation during yeast and hyphal growth. 5’ RACE-PCR identified a notably short 5’ UTR of 50 bp, suggesting a TATA box within the first 110 bp upstream of the start codon. Next, various fragments of the intergenic region consisting of the first 500 bp up to the whole length region were fused to GFP and ectopically integrated into the NEUT5L locus. The resulting mutants were screened microscopically for GFP signals and mRNA levels were determined via qRT-PCR. 1200 bp were determined as the minimum size required for a weak fluorescence signal, while 1500 bp and larger fragments induce bright signals, an observation which correlated with detected mRNA amounts. However, a deletion of the first 1000 bp led to the absence of detectable GFP signals, indicating their importance for full activation of ECE1. We further analysed mutants lacking transcription factors potentially binding to the promoter region. Amongst this set Ahr1 turned out as a novel regulator required for high-level transcription of ECE1. Taken together, these findings allow further experiments to define the possible interaction pattern and binding behaviour of transcription factors at this promoter.

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The in vivo efficiency of anidulafungin and tigecycline against polymicrobial catheter-associated peritonitis

Ona Rogiers, Patrick Van Dijck, Sona Kucharikova VIB-KU Leuven Center for Microbiology, KU Leuven Laboratory of Molecular Cell Biology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium

The emerging use of medical devices encounters an increased occurrence of biofilms-related infections. Both the bacterium Staphylococcus aureus and the yeast Candida albicans are key players in the cause of hospital-acquired infections with an extreme ability to inhabit diverse host niches especially in immunocompromised individuals. It is crucial to study and understand the behavior of these pathogens when coexisting together and to discover a viable option for treatment of not only single species but also mixed species biofilms.

In the present study we focused for the first time on C. albicans - S. aureus device- associated intraperitoneal biofilm infections. With the development of a new in vivo model to study catheter-associated peritonitis caused by C. albicans and S. aureus we provided an insight into the pathogenesis of this dual-species biofilm-associated infection and elucidated possible options for treatment. Both anidulafungin and tigecycline showed promising results against intraperitoneal catheter-associated C. albicans – S. aureus biofilm infections when used in appropriate doses (1mg/kg of body weight/day vs 10mg/kg of body weight/day). Further we examined the efficacy of combination treatment consisting of anidulafungin and tigecycline against mature C. albicans – S. aureus intraperitoneal device-related biofilm infections. All together, these results are promising and suggest that in combination anidulafungin drives tigecycline to bactericidal activity. Currently the in vivo studies are conducted and the results will be presented in the poster.

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Temporal and spatial control of fungal filamentous growth

Patrícia M. Silva, Martine Bassilana, Robert A. Arkowitz Université Côte d’Azur, CNRS, Inserm, iBV, Nice, France

Candida albicans is a human pathogen that can cause life-threatening infections in immunocompromised patients, in part, due to its ability to switch between an oval budding form and a hyphal form. The small-Rho GTPase Cdc42 plays a crucial role in hyphal growth and, in its active state, localizes as a persistent tight cluster at the tips of growing hyphal filaments (1). Our goal is to investigate the importance of this site-specific location and whether it is crucial for initiation and maintenance of hyphal growth. Using a light-activated membrane recruitment system, we are able to control the recruitment of constitutively active Cdc42 to the plasma membrane. We are investigating how such a photo-recruitment disrupts hyphal growth and where, when and how is new growth subsequently initiated. Our results demonstrate that, upon photo-recruitment of active Cdc42, filament extension is abrogated and new growth can occur throughout the cell. Location of new growth correlates with the length of the filament, which may reflect how well- established the growth site is. Before a new growth site appears, the initial filament stops extending, concomitant with the disruption of the cluster of endogenous active Cdc42. We are currently investigating the molecular mechanisms that underlie this disruption at the initial hyphal growth site and the subsequent site- specific initiation of new hyphal growth.

(1) Corvest, Bogliolo, Follette, Arkowitz & Bassilana (2013). Mol Microbiol. 89: 626-48.

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The role of APSES transcription factors in Histoplasma capsulatum virulence and adaptation to stress conditions

Larissa V G Longo1 ,2, Rosana Puccia1, Chad A Rappleye2 1Escola Paulista de Medicina - Universidade Federal de São Paulo - UNIFESP, São Paulo, SP, Brazil, 2The Ohio State University - OSU, Columbus, OH, USA

The APSES proteins belong to a fungal-specific family of transcriptional factors of the basic helix-loop-helix (bHLH) class. They function as key regulators of development and other biological processes, such as stress response and control of virulence traits in various fungi. Our work investigates the role of the APSES transcription factors in the biology and virulence of the thermodimorphic pathogenic fungus Histoplasma. capsulatum. The H. capsulatum encodes four APSES proteins: Stu1, Aps2, Mbp1, and Swi6. None of these factors showed increased expression under nitrosative or oxidative stress or during macrophage infection. However incubation at pH 4 caused a 2-fold down-regulation of Swi6 and Mbp1 and a 4-fold up-regulation of Stu1 compared to yeasts at pH 7, suggesting roles in pH-dependent responses. To determine their functional roles in Histoplasma biology, RNA-interference (RNAi) was used to knocked-down APSES protein levels. Lines were generated that had more than 90% silencing of Stu1, Aps2, and Swi6 as determined by co-silencing of the GFP sentinel. However, only 50% knock down was achieved for Mbp1, suggesting some essentiality of Mbp1. All knocked-down lines showed normal growth kinetics of yeasts and virulence in P388D1 macrophages. In a mouse model of histoplasmosis, Stu1- and Aps2-deficient strains were strongly attenuated. In addition, the fungal transition from yeast to mycelial phase was impaired in APSES-deficient cells. These data indicate Histoplasma virulence in vivo requires APSES-family transcription factors and further experiments to assess what aspect of infection is defective are being performed.

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Fungal Transformers: Tracking a Moving Target

Delma Childers1, Gabriela Avelar1, Judith Bain1, Arnab Pradhan1, Daniel Larcombe1, Mihai Netea2, Lars Erwig1 ,3, Neil Gow1, Alistair Brown1 1University of Aberdeen Centre for Medical Mycology, Aberdeen, UK, 2Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands, 3GlaxoSmithKline, London, UK

In human hosts, the opportunistic fungal pathogen Candida albicans often proliferates in sugar-poor niches (e.g. the gut), but is exposed to sugars in other niches (e.g. the bloodstream). Carbon sensing regulates several aspects of fungal physiology including metabolism, cell wall architecture and elasticity, and virulence. In addition, yeast cell division exposes pathogen-associated molecular patterns (PAMPs) at the cell surface that are known to be immune-stimulatory (e.g. β-glucan). Host niches, morphogenesis and antifungal drugs have also been implicated in PAMP exposure. However, little is known about which host signals influence PAMP exposure and the molecular mechanisms that modulate this exposure. We have recently shown that lactate, an alternative carbon source present in mucosal niches and produced by activated innate immune cells, acts as a signalling molecule to modulate β-glucan exposure (Ballou et al., Nature Micro, 2016). Lactate-induced β-glucan masking is driven via a non-canonical signal transduction pathway resulting in a calcineurin-independent activation of the transcription factor Crz1. However, it remains to be elucidated whether the reduction in β-glucan exposure is the result of PAMP camouflaging by other cell wall components, by PAMP modification, or a combination of both. Therefore, we are characterizing the signalling pathways and downstream effectors that affect PAMP exposure in response to different carbon sources and other key environmental inputs that C. albicans encounters in host niches. We are also examining the impact of PAMP exposure on human disease.

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Fast in vivo super-resolution microscopy and background reduction in Candida albicans.

Wouter Van Genechten, Liesbeth Demuyser, Peter Dedecker, Patrick Van Dijck KU Leuven, Leuven, Belgium

Optical and electron microscopy are extremely valuable tools in life sciences, but both techniques also have their disadvantages. One of the drawbacks of optical microscopy is the restriction on resolution enhancement due to the diffraction limit of light. Electron microscopy on the other hand, can only be performed on fixed, and thus dead, cells impairing real-time visualization of the subject molecule.

State-of-the-art microscopy techniques such as super-resolution optical fluctuation imaging (SOFI) combine solutions for both problems. They overcome the diffraction limit of light using temporal fluctuations of photoswitchable fluorescent proteins visualized with normal in vivo imaging techniques. So far these techniques have been performed and developed for use in mammalian cells yet not, as far as we know, in any of the Candida species. In this work we present the development and application of photoswitchable fluorescent proteins (PSFP) for use in the major human fungal pathogen Candida albicans.

Two Candida-optimized variants of a green PSFP have shown to be very promising for use in super-resolution microscopy. Attaching these fluorescent proteins to several structural and signaling components showed precise localization. A second major advantage of this system is the ability to reduce the background fluorescence of C. albicans. The latter is a well-known problem when visualizing proteins with low abundancy. Combining the increase in resolution and consequent possibility to do accurate co-localization assays with the reduction of the autofluorescence, renders this an extremely valuable tool for any researcher performing fluorescence microscopy on C. albicans.

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Mitochondrial proline metabolism is key to arginine-induced morphogenesis in Candida albicans

Fitz Gerald Silao, Kicki Ryman, Per Ljungdahl Stockholm University, Stockholm, Sweden

Amino acids (AA) are among the best-established inducers of morphogenesis in the human fungal pathogen Candida albicans, yet a clear mechanistic understanding of how AA initiate the yeast-to-filamentous switch is lacking. Using a combination of genetic and biochemical approaches we have shown that the inducing signal from several AA is derived from their catabolism within cells rather than being sensed in the extracellular environment. Specifically, proline, one of the most potent inducers of morphogenesis, is exclusively metabolized in the mitochondria, generating the reduced electron carriers NADH and FADH2 as catabolic by-products. These reduced carriers are oxidized by the mitochondrial electron transport chain, a process coupled to the generation of ATP. Interestingly, strongly inducing AA such as arginine and ornithine are also catabolized through proline utilization pathway. These results align well with a recent model that elevated intracellular levels of ATP is key to the induction of the Ras1/cAMP/PKA signaling cascade; this pathway positively controls the activity of the morphogenic effector transcription factor Efg1. Our findings are inconsistent with the accepted model that arginine induces morphogenesis primarily through the generation of CO2. Taken together, our results provide the basis of an alternative unifying model in which the catabolism of arginine (and ornithine) to glutamate via proline in the mitochondria leads to elevated ATP levels, which in turn stimulate the Ras1/cAMP/PKA pathway, positively activing Efg1 and thereby inducing the genetic program that engages the yeast-to-filamentous switch.

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CRISPR-Cas9-engineered C. glabrata strains for the study of virulence factors in animal models.

Yacine Dahman1, Ludovic Enkler1, Julie Denis2, Delphine Richer1, Ermanno Candolfi2, Fabrice Jossinet1 1Institut de Biologie Moleculaire et Cellulaire (I.B.M.C), UPR 9002, Strasbourg, France, 2Institut de Parasitologie et de Pathologie Tropicale de Strasbourg, EA 7292, Strasbourg, France

Recent estimates suggest that about 1 billion people worldwide suffer from disease related to fungi. Among them, the opportunistic fungal pathogen Candida glabrata is a major public health concern. This increases the need to develop innovative therapeutic strategies to circumvent its high rates of resistance to antifungals. To better understand the pathogenicity of C. glabrata, we studied its transcriptional landscape during systematic infection in immunocompromised Drosophila melanogaster. Using RNAseq, we identified several gene candidates differentially expressed and potentially involved in the host adaptation and/or the infection process. To validate these genes as potential virulence factors, we developed a recombinant strain of C. glabrata expressing the CRISPR-Cas9 system. Several candidates have been knocked-out and were challenged for infection fitness in flies. In parallel, we’re using this genetic tool to investigate one of the most striking molecular adaptation of C. glabrata. It has been observed that the RNA component of its nuclear RNase P harbors three large additional domains. Such insertions have already been observed as a driving force in the evolution of non-coding RNAs. Frequently, they allow the interaction with proteins leading to novel biological functions. In order to test this hypothesis, we have produced a CRISPR-Cas9 variant of this RNase P. It allows us to purify the ribonucleoprotein complex and to identify its protein components by mass spectrometry. We have tested and compared the composition of this complex between several experimental conditions: liquid cultures and during infection of models such as human macrophages and Drosophila melanogaster.

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Adventures in Antifungal Drug Development

Damian Krysan University of Rochester, New York, USA

The species H. sapiens has developed a total of three classes of antifungal drugs that are generally applicable to the treatment of life-threatening fungal infections. The most recent addition to this trio, the echinocandins, was initially discovered in the early 1970’s and was finally introduced into clinical practice in 2002. This glacially slow pace of antifungal drug development is starkly contrasted by the steadily increasing global importance of fungal infections as causes of human death and suffering. There are a number of challenges inherent in antifungal drug development and these will be discussed. My laboratory has been interested in applying different strategies to addressing the lack of antifungal drugs including re-purposing existing drugs, applying novel screening assays to the identification of new chemical matter, and targeting novel cellular processes. In this presentation, I will discuss examples of each of these approaches including the repurposing of tamoxifen as an adjuvant therapy for cryptococcal meningitis, the identification of a new class of fungicidal anti-cryptococcal small molecules through high throughput screening, and the exploration of acetyl CoA synthetase as a new antifungal target.

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New treatment strategies for cryptococcosis?

Francoise Dromer Institut Pasteur, Paris 75015, France

Management of invasive fungal infection is far from optimal as shown by their increased incidence over time and mortality rates that are still too high despite antifungal therapies. We will focus on cryptococcosis that remains a concern in Sub-Saharan Africa since it is a major cause of death in HIV-infecting patients in these countries despite access to antiretroviral therapy. New strategies are developed including the implementation of systematic screening of capsular polysaccharide in asymptomatic patients with low CD4 counts, and the use of different therapeutic regimen for induction therapy. We will discuss if the development of a cryptococcal vaccine could be envisioned in this setting knowing that infection by C. neoformans occurs early in life and that individuals who develop the disease are severely immunocompromised.

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Plant defensins and linear plant defensin-derived peptides increase caspofungin’s antibiofilm against Candida biofilms in vitro and in vivo

Tanne Cools1, Caroline Struyfs1, Jan Wouter Drijfhout2, Celia Lobo Romero3 ,4, Sona Kucharikova3 ,4, Patrick Van Dijck3 ,4, Bruno Cammue1 ,5, Karin Thevissen1 1CMPG, KU Leuven, Leuven, Belgium, 2Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, The Netherlands, 3Department of Molecular Microbiology, VIB, Leuven, Belgium, 4Laboratory of Molecular Cell Biology, KU Leuven, Leuven, Belgium, 5Department of Plant Systems Biology, VIB, Gent, Belgium

Public health problems are frequently associated with device-associated biofilm infections, with Candida albicans being the major fungal pathogen in this respect. We identified potent antibiofilm combination treatment in which caspofungin, the preferred antimycotic to treat such infections, is co-administered with a peptide, either a native antifungal plant defensin such as HsAFP1 isolated from the plant Heuchera sanguinea or linear HsAFP1-derived peptides. In this regard, we identified the 19-mer peptide HsLin06_18, which is the smallest linear HsAFP1-derived peptide that can still increase caspofungin’s but also anidulafungin’s antibiofilm activity. We found that a combination of caspofungin and HsLin06_18 significantly reduced survival of C. glabrata and C. albicans biofilms grown on catheters, which was confirmed in a subcutaneous rat catheter model. Mode of action research on the caspofungin-HsLin06_18 combination points to increased uptake of HsLin06_18 upon treatment with subinhibitory caspofungin doses, resulting in immediate membrane permeabilization. All these findings point to broad-spectrum antibiofilm activity of a combination of HsLin06_18 and caspofungin and to its clinical potential to treat fungal biofilm-based infections.

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Selective BET bromodomain inhibition as a potential antifungal therapeutic strategy

Flore Mietton1 ,2, Elena Ferri3, Morgane Champleboux2, Ninon Zala2, Danièle Maubon4, Yingsheng Zhou3, Mike Harbut5, Didier Spittler1, Cécile Garnaud4, Marie Courçon2, Murielle Chauvel6, Christophe d’Enfert6, Boris Kashemirov3, Mitchell Hull5, Charles McKenna3, Jérôme Govin2, Carlo Petosa1 1Institut de Biologie Structurale, Université de Grenoble Alpes, CEA, CNRS, Grenoble, France, 2Institut de Biosciences et Biotechnologies de Grenoble, Université de Grenoble Alpes, CEA, Inserm, Grenoble, France, 3Department of Chemistry, University of Southern California, Los Angeles, CA, USA, 4Institut de Biologie et Pathologie, CHU Grenoble Alpes, Grenoble, France, 5California Institute for Biomedical Research, La Jolla, CA, USA, 6Institut Pasteur, INRA, Unité Biologie et Pathogénicité Fongiques, Paris, France

Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. We investigated a chromatin-interacting module, the bromodomain from the BET family of proteins, as a potential antifungal target in Candida albicans. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two bromodomains result in a loss of viability in vitro and decreased virulence in mice. Using high-throughput chemical screening, we identified compounds that inhibit C. albicans Bdf1 in vitro with high selectivity over human bromodomains. Crystal structures of the Bdf1 bromodomains reveal binding modes for these inhibitors that are sterically incompatible with the human BET binding pockets. Furthermore, we identified a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 bromodomain-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.

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Commensal anaerobic gut microbiota promote Candida albicans colonization resistance

Laura Coughlin, Sarah Hussain, Andrew Koh University of Texas Southwestern Medical Center, Dallas, TX, USA

Background

Candida albicans (CA) colonization is required for invasive disease. We have demonstrated that commensal anaerobic bacteria are critical for maintaining CA colonization resistance in mice (Fan et. al. Nat Med 2015). Activation of the transcription factor HIF-1α and the antimicrobial peptide LL-37 (CRAMP in mice) by commensal bacteria is critical for inducing protection against CA colonization of the gut.

Methods:

C57BL/6 mice (female, 6-8 wks; Jackson, Taconic, or Envigo) were given oral antibiotics (drinking water) for 7 days and then orally gavaged with CA strain SC5314. CA colonization levels were assessed by culturing stool homogenates on selective media. 16S rRNA sequencing was performed on fecal pellets. Separate mice were pre-treated with antibiotics, colonized with CA, and then challenged with Bifidobacterium longum and Bifidobacterium infantis. Bacterial and fungal levels were measured weekly. Mouse colon HIF-1α and CRAMP mRNA and protein levels were then measured.

Results:

Untreated (no antibiotics) mice were resistant to CA colonization. Penicillin-treated mice were susceptible to CA colonization. Streptomycin-treated mice showed variable CA colonization phenotypes. 16S rRNA sequencing revealed an expansion of Bifidobacteria in streptomycin-treated Envigo mice resistant to CA colonization. Addition of Bifidobacteria longum to mice colonized with CA promoted CA colonization resistance. B. longum induced both HIF-1α and CRAMP mRNA and protein expression in the mouse colon. Mice co-colonized with B. longum and CA had significantly decreased mortality from infection after immunosuppression compared to mice colonized with CA only. HIF-1α and CRAMP were required for B. longum-induced protection against C. albicans colonization of the gut.

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Forbidden Kingdom: role of the mycobiome and fungus-host interactions in human skin disease.

Thomas Dawson1 ,2 1Agency for Science, Technology, and Research, Institute of Medical Biology, Singapore, Singapore, 2Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC, USA

While the gut bacterial microbiome has achieved virtual celebrity status, the human skin microbiome remains elusive and poorly investigated. Only recently have investigations focused on the skin biome and inclusive metagenomic or ITS studies which move beyond bacteria. Nearly a billion people worldwide are affected by fungal mediated disease, and multiple studies indicate a likely causative role for fungi in skin disorders such as pityariasis versicolor and seborrheic dermatitis, and a role in exacerbation of others including atopic dermatitis, atopic eczema, and psoriasis. However, it remains unclear what role the microbiome would play in maintenance of skin health. Recent work in our labs indicates that the most common skin fungi, Malassezia, communicate with the host immune system through specific lipid mediators. Study of the skin microbiome should provide a window into interaction of commensal microbes with the immune system from a more easily accessed compartment (the skin surface) which is also more easily influenced (via topical treatment). However, future detailed work will be required to elucidate the role of and treatment for the skin microbiome in human health and disease.

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Gut Fungal Microbiota: The Yin and Yang of Inflammatory Bowel Disease

Mathias L. Richard1, Harry Sokol2 ,3 1INRA, Micalis Institute, Jouy en Josas, France, 2INSERM, U1157, Paris, France, 3Gastroenterology Department, St Antoine Hospital, Paris, France

The prevalence of inflammatory bowel diseases (IBD) has been steadily increasing since 1960. They are widespread throughout Europe, North America, China, and Japan and are emerging as a global disease. The equilibrium among epithelial cells, the immune system, and the related microbiota seems to be paramount in ensuring the absence of these IBD. The role of bacteria in the setting of the gut microbiota has been thoroughly documented, but the role of fungi, which are less abundant, needs to be investigated. Faecal and mucosa-associated fungal microbiota was thus investigated in different cohorts of healthy and IBD patients. Barcoding data showed a clear fungal and bacterial dysbiosis. We also identified many correlations between bacterial and fungal components with differences between IBD and healthy subjects, suggesting the existence of disease-specific inter-kingdom alterations. Specific mouse lines (Card9, Dectin1/2) and conventional or germ free models are now used to decipher the mechanisms underlying these observations.

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The regulation of host resistance to fungi by the microbiota: towards a systems-level understanding of fungal diseases

Giuseppe Paolicelli1, Matteo Puccetti2, Teresa Zelante1, Luigina Romani1 1University of Perugia, Department of Experimental Medicine, Perugia, Italy, 2University of Perugia, Department of Pharmaceutical Sciences, Perugia, Italy

An increased understanding of the importance of microbiota in shaping the host’s immune and metabolic activities has rendered fungal interactions with their hosts more complex than previously appreciated. It is now clear that a three-way interaction between host, fungi, and microbiota dictates the types of host-fungus relationship. Indeed, microbial dysbiosis predisposes to a variety of chronic fungal infections and diseases at local and distant sites. We have explored metagenomics for the purpose of deciphering the contribution of the microbiota to fungal infectivity. By correlating changes in metabolite profiles with microbiota metagenomic composition, we have defined several functional nodes by which certain bacteria species contribute to or subvert host-fungal symbiosis and mucosal homeostasis in the gut and lung. Distinct microbiota metabolites contribute to inflammation or tolerance to Candida albicans or Aspergillus fumigatus in different experimental settings and contribute to regulation of unwanted immune response at distant sites. These findings provide evidence for the direct link between the microbiota and the host immune response to fungi via metabolic inflammation and tolerance.

This work is supported by the Specific Targeted Research Project FUNMETA (ERC-2011-AdG-293714).

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IL-17 signal transduction and immunity to Candida albicans

Sarah Gaffen university of Pittsburgh, Pittsburgh, USA

Candida albicans is a commensal fungus responsible for opportunistic oropharyngeal candidiasis (OPC, thrush) in settings of immunodeficiency such as HIV/AIDS and chemotherapy. C. albicans also causes systemic and dermal infections. in The cytokine IL-17 is essential for protective immunity to C. albicans, as impairments in IL-17 receptor (IL-17R) signaling components (IL-17RA, IL-17RC, Act1) cause mucosal candidiasis in both murine models of infection as well as in humans with defects in the IL-17 receptor signaling pathway. The IL-17R complex is ubiquitously expressed, and antifungal signaling by IL-17 has been documented in cells of mesenchymal and epithelial origin but also in hematopoietic cells. This talk will focus on multiple mechanisms by which IL-17 drives immunity to C. albicans, including (i) tissue-specific effects of IL-17 signaling, (ii) sources of IL-17 that drive immunity to mucosal candidiasis, and (iii) fungal-derived factors that drive IL-17 production in vivo in the context of infection.

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Innate immune memory and fungal infections

Jessica Quintin Institut Pasteur, Paris, France

The innate arm of the immune system is generally considered as primitive and non-specific and in contrast to the adaptive immune arm, to be devoid of memory. However in plants and invertebrate animals that lack adaptive immunity, innate immunity exhibits a prolonged enhanced functional state after adequate priming. Similar enhancement of the innate immune functions has occasionally been observed in vertebrates, including humans. Non-specific protective effects against reinfection have been described following infection with Candida albicans, a phenomenon we coined as trained immunity. Mice defective in functional T and B lymphocytes are protected against reinfection. Monocytes and macrophages are among the main cells of the innate immune arm that can be primed. C. albicans and beta-glucans induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. The immunological imprinting after a primary infection determines the functional fate of monocytes and macrophages, and the susceptibility of the host to secondary infections.

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Novel molecular mechanisms regulating phagosome biogenesis during interplay of airborne filamentous fungi with the mammalian phagocytes

Georgios Chamilos University of Crete, and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Crete, Greece

Airborne filamentous fungi are emerging causes of life threatening infections in the expanding population of immunocompromised patients. In the immunocompetent host clearance of inhaled fungal conidia occurs on a daily basis via a highly coordinated immune response that confers pathogen elimination while preventing inflammatory lung damage. Efficient elimination of fungal conidia by professional phagocytes occurs relatively late (within hours) inside acidified phagolysosomes via incompletely characterized mechanisms. Work from our group revealed that a specialized autophagy pathway termed LC3 associated phagocytosis (LAP) is a master regulator of phagosome biogenesis and intracellular killing of Aspergillus fumigatus by monocytes/macrophages ex vivo and in vivo. Notably, LAP has distinct signaling requirements than canonical (macro) autophagy including activation of a unique VPS34 PI3K III complex for sustained PtdIns (3) P production on the phagosome, stabilization of NADPH oxidase complex and subsequent LC3 II recruitment. From the pathogen perspective, A. fumigatus cell wall melanin targets LAP to promote virulence. Notably, LAP blockade is a shared property of melanin pigments mediated via the disruption of NADPH oxidase assembly on the phagosome membrane. Interference with a novel Ca2+ signaling pathway upstream of VPS34 and is the predominant mechanism of melanin induced inhibition of LAP. Physiologically, melanin removal during intracellular swelling of A. fumigatus conidia is a fundamental requirement for activation of LAP and fungal killing. Importantly, the ability of other filamentous fungi for surface retention of melanin induces phagosome maturation arrest and prolonged intracellular persistence. Overall, harnessing antifungal autophagy is an appealing therapeutic strategy to combat fungal diseases.

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The Genetic Architecture of Innate Antifungal Immunity

Agostinho Carvalho University of Minho, Braga, Portugal

Recent progress in medical care has, paradoxically, contributed to an increased prevalence of life-threatening susceptibility to severe forms of fungal infection, such as invasive aspergillosis (IA). Despite remarkable progress in the diagnosis and treatment of IA, it remains a leading cause of morbidity and mortality among immunocompromised hosts. Given the variable risk of infection and its clinical outcome even among patients with comparable predisposing clinical factors and microbiological exposure, susceptibility to IA is thought to rely largely on genetic predisposition. Resolving the mechanisms by which genetic variation contributes to susceptibility to infection is therefore of intrinsic biological interest and fundamental to translate ongoing efforts into clinical utility. Innate protection against fungi is conferred mainly through phagocytes that recognize molecular motifs from pathogens through pattern recognition receptors, and through humoral recognition molecules, which by retaining antibody-like effector functions, contribute to pathogen recognition and opsonization, and tuning of inflammation. Given their key role at the crossroads of innate and adaptive antifungal immunity, several genetic variants in these receptor families have been pinpointed as key risk factors for IA. Although the overall weight of the antifungal immune response clearly results from adding effects of single genetic factors and their complex interactions with clinical immune dysfunctions, a few variants have been validated as robust genetic markers. These consistent findings are expected to lay the foundations for well-designed prospective trials, ultimately endorsing genetic testing in risk stratification approaches for IA.

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S5/5Fungal Effector Mechanisms: Direct Killing by Cytolytic Lymphocytes

Christopher Mody University of Calgary, Calgary, Canada

We can argue whether fungi or malignant tumours provided the first and greatest evolutionary pressure for advanced species. Nevertheless, host defence to both of these invaders is paramount to survival. Perhaps it is not a surprise that vertebrates developed a highly sophisticated and targeted innate mechanism of host defence that shares elements to kill both fungi and malignant cells. Both T lymphocytes and Natural Killer (NK) cells have the ability to directly recognize and kill fungi. For this purpose, NK cells use a new class of pattern recognition receptors called the Ig-like cytolytic receptors. Ligation of these cytolytic receptors results in the formation of a synapse between the fungal and cytolytic host cell. The receptor then triggers a unique signaling pathway resulting in distinct cytolytic granule mobilization to the area of fungal contact, degranulation, release of cytolytic effectors and fungal killing. In this lecture I will review the initial work leading to the concept of direct fungal killing by cytolytic T cells and NK cells. I will contrast it to the killing of intracellular microbial pathogens by cytolytic lymphocytes. I will review the recent literature, including the concept of cytolytic pattern recognition receptors and their fungal ligands. I will present new information on the unique features of NK cell signal transduction and cytolytic granule trafficking to the fungal synapse leading to fungal cell death.

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The intraspecies diversity of C. albicans triggers qualitatively and temporally distinct hostresponses that determine the balance between commensalism and pathogenicity.

Salome Leibundgut Universtiy of Zurich, Zurich, Switzerland

The host immune status is critical for preventing opportunistic infections with C. albicans. Whether the natural fungal diversity that exists between C. albicans isolates also influences disease development remains unclear. Here, we used an experimental model of oral infection to probe the host response to diverse C. albicans isolates in vivo and found dramatic differences in their ability to persist in the oral mucosa, which inversely correlated with the degree and kinetics of immune activation in the host. Strikingly, the requirement of IL-17 signaling for fungal control was conserved between isolates, including isolates with delayed induction of IL-17. This underscores the relevance of IL-17 immunity in mucosal defense against C. albicans. In contrast, the accumulation of neutrophils and induction of inflammation in the infected tissue was strictly strain-dependent. The dichotomy of the inflammatory neutrophil response was linked to the capacity of fungal strains to cause cellular damage and release of alarmins from the epithelium. The epithelium thus translates differences in the fungus into qualitatively distinct host responses. Together, this study provides a comprehensive understanding of the antifungal response in the oral mucosa and demonstrates the relevance of evaluating intraspecies differences for the outcome of fungal-host interactions in vivo.

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Calcineurin orchestrates lateral transfer of Aspergillus fumigatus during macrophage cell death

Anand Shah, Shichina Kannambath, Susanne Herbst, Matt Fisher, Serge Mostowy, Darius Armstrong-James Imperial College London, United Kingdom, UK

Aspergillus fumigatus (Af) is a lethal fungal pathogen in transplant recipients. We report a major role for the calcineurin pathway, which is the primary target of transplant immunosuppression, in initial fungal control of Af in human macrophages. Calcineurin was required for phagocytosis, reactive oxygen species production, and killing of Af. Ultimately, progressive fungal germination within macrophages triggered calcineurin-dependent necroptotic cell death, and was coupled to lateral transfer (metaforosis) of Af to recipient macrophages to enable fungal control. Metaforosis occurred through a vasodilator-stimulated phosphoprotein (VASP)-actin encapsulated late endosomal compartment. We confirmed the relvance of this phenomenom in vivo in a zebrafish model of invasive aspergillosis. These observations with a model mould pathogen identify an entirely novel calcineurin-dependent form of programmed cell death as a primary innate response to fungi in the lung, which facilitates cooperative control of infection by macrophages through a new cell-cell transfer mechanism know as metaforosis. To our knowledge this is the first description of a host-mediated pathogen cell to cell transfer mechanism. These findings yield novel insights into host innate immunity to Aspergillus fumigatus in the lung, extend current understanding of the pathogenesis of pulmonary aspergillosis in organ transplantation, and have broader relevance to transplant immunity and chronic inflammatory lung disease.

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Dermal Fibroblasts Play a Central Role in Skin Model Protection against C. albicans Invasion

Andreas Kühbacher1, Philip Stevens3, Helena Henkel1, Christian Grumaz1, Doris Finkelmeier1, Anke Burger-Kentischer1 ,2, Kai Sohn1, Steffen Rupp1 ,2 1Fraunhofer IGB, Stuttgart, Germany, 2University of Stuttgart, Stuttgart, Germany, 3Medical University of Vienna, Vienna, Austria

Research efforts in the last decades have shed light on mechanistic interactions of fungal pathogens with host organisms down to the molecular level. Although considerable progress has been made in the last decades, the function of the individual components of epithelial barriers with regard to host-microbial interaction is not fully understood.

The fungal pathogen Candida albicans colonizes basically all human epithelial surfaces including the skin. Under certain conditions, such as immunosuppression, invasion of the normally protected epithelia occurs. In natural skin these infections are only superficial but not much is known about defense mechanisms against C. albicans in subepithelial layers such as the dermis.Using immune cell-supplemented 3D skin models we could define a new role for fibroblasts in the dermis and identify a minimal set of cell types for skin protection against C. albicans invasion. Dual RNA-Seq of individual host cell populations and C. albicans revealed that dermal invasion is directly impeded by dermal fibroblasts. They are able to integrate signals from the pathogen and CD4+ T cells and shift towards an antimicrobial phenotype with broad specificity that is dependent on TLR2 and IL-1β. Skin model protection could be induced by addition of IL-1β even in the absence of T cells. TLR2 activation in dermal fibroblasts which occurs through C. albicans, results in induction of IL-1β expression, but it is secreted only in the presence of CD4+ T cells. These results highlight a central function of dermal fibroblasts for skin protection opening new possibilities for treatment of infectious diseases.

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Host factor adenosine modulates inflammatory responses in systemic candidiasis

Ava Hosseinzadeh1, Munender Vodnala1, Madhu Shankar1, Mattias Carlström2, Anders Hofer1, Constantin Urban1 1Umeå University, Umeå, Sweden, 2Karolinska Institute, Stockholm, Sweden

Invasive fungal infections may be accompanied by overwhelming inflammatory responses in the host, often at the time of immune recovery, resulting in immune reconstitution inflammatory syndrome (IRIS). In search for host factors which reduce inflammatory responses in systemic mycoses we investigated adenosine. The nucleoside is produced in many tissues, such as for instance by endothelium. Adenosine is considered as anti-inflammatory molecule conveying function via specific receptors, classified in four different subtypes A1, A2A, A2B and A3. The role of adenosine in regulating inflammatory processes during microbial infection is poorly understood. We showed that adenosine levels increase more than ten-fold in plasma of mice systemically infected with Candida albicans. Neutrophils are recruited to sites of C. albicans infection in large numbers and contribute to an inflammatory milieu by production of ROS, secretion of pro-inflammatory cytokines and release of neutrophil extracellular traps (NETs). Interestingly, we found that adenosine-treated neutrophils released less amounts of cytokines and NETs upon Candida stimulation. Adenosine-triggered regulation was mediated via adenosine receptor A3 expressed on neutrophils. The signal was further conveyed by PI3 kinase and protein kinase B (AKT). Pharmacological activation of A3 in a murine model of systemic candidiasis reduced inflammation and markers for organ damage in plasma. We are currently investigating A3 knockout mice for confirmation of pharmacological data. In summary, our findings identify adenosine as important modulator of inflammation during systemic candidiasis and suggest that pharmacological targeting of A3 receptor could be a promising adjunct therapy for IRIS during episodes of invasive mycoses.

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Understanding How the Hsp90 Kinase Interactome Affects Candida albicans Virulence

Leenah Alaalm1 ,2, Carolyn Williamson1 ,2, Kangzhen Dong1 ,2, Stephanie Diezmann1 ,2, Ranjith Rajendran3, Gordon Ramage3 1Department of Biology & Biochemistry, University of Bath,, Bath, UK, 2The Milner Centre for Evolution, University of Bath,, Bath, UK, 3Glasgow Dental Hospital & School, University of Glasgow Dental School, Glasgow, UK

Systemic infections caused by Candida albicans are often life-threatening and difficult to treat due to therapeutic limitations. Here, we are focusing on the essential and central regulator, the heat shock protein 90 (Hsp90) hence it plays a key role in regulating several virulence traits of C. albicans. Yet, treatment combining standard antifungal therapy and Hsp90 inhibition resulted in severe host toxicity in the murine model of systemic candidiasis. Thus, novel drug targets are urgently needed. A chemical genomic screen revealed that Hsp90 regulates 226 C. albicans genes, with 35 encoding kinases, which are considered attractive drug targets. We propose that combinatorial therapy comprising Hsp90 inhibitors, which are already in clinical trials as anti-cancer drugs, and C. albicans-specific kinase inhibitors could provide an effective therapeutic strategy for the treatment of systemic candidiasis. To identify suitable kinases, we study two sets, those that regulate Hsp90 function (Ck2 kinase complex) and those that require Hsp90 for proper function (Hsp90-target kinases). So far, we identified novel physical interactions between Hsp90 and its kinase-targeting co-chaperone Cdc37 and the Ck2 kinase complex. We selected nine Hsp90-target kinases, four highly conserved and five uncharacterized, fungal-specific for further study of their virulence potential. Gene deletion mutants are characterized in a series of in vitro and in vivo assays. So far, we identified three kinases affecting morphogenesis, and two kinases regulating Fluconazole resistance in an Hsp90-dependent fashion. Understanding how the Hsp90-kinase circuitry regulates virulence will be a crucial milestone in our efforts to identify novel drug targets.

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Uncovering new links between mitochondria and hyphal growth of Candida albicans

Barbara Koch, Adele Barugahare, Traude Beilharz, Ana Traven Monash University, Melbourne, Australia

The morphological switch between yeast and hyphae is a key virulence factor for Candida albicans. Here we investigated the roles of mitochondrial dynamics in hyphal growth of C. albicans using a small molecule inhibitor. Strikingly, the inhibitor represses both the yeast-to hyphae transition and ongoing filamentation, and its effects on morphogenesis can be uncoupled from general growth inhibition. RNAseq experiments of inhibitor-treated cells coupled with Candida mutant analyses suggest the existence of a novel mechanism of action to represses hyphal growth. The inhibitor was protective to host cells, increasing the survival of bone-marrow derived macrophages in ex vivo macrophage-Candida infection assays, indicating it has potential as a therapeutic. Our work has implications for uncovering novel biology that governs C. albicans filamentation. Our study is also relevant for identifying new therapeutic avenues with distinct mechanism of action compared to the antifungal drugs used in the clinic at present. Since Candida sp cause an estimated 400, 000 life-threatening infections per year worldwide, and the mortality remains in the range of 40%, identifying new avenues for treatment is an urgent need

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The Kennedy Pathway: A Rheostat for Virulence in Candida albicans

Robert Tams1, Chelsi Cassilly1, Sanket Anaokar2, William Brewer1, Justin Dinsmore1, Joseph Chen3, Jana Patton-Vogt2, Todd Reynolds1 1University of Tennessee, Knoxville, Tennessee, USA, 2Duquesne University, Pittsburgh, Pennsylvania, USA, 3National Taiwan University, Taipei, Taiwan

Phospholipids are important structural lipids of cellular membranes that are also involved in cell signaling. We previously reported that phosphatidylethanolamine (PE) synthesis by the de novo biosynthetic pathway is required for virulence in Candida albicans. Further downstream, PE can be methylated to form phosphatidylcholine (PC), and the role of PC synthesis in virulence is unknown. We hypothesized that PE conversion to PC would be required for virulence. However, we found that a mutation that blocks PE conversion to PC increased virulence. This led to the hypothesis that increased PE synthesis may cause an increase in virulence. To test this, we developed a strategy to increase or decrease PE/PC synthesis independently of the de novo pathway using the Kennedy pathway. The Kennedy pathway involves three successive enzymatic steps that synthesize PE or PC from diacylglycerol and exogenous ethanolamine or choline, respectively. By modulating the gene for the final step in the pathway, EPT1, we have decreased or increased PE/PC synthesis. We have found that an ept1Δ/Δ knockout strain has decreased cytotoxicity to FaDu tissue culture cells, and decreased fungal burden in the kidneys of infected mice. In contrast, overexpression of EPT1 causes increased cytotoxicity to FaDu cells, and mice infected with the EPT1 overexpression mutant succumb to infection more rapidly than those infected with the wild type. Thus, tuning the levels of PE/PC correlates with levels of virulence, suggesting that this pathway is able to regulate virulence in mice. We are currently exploring the mechanism by which PE/PC regulation impacts virulence.

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Investigating the mechanisms by which the transcription factor Hsf1 regulates Candida albicans morphogenesis

Amanda Veri1, Teresa O’Meara1, Rebecca Shapiro2, Faiza Tebbji3, Sang Hu Kim1, Juan Colazo1, Nicole Robbins1, Malcolm Whiteway4, Koon Ho Wong5, Leah Cowen1 1University of Toronto, Toronto, Ontario, Canada, 2Broad Institute, Boston, Massachusetts, USA, 3Université Laval, Québec City, Québec, Canada, 4Concordia University, Montréal, Québec, Canada, 5University of Macau, Macau SAR, China

Invasive fungal infections caused by Candida albicans are among the leading causes of death in immunocompromised individuals. One key virulence trait of C. albicans is its ability to transition from yeast to filamentous growth, which is typically regulated by environmental cues at the physiological temperature of 37°C. The heat shock transcription factor Hsf1 orchestrates the C. albicans temperature response in part through regulation of Hsp90, a molecular chaperone involved in governing morphogenesis. Here, we focus on elucidating the role of Hsf1 in the yeast-to-filament transition. We discovered that overexpression and depletion of HSF1 induces filamentation independent of the temperature requirement of 37°C. However, the mechanisms by which differing Hsf1 levels regulate the morphogenetic program appear distinct. HSF1 depletion compromises Hsp90 function even when HSP90 expression is driven by a constitutive Hsf1-independent promoter. This suggests that HSF1 depletion alters the expression of a critical regulator of Hsp90, leading to compromised chaperone function and filamentous growth. Overexpression of HSF1 tightly links morphogenesis with temperature as moderate overexpression allows filamentation at 34°C while strong overexpression drives filamentation at 30°C. HSF1 overexpression enables morphogenesis through overexpression of positive regulators of filamentation such as Brg1 and Ume6. To determine if these are direct Hsf1 targets and to provide a global analysis of Hsf1-targets critical for morphogenesis, we are performing chromatin immunoprecipitation coupled to sequencing and RNA sequencing analyses. This work illuminates a novel role for Hsf1 in regulating morphogenesis and provides a fascinating example of a gene whose overexpression and depletion induces C. albicans filamentation.

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Iron restriction inside the phagosome of macrophages is an essential host defense mechanism against Mucorales

Angeliki M. Andrianaki1 ,6, Irene Kyrmizi1 ,5, George Samonis6, Dimitrios P. Kontoyiannis2, Ashraf S. Ibrahim3 ,4, George Chamilos1 1Department of Clinical Microbiology and Microbial Pathogenesis, School of Medicine, University of Crete, Heraklion, Crete, Greece, 2Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA, 3Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA, 4David Geffen School of Medicine at UCLA, Los Angeles, California, USA, 5Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Crete, Greece, 6Department of Internal Medicine, Infectious Diseases Unit, University of Crete Medical School, Heraklion, Crete, Greece

Mucorales are emerging airborne fungal pathogens with unique virulence attributes and incompletely understood pathogenesis. We found that as opposite to Aspergillus fumigatus, Mucorales have a selective “tropism” for alveolar macrophages (AMs), which accounts for the establishment of prolonged intracellular “persistence” in the lungs of immunocompetent mice. Although Mucorales conidia are fully susceptible to the effector mechanisms of macrophages, they avoid killing and establish intracellular “persistence” via the induction of phagosome maturation arrest. Mechanistically, lack of intracellular cell wall swelling of Mucorales conidia leads to surface retention of melanin and subsequent blockade in early critical steps of phagosome biogenesis. On the other site, inhibition of germination of Mucorales conidia inside macrophages is an essential host defense mechanism, as AM depletion significantly increases susceptibility of mice to mucormycosis. Nutritional immunity via iron restriction is the principal mechanism of inhibition of Mucorales growth inside arrested phagosomes of macrophages. Importantly, increased iron availability via iron or deferoxamine supplementation leads to germination of Mucorales conidia inside macrophages, which is reversed upon genetic ablation of essential components of iron assimilation pathways of the fungus. Collectively, our findings reveal novel aspects of cellular immunity against Mucorales that are critical in pathogenesis of mucormycosis and represent future potential targets for therapeutic interventions.

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Sialic acids on the surface of Aspergillus fumigatus conidia impacts pathogen recognition and anti-fungal response by PBMCs

Intan M Dewi1 ,2, Martin Jaeger1, Berenice Roesler1, Mark S Gresnigt1, Frank L van de Veerdonk1 1Radboud University Medical Center, Nijmegen, The Netherlands, 2Universitas Padjadjaran, Bandung, Indonesia

Aspergillus fumigatus is a ubiquitous fungus that cause invasive pulmonary aspergillosis, ABPA, and CPA. In the past decade there have been reports on IPA associated with influenza. Influenza virus harbors neuraminidase on its surface, and most of these patients had received neuraminidase-inhibitors during the course of the influenza infection.

Previous studies have identified the presence of sialic acids in A. fumigatus conidial surface, which mediate adhesion to fibronectin and basal lamina in pulmonary epithelial cells. We hypothesized that sialic acids could also be important for immune recognition of this pathogen. Our study aims to determine whether the removal of sialic acids from A. fumigatus conidia and the presence of neuraminidase-inhibitors affects the host’s immune response.

Peripheral blood mononuclear cells were stimulated with A. fumigatus conidia pre-treated with neuraminidase. In a different set of experiments, Oseltamivir was pre-incubated with PBMCs for 2 hours prior to stimulation with conidia. TNFα, IL-1β, and IL-6 were measured from culture supernatants after 24 hours of incubation at 37˚C. Removal of sialic acids of A. fumigatus conidia significantly increased the capacity to induce TNFα, IL-6 and IL-1β in human PBMCs. Reversibly, pre-exposure of PBMCs to neuraminidase-inhibitor prior to stimulation with live conidia decreased the cytokine response by these cells.

These findings indicated that sialic acid removal from A. fumigatus conidia prior to stimulation may increase pro-inflammatory cytokines by PBMCs and possibly affect the host’s anti-fungal defense. This study might help us to understand further about the role of fungal sialic acids in the host-pathogen interface.

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Identification of mediators of antifungal tolerance in Candida albicans

Eric Delarze1, Fabio Maranzano1, Marion Patxot-Bertran1, Sadri Znaidi2, Mélanie Legrand2, Maiken Cavling Arendrup3, Carol Munro4, Christophe d’Enfert2, Dominique Sanglard1 1University of Lausanne and University Hospital Center, Lausanne, Vaud, Switzerland, 2Pasteur Institute, Paris, France, 3Statens Serum Institut, Copenhagen, Denmark, 4University of Aberdeen, Aberdeen, UK

Antifungal tolerance can be defined as the ability of C. albicans cells to survive at high drug concentrations but without acquiring mutations associated with resistance. The mechanisms mediating drug tolerance are still not well understood. In this study we aimed to identify mediators of tolerance to fluconazole (FLC) using two different approaches. First, the tolerance profile of 27 clinical C. albicans isolates against FLC using the EUCAST drug susceptibility assay was assessed. Tolerant strains were identified by their ability to sustain residual growth after 24h at a drug concentration above the FLC resistance breakpoint (> 4µg/ml). This screening revealed different profiles grouped from low to high tolerance. These FLC-tolerant strains are candidates for further characterization by genetic and transcriptomic approaches and will be used to test the importance of drug tolerance in animal models. In a second approach, in order to identify genetic mediators of tolerance, we used a collection of 582 tetracycline-inducible overexpression barcoded strains, which were pooled and maintained under FLC pressure for five days of repeated subcultures. This strategy was used to enrich the pool in FLC-resistant and/or FLC-tolerant strains. After amplification and sequencing of all barcodes from the cultures, the fractional index of each strain in the population was calculated. The FLC susceptibility profile of enriched strains yielded the identification of two potential tolerance mediators. When overexpressed, these genes contributed to increase tolerance to FLC as compared to controls. The potential role of these two genes upon tolerance is now under investigation.

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The long pentraxin PTX3 regulates the pathogenic potential of Trichosporon asahii via differential induction of IL-10

Cláudio Duarte-Oliveira1 ,2, Samuel Gonçalves1 ,2, Fernando Rodrigues1 ,2, Cecilia Garlanda3, Gustavo Goldman4, Alberto Mantovani3 ,5, Agostinho Carvalho1 ,2, Cristina Cunha1 ,2 1Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal, 2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal, 3Humanitas Clinical and Research Center, Rozzano (Milan), Italy, 4Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil, 5Humanitas University, Rozzano (Milan), Italy

Most species of the Trichosporon genus are commensals of the human skin and gastrointestinal tract. Trichosporon asahii is however an increasingly recognized causative agent of superficial and invasive fungal disease in humans. How T. asahii interacts with and subverts the host immune system remains largely unknown. The long pentraxin 3 (PTX3) is a soluble pattern-recognition receptor and an essential component of the humoral arm of innate immunity. Because PTX3 plays a nonredundant role in resistance against selected microbial pathogens, we evaluated its contribution to the immune response to T. asahii by resorting to cellular and in vivo models of infection. We found that PTX3 expression was induced during infection and that PTX3 could bind the cell surface of blastoconidia from T. asahii. Accordingly, bone marrow-derived macrophages (BMDM) from Ptx3-/- mice and human macrophages from PTX3-deficient individuals displayed a marked defect in fungal internalization and killing. Cytokine production upon infection in these cells remained largely intact, apart from IL-10, which was almost undetectable. In a model of invasive trichosporonosis, Ptx3-/- animals were highly susceptible to disseminated infection, with increased fungal burdens in the liver, kidney, and spleen, and extensive inflammatory pathology in these target organs. Decreased levels of IL-10 were observed in organ homogenates, whereas proinflammatory cytokines presented comparable levels to the wild-type controls. Finally, the treatment of Ptx3-/- animals with recombinant PTX3 reverted the IL-10 deficiency and susceptibility to trichosporonosis. Our results suggest that PTX3 regulates IL-10 production during T. asahii infection, eliciting mechanisms of antifungal resistance with controlled immunopathology.

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Fingerprinting of Candida spp. blood infections

Philipp Kämmer1, Sylvie Schulze2, Thomas Wolf2, Kerstin Hünniger3, Jörg Linde2, Oliver Kurzai3, Reinhard Guthke2, Bernhard Hube1 ,4, Sascha Brunke1 1Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany, 2Research Group Systems Biology and Bioinformatics, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany, 3Septomics Research Center, Friedrich Schiller University and Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany, 4Friedrich Schiller University, Jena, Germany

Candida species are important agents of invasive fungal infections. Among them, Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis account for the majority of all systemic Candida infections. To mimic the initial phase of systemic Candida infections, we here established an ex vivo whole blood infection model for all these species. This model allowed the investigation of interdependent transcriptional responses of both human host and fungal pathogen, based on a dynamic dual-species RNAseq approach.

We found a predominantly uniform human response to infection with the four species, with very few species-specific regulations. These strongly time-dependent transcriptional changes are governed by processes of the immune system response, in particular by genes involved in TNF and NF-κB signaling.

On the fungal side, the three more closely related species C. albicans, C. tropicalis, and C. parapsilosis each regulate a noticeable fraction of their genomes in response to blood exposure, while C. glabrata surprisingly only reacts with minor transcriptional adjustments. Common fungal responses comprise the shutdown of protein biosynthesis and the activation of heat shock responses. In contrast, genes involved in oxidative stress response are broadly up-regulated by C. albicans, C. tropicalis, and C. parapsilosis, but not by C. glabrata. The regulation of several other processes, like iron homeostasis or cell cycle progression, is even more species-specific. This led us to the conclusion that different Candida species pursue discrete strategies to survive within the harsh bloodstream environment, while the transcriptional host response is much more uniform.

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The absence of TLR3 controls paracoccidioidomycosis with increase of cytotoxic CD8+ T cells

GRASIELLE JANNUZZI1, JOSÉ ROBERTO DE ALEMIDA1, GILBERTO KAIHAMI2, LAVINIA ROMERA1, SANDRO ROGÉRIO DE ALMEIDA1, KAREN FERREIRA3 1Faculty of Pharmaceutical Sciences, University of São Paulo, SÃO PAULO, Brazil, 2IQ-USP - Institute of Chemistry, University of São Paulo, SÃO PAULO, Brazil, 3Institute of Environmental Sciences, Chemical and Pharmaceutical, of Federal University of São Paulo, DIADEMA,SÃO PAULO, Brazil

Toll-like receptors comprise the best-characterized pattern-recognition receptor family that can activate different immune responses, depending on which receptor and adaptor set are utilized. TLRs, such as TLR2, TLR4 and TLR9, and their signaling have been implicated in the recognition of P. brasiliensis and regulation of the immune response, however, the role of TLR3 remains unclear. Thus, understanding the endosomal function of TLR3 in experimental PCM is crucial. We used in vitro and in vivo models of infection by P. brasiliensis, WT and TLR3-/- mice, to assess the contribution of TLR3 on development of infection. We show absence of TLR3 leads to increase nitric oxide and phagocytic ability by macrophages, the first 4 hours of interaction with yeast P. brasiliensis. We further show that TLR3-/- mice plays a protective role after 30 days of intratracheal infection with P. brasiliensis, showing decrease of CFU grown, increase of pro-inflammatory cytokines production, as well as increase of cytotoxic T CD8+ cells producers of IFN-γ and IL-17. In early stages, we observed increase tlr9 expression in lungs of TLR3-/- mice in first 4 hours post infection and later (72 hours) enhanced recruitment of neutrophil in the lung followed by increased inflammatory cytokines. Recent studies indicate that TLR3 is important receptor for the immune response in mycosis and its absence favors the fungal infection. In contrast, our findings show that in the case of PCM, the TLR3 is deleterious to the host, suggesting that activation of TLR3 can be a possible escape mechanism of P. brasiliensis.

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Contact-sensing and directional growth regulation in Candida albicans hyphae

Alexandra Brand, Tina Bedekovic, Mariana Almeida University of Aberdeen, Aberdeen, UK

The production of hyphae is strongly linked to pathogenesis during superficial mucosal infections and the life-threatening disseminated disease, invasive candidiasis. Hyphae constitute the ‘Special Weapons And Tactics’ capability deployed by C. albicans during mucosal and endothelial cell layer invasion. Hyphae are equipped with adhesins and secreted effectors but these are only effective if hyphal guidance mechanisms are operational to direct penetrative growth into host tissue. We have identified three distinct hyphal phenotypes in which the ability to respond normally to external cues is attenuated or lost. Each of the three phenotypes is produced by specific grouping of mutant or gene deletion strains. The functional links within some groupings are relatively clear. For example, the sinusoidal growth morphology is observed when Pxl1 or its kinase, Ptk2, are deleted. However, in other cases the relationship between the proteins in a grouping is less clear but potential pathways are emerging. Our aim is to elucidate these signalling pathways and generate an integrated model of how hyphal guidance signals influence the molecular machinery that drives polarised growth.

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A novel Blastomyces ligand for Dectin-2 with adjuvant activity for vaccination against fungi

Bruce Klein, Huafeng Wang, Taek-Jin Lee, Richard Merkhofer, Robert Zarnowski, Tristan Brandhorst, Kevin Galles, Wuethrich Marcel University of Wisconsin-Madison, Madison, WI, USA

The development of vaccines against fungi and other intracellular microbes is impeded in part by the lack of suitable adjuvants. While most current vaccines against infectious diseases preferentially induce production of antibodies, cellular immunity is essential for the resolution of fungal infections. Resistance to intracellular microbes such such as fungi requires Th17 and Th1 cells, which are dependent on C-type lectin receptors including Dectin-2. Here, we identified a novel ligand for Dectin-2, the glycoprotein Blastomyces Eng2 (Bl-Eng2). Bl-Eng2 triggers signaling in Dectin-2 reporter cells and induces IL-6 in human PBMC and BMDC from wild type but not Dectin-2-/-and Card9-/- mice. The addition of Bl-Eng2 to a subunit vaccine primed increased numbers Ag-specific Th17 and Th1 cells, and protected mice after fungal challenge indicating its potency as a vaccine adjuvant. Thus, Bl-Eng-2 could be harnessed as an adjuvant to protect against infectious diseases requiring cellular immunity.

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Putting Fungal Virulence in Context

Sven Krappmann Institute of Clinical Microbiology, Immunology and Hygiene, University Hospital Erlangen and Friedrich-Alexander University (FAU), Erlangen, Germany

The pathogenesis of mycoses relies on a plethora of factors and is influenced by the intimate pathogen-host interplay. A fundamental theme is the quest for nutritional sources by the invading pathogen to support its growth, while the host restricts access to several elementary factors. The metabolic versatility of a fungal pathogen to circumvent this ‘nutritional immunity’ has to be considered as virulence determinant and comprehensive knowledge about the pathways and regulatory circuits that are operative during infection is of importance, given the specificity of fungal metabolism and the limited number of established antifungal targets. Moreover, competitive traits that have evolved at the primary ecological niche may further support virulence, as infection of a susceptible host might have to be seen in the context of microbial competitors. The omnipresent mold Aspergillus fumigatus has emerged as leading cause of fungal infections during an underlying immunodeficiency. To assess the role of metabolic versatility for virulence, we have addressed aspects of its primary metabolism, among them sulfur assimilation or biosynthesis of aromatic amino acids. Current studies aim at a more comprehensive assessment by metabolic network modelling with the long-term goal to spot novel angles for antifungal therapy, as this approach allows to predict candidates in a systematic way and to identify non-redundant targets. As further aspects, we address the impact of A. fumigatus sexuality on secondary metabolism by detailed inspection of its bipolar mating-type system and the role of a conidial peptide effector with respect to antimicrobial activity.

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Cell Wars: The battle between Cryptococcus titan cells and the immune system

Kirsten Nielsen University of Minnesota, Minneapolis, Minnesota, USA

Upon inhalation into the lungs, a subset of Cryptococcus neoformans cells transform into enlarged titan cells. While typical C. neoformans cells are 5-7 μm in diameter, the titan cells can be up to 100 μm in diameter. Along with their large size, titan cells also exhibit alterations in their cell wall and capsule structure. These morphological changes impact how the host immune system perceives and responds to the C. neoformans infection and ultimately leads to increased disease. In addition to morphological changes, titan cells are also polyploid. Typical C. neoformans cells are haploid (1C) but titan cells are tetraploid (4C), octoploid (8C), 16C, etc. with ploidies as high as 312C readily observed. Surprisingly, the polyploid titan cells divide by budding to produce haploid or aneuploid daughter cells that can have novel traits such as increased drug resistance and tolerance to various stresses. Current studies aim to understand how C. neoformans senses the host environment to trigger titan cell formation as well as the molecular processes underpinning the unique morphological and genomic changes observed in titan cells, and how these titan cells impact infection and disease.

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Epithelial activation by Candidalysin

Julian Naglik Mucosal & Salivary Biology Division, The Dental Institute, King’s College London, London, UK

Cytolytic proteins and peptide toxins are critical virulence factors of bacterial pathogens and play a major role in bacterial disease. Human pathogenic fungi were not known to possess such toxins. Recently, we discovered that the hyphal form of Candida albicans produces a cytolytic peptide toxin (Candidalysin) that is critical for mucosal pathogenesis and immune activation. Candidalysin is a 31 amino acid peptide derived from its parent protein, Ece1p. Upon secretion, Candidalysin intercalates into and destabilises the structural integrity of epithelial cell plasma membranes. As a result, alarmins are released, which leads to epithelial activation via the epidermal growth factor receptor (EGFR). EGFR activation triggers mitogen-activated protein kinase (MAPK) signalling and, ultimately, a strong inflammatory response. The secretion of inflammatory mediators recruit neutrophils and activated resident innate Type 17 cells, which together help clear the fungal infection.

93

W4/1

Phagosomal neutralization by Candida albicans induces macrophage pyroptosis

Slavena Vylkova1 ,2, Michael Lorenz1 1The University of Texas Health Science Center at Houston, Houston, TX, USA, 2Research Group Host Fungal Interfaces, Friedrich Schiller University, Jena, Germany

The interaction of Candida albicans with the innate immune system is the key determinant of the pathogen/commensal balance and has selected for adaptations that facilitate the utilization of nutrients commonly found within the host, including proteins and amino acids; many of the catabolic pathways needed to assimilate these compounds are required for persistence in the host. We have shown that C. albicans co-opts amino acid catabolism to generate and excrete ammonia, which raises extracellular pH, both in vitro and in vivo and induces hyphal morphogenesis. Mutants defective in the uptake or utilization of amino acids, such as those lacking Stp2p, a transcription factor that regulates the expression of amino acid permeases, are impaired in multiple aspects of fungal-macrophage interactions resulting from an inability to neutralize the phagosome. Here we identified a novel role in amino acid utilization for Ahr1p, a transcription factor previously implicated in regulation of adherence and hyphal morphogenesis. Mutants lacking AHR1 were defective in growth, alkalinization and ammonia release on amino acid-rich media, similar to stp2Δ and ahr1Δstp2Δ cells, and occupied more acidic phagosomes. Notably, ahr1Δ and stp2Δ strains did not induce pyroptosis as measured by Caspase-1-dependent IL-1β release, though this phenotype could be suppressed by pharmacological neutralization of the phagosome. Altogether, we show that C. albicans-driven neutralization of the phagosome promotes hyphal morphogenesis, sufficient for induction of Caspase-1-mediated macrophage lysis.

94

W4/2

Candida albicans takes a licking during infection

Alex Hopke1, Nadine Nicke1, Erica Hidu1, Genny Degani2, Laura Popolo2, Robert Wheeler1 1University of Maine, Orono, ME, USA, 2University of Milan, Milan, Italy

Pathogens hide immunogenic epitopes from the host to evade immunity, persist and cause infection. Candida albicans, which can cause fatal disease in immunocompromised patient populations, masks the inflammatory epitope β-glucan in its cell wall from host recognition. While β-glucan becomes exposed during infection in vivo, the mechanism behind this exposure was unknown. Here, we show that this unmasking involves neutrophil extracellular trap (NET) mediated attack, which triggers changes in fungal cell wall architecture that enhance recognition by the Dectin-1 β-glucan receptor in vitro. Furthermore, using a mouse model of disseminated candidiasis, we demonstrate the requirement for neutrophils in triggering these fungal cell wall changes in vivo. Importantly, we found that fungal epitope unmasking requires an active fungal response in addition to the stimulus provided by neutrophil attack. NET-mediated damage initiates fungal MAP kinase-driven responses, particularly by Hog1, that dynamically relocalize cell wall remodeling machinery including Chs3, Phr1 and Sur7. Neutrophil-initiated cell wall disruptions augment some macrophage cytokine responses to attacked fungi. This work provides insight into host-pathogen interactions during disseminated candidiasis, including valuable information about how the C. albicans cell wall responds to the biotic stress of immune attack. Our results highlight the important but underappreciated concept that pattern recognition during infection is dynamic and depends on the host-pathogen dialog.

95

W4/3

Mechanistic basis of epithelial damage in response to A. fumigatus infection

Elaine Bignell, Uju icheoku, Margherita Bertuzzi, Norman van Rhijn, Takanori Furukawa, Zorana Carter, Lea Gregson, Sayema Khan, Panagiotis Papastamoulis, Magnus Rattray, Michael Bromley, Magnus Rattray University of Manchester, Manchester, UK

The human lung is continually exposed to spores of the airborne mould Aspergillus fumigatus. Inhaled spores are small enough to bypass mucociliary clearance mechanisms and reach the alveoli of the lung where interaction between host and pathogen cells can lead to fungal clearance, or to development of inflammatory or invasive fungal diseases. In order to find out why A. fumigatus is cytotoxic to human epithelia we have observed the interaction between host and pathogen in laboratory culture, and measured epithelial decay, lytic death of host cells, host cell signalling and cytokine degradation in response to fungal challenge. This research has revealed multiple mechanisms involved in eliciting epithelial damage, occurring at different stages of the host-pathogen interaction and involving different fungal morphotypes. We have used laboratory experimentation to ask the following questions: Why is A. fumigatus cytotoxic to human cells? Does the host or the pathogen drive epithelial damage and how? How do pathogen-derived proteins shape the immune environment? How is damage driven in the whole animal host? Which elicitors of host damage are produced by A. fumigatus, and have they recently evolved? We also introduce a new high throughput study addressing the A. fumigatus regulatory network driving epithelial damage in cultured human lung cells.

96

W4/4

Therapeutic Implications of Candida albicans and Staphylococcus aureus Mixed Biofilms

Eric Kong1, Christina Tsui1, Sona Kucharicova2, Patrick Van Dijck2, Mary Ann Jabra-Rizk1 1University of Maryland, Baltimore MD, USA, 2KU Leuven, Leuven, Belgium

Biofilm associated fungal-bacterial mixed infections are responsible for significant morbidity and tend to be challenging to treat. Candida albicans and Staphylococcus aureus are leading microbial pathogens mainly due to their ability to form biofilms on implanted medical devices. However, the impact of mixed species biofilm growth on therapy remains largely understudied. We recently demonstrated in vitro that in mixed biofilms, C. albicans conferred S. aureus with significantly enhanced tolerance to antibacterials. As these findings may be indicative of therapeutic outcomes in a co-infected host, we developed a clinically relevant subcutaneous catheter mouse model to explore the phenomenon of enhanced antimicrobial tolerance in mixed infections under in vivo conditions. In this model, catheters infected with S. aureus, C. albicans or in combination were implanted subcutaneously in mice and biofilms were allowed to form for 48 hours prior to initiation of vancomycin therapy, which was administered for 6 days. Harvested catheters were assessed for microbial recovery and scanning electron microscopy analysis. Results demonstrated that where vancomycin significantly reduced S. aureus recovery in mice infected only with S. aureus, in co-infected animals, vancomycin had no impact on S. aureus recovery. Microscopic images revealed a thick biofilm matrix formed within catheter lumen composed of C. albicans hyphae and embedded S. aureus. These findings underscore the clinical and therapeutic implications of mixed biofilm-associated infections in a host. Understanding microbial interactions central to the persistence and resistance of polymicrobial infections will greatly aid in overcoming limitations of current therapies and in defining potential new drug targets.

97

W4/5

Oxidative stress resistance and modulation of macrophage pro-inflammatory response by Candida albicans MAM33

Manju Chauhan, Selvakumar Subbian, Neeraj Chauhan Rutgers, the State University of New Jersey, Newark, NJ, USA

Mitochondria are key contributors to fungal pathogenesis. However, the biological role of most Candida albicans mitochondrial proteins is poorly understood. In the current study, we present data on the characterization of, Mam33, a putative mitochondrial protein of C. albicans. The subcellular localization of Mam33 was confirmed by fluorescence microscopy. Disruption of MAM33 causes reduced resistance to oxidative stress and hypersensitivity to SDS, suggesting cell surface defects in these mutants. To determine the role of Mam33 in interaction with host immune cells, we performed invasion (10 min) and entry/survival (90 min) assays by using the murine J774 macrophage like cells. Our results show that compared to the wildtype, mam33∆/∆ null mutant strains were defective for invasion and/or entry/survival. We observed a reduction in the level of expression of proinflammatory molecules, including Ccl2, Cxcl10, IL6, IL12a, IL12b and Tnfa, in J774 cells infected with mam33∆/∆ compared to the wildtype. Consistent with the down regulation of proinflammatory markers, expression of IL10, an immune-suppressing cytokine, was upregulated in J774 cells upon infection by mam33∆/∆. In addition, transcriptional profiling revealed significant down-regulation of genes involved in the glycolytic and N-acetylglucosamine metabolism pathways. However, genes involved in the TCA cycle and ATP synthesis were enriched more than 11-folds in mam33∆/∆, relative to the wildtype. To provide, further insight into the role of MAM33 in fungal virulence, current experiment using a murine model of invasive candidiasis and metabolomics approaches are on-going. Together, these results reveal a novel role of fungal mitochondrial proteins in host-pathogen interactions.

98

Posters

P1A

Targeting synthetically lethal partners of antimicrobial resistance alleles in Candida glabrata to minimize emergence of drug resistant infection

Jane Usher, Ken Haynes University of Exeter, Exeter, UK

Drug resistance has emerged as a huge problem in many areas of medicine from cancer to infectious diseases. This is driving the development of novel therapeutic strategies. One that is gaining ground, is multi-target therapy, where combinations of drugs targeting different components of a disease network are deployed. A major impediment to this approach is the characterization of suitable targets for combination therapies. To date most combinatorial therapy targets have been selected based on previous biological knowledge of drug mode of action and/or mechanisms of resistance, severely constraining the number of proteins that can be targeted.

Unbiased genome-wide screens will reveal many more components of the interaction networks of known drug targets, which could then be targeted in combination therapies. To test this principle in the context of antimicrobial resistance we have implemented an unbiased genome-wide screening technology, SGA analysis, facilitating characterization of pair-wise synthetic genetic interactions. We performed an unbiased SGA screen with a C. glabrata PDR1+ gain of function allele. PDR1 encodes a transcriptional regulator and gain of function mutations in this gene, are the principal mediators of fluconazole resistance in C. glabrata. We identified a gcn5 null mutation as one negative synthetic interaction with PDR1+. We showed that deletion of GCN5 and/or chemical inhibition of the protein Gcn5, are synthetically lethal with PDR1+. These data demonstrate that deletion or chemical inactivation of a PDR1+ synthetically lethal gene results in cellular death if wild-type PDR1 mutates to a PDR1+ FLZ resistance conferring allele.

99

P2A

Phagosomal neutralization by Candida albicans induces macrophage pyroptosis

Slavena Vylkova1 ,2, Michael Lorenz1 1The University of Texas Health Science Center at Houston, Houston, TX, USA, 2Research Group Host Fungal Interfaces, Friedrich Schiller University, Jena, Germany

The interaction of Candida albicans with the innate immune system is the key determinant of the pathogen/commensal balance and has selected for adaptations that facilitate the utilization of nutrients commonly found within the host, including proteins and amino acids; many of the catabolic pathways needed to assimilate these compounds are required for persistence in the host. We have shown that C. albicans co-opts amino acid catabolism to generate and excrete ammonia, which raises extracellular pH, both in vitro and in vivo and induces hyphal morphogenesis. Mutants defective in the uptake or utilization of amino acids, such as those lacking Stp2p, a transcription factor that regulates the expression of amino acid permeases, are impaired in multiple aspects of fungal-macrophage interactions resulting from an inability to neutralize the phagosome. Here we identified a novel role in amino acid utilization for Ahr1p, a transcription factor previously implicated in regulation of adherence and hyphal morphogenesis. Mutants lacking AHR1 were defective in growth, alkalinization and ammonia release on amino acid-rich media, similar to stp2Δ and ahr1Δstp2Δ cells, and occupied more acidic phagosomes. Notably, ahr1Δ and stp2Δ strains did not induce pyroptosis as measured by Caspase-1-dependent IL-1β release, though this phenotype could be suppressed by pharmacological neutralization of the phagosome. Altogether, we show that C. albicans-driven neutralization of the phagosome promotes hyphal morphogenesis, sufficient for induction of Caspase-1-mediated macrophage lysis.

100

P3A

Hyphal maintenance is dispensable for virulence of Candida albicans in a systemic mouse model

Christine Dunker, Melanie Polke, Bianca Schulze, Ilse Jacobsen Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany

The ability to form hyphae is a prominent virulence feature of Candida albicans. C. albicans mutants unable to form hyphae are usually strongly attenuated in virulence in both systemic and mucosal infection models. C. albicans EED1 deletion mutants are able to form germ tubes but fail to maintain filamentation and instead revert to yeast growth within a few hours. This is associated with a significantly reduced capability to damage epithelial cells in vitro and attenuated virulence in an intraperitoneal mouse model. Surprisingly however, both deletion of EED1 and abrogation of expression using a tet-regulable promoter did not affect virulence in a murine model of systemic candidiasis. To understand how this mutant compensates the lack of filamentation, we analyzed the course of murine systemic infection in detail. Onset and severity of clinical symptoms as well as alterations of physiological parameters and systemic inflammation marker were comparable between mice infected with either mutant or wild type. However, fungal burden was significantly higher in mice infected with EED1 deficient C. albicans cells. This coincided with lower numbers of leukocytes and less cytokines early after infection but increased amounts at later stages. We furthermore analyzed the interaction of wild type and EED1 mutant yeast cells with macrophages in vitro. Surprisingly, the EED1 mutant was able to survive and replicate within macrophages without filament formation, resulting in macrophage damage comparable to the wild type but lower cytokine induction. Thus, increased proliferation/survival appears to compensate for the hyphal maintenance defect of EED1 mutants during systemic infection.

101

P4A

A giant impact: elucidating the role of Candida albicans cellular gigantism on host-pathogen interactions

Dhara Malavia, Laura Lehtovirta, Omran Alamir, Neil Gow, Duncan Wilson University of Aberdeen, Aberdeen, UK

Candida albicans is a major human fungal pathogen, responsible for superficial, as well as life-threatening disseminated infections. Polymorphism in C. albicans is an established virulence attribute and fungal cells must acquire essential nutrients from the host in order to proliferate. However, the host immune system has evolved complex mechanisms to actively withhold essential nutrients like zinc in a process known as nutritional immunity. Therefore, in the battle of pathogenicity versus nutritional immunity, the fungus must have developed strategies to survive and thrive within the host under the restrictive conditions of nutritional immunity. We have observed that C. albicans transforms to a giant ‘Goliath’ yeast morphology upon zinc starvation. This response is also seen in multiple Candida albicans clinical isolates and in the related species, Candida dubliniensis and Candida tropicalis. Investigations into the physiological properties of Goliath cells has revealed cell wall alterations, increased lipid droplets, enlarged vacuoles, delayed hyphae formation and increased adhesion to abiotic surfaces. With such prominent differences in virulence-associated properties, we are now exploring the interactions of Goliath cells with human epithelia and immune cells.

102

P5A

Candidalysin is the Candida albicans haemolytic factor

Selene Mogavero1, Sarah Höfs1, Alexa Lauer1 ,2, Bernhard Hube1 ,2 1Hans Knöll Institute, Jena, Germany, 2Friedrich Schiller University, Jena, Germany

Candida albicans has recently been discovered to be able to produce an important virulence factor, the hyphae-associated secreted peptide toxin Candidalysin. This toxin has proven to be crucial for the establishment of a mucosal infection (1). But what other roles might this toxin have? C. albicans has long been known to be haemolytic, but the haemolytic factor has never been clearly identified. Previous work suggested that it probably is a secreted mannoprotein (2). Might the yet-not-clearly-identified haemolytic factor actually be Candidalysin? Using both C. albicans mutants and synthetic peptides in haemolysis assays, we can now clearly attribute the full haemolytic activity of C. albicans to its secreted Candidalysin. We further elucidated the mechanism of the toxin-induced red blood cell (RBC) lysis, and found that pH, cholesterol, divalent cations, and purinergic receptor activation play a central role. We also observed a significant variation of our results depending on blood donor, which opens new questions about the mechanism of RBC lysis by Candidalysin. For example, might there be a differential role of cell-surface antigens for Candidalysin susceptibility? Our results shed some new light on C. albicans virulence, with important clinical implications, especially for patients suffering from candidaemia, condition that has been rising in incidence in the past decades, due to the increase in immunocompromised patients.

1. Moyes DL et al., 2016. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature. 532 (7597): 64-68.

2. Watanabe T et al., 1999. Characterization of a haemolytic factor from Candida albicans. Microbiology. 145 (Pt 3): 689-694.

103

P6A

DUB module of SAGA complex regulates H2B deubiquitination and morphological transition

Wencheng Zhu, Xueyi Fan, Hongyu Wu, Jiangye Chen Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

Candida albicans is a major opportunistic human fungal pathogen. Its pathogenicity is mainly caused by its capability of conversion between yeast and hyphae. This morphological conversion in C. albicans is regulated by multiple transcriptional regulators sensing many signal pathways. Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is an evolutionarily conserved multifunctional transcription coactivator complex and plays a critical role in regulating transcription by modifying chromatin and identifying modification of histones. SAGA complex consists of five functional modules including histone acetyltransferase (HAT) module, deubiquitination (DUB) module, TATA-binding protein (TBP) regulatory module, structural module and transcriptional activator interaction module, which may be involved in the regulation of C. albicans morphogenesis. In this study, we focused on the functional roles of DUB module which consists of four components, Ubp8, Sgf73, Sus1 and Sgf11 in C. albicans. Deletion of the four components in DUB module compromised hyphal development. Each of them has different contribution to C. albicans morphologenesis. Our biochemical and morphological data demonstrates that the H2B deubiquitination is dynamically regulated via Ubp8 during transition of yeast and hyphae, and controls morphological plasticity of C. albicans.

104

P7A

Contribution of Wor1 promoted melanin synthesis in Candida albicans commensalism

Baodi Dai, yinxing Xu, Ning Gao, Jiangye Chen Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

Candida albicans is the most common human fungal pathogen but also a normal commensal in human gastrointestinal microbiome. It can exist in multiple morphological states and its ability to switch between different morphological forms is important to its pathogenesis and commensalism. The white and opaque cells of C. albicans have different properties including cell shape, colony colour, metabolic preference, gene expression pattern, and host tissue preference, and the switch between the two types of cells are associated with the pathogenesis. Wor1 (white-opaque switching regulator 1) is a master regulator to control the white-opaque switching and is also found to regulate a phenotypic switch that promotes commensalism when passing through the mammalian gut in MTLa/α cell types. In this study, we report the regulatory role of Wor1 in melanin synthesis. Highly induced WOR1 expression promoted melanin production via two putative laccases Fet31 and Fet99. Interestingly, melanin synthesis of C. albicans is correlated with cell surface morphology of opaque and GUT. We also demonstrate that the melanin production is important for C. albicans commensalism in human niche.

105

P8A

Synergistic interactions between C. albicans mutant strains and Oral bacteria during the Formation of Mixed Biofilms

Daniel Montelongo-Jauregui1, Anand Srinivasan2, Anand K. Ramasubramanian3, Stephen P. Saville1, Jose L. Lopez-Ribot1 1The University of Texas at San Antonio, San Antonio, Texas, USA, 2BioBridge Global, San Antonio, Texas, USA, 3San Jose State University, San Jose, California, USA

Fungal and bacterial populations coexist in the oral cavity including oral tissues and implants, where they commonly form biofilms resulting in elevated levels of antibiotic resistance that further complicates treatment against these infections. Unfortunately, the bidirectional interactions between these microbial populations are poorly understood. Using our previously developed in vitro model for co-culture of fungal-bacterial biofilms of Candida albicans and Streptococcus gordonii on 96-well microtiter plates, we attempt to elucidate at the molecular level important factors responsible for the interaction between these microorganisms in mixed biofilms. Here, we co-cultured C. albicans ∆efg1 and ∆brg1 mutants, which are highly defective in both filamentation and biofilm formation, as well as adhesin-deficient mutants ∆als3 and ∆bcr1 alone and in combination with S. gordonii wild-type strain DL1.1. Biofilms were grown in either microbiological media or BMM synthetic saliva. The 3D architecture of biofilms was analyzed using confocal microscopy. Results revealed that growth in BMM or addition of mucin, as well as co-culture with the bacterium, restored the biofilm forming ability of the C. albicans ∆als3 and ∆bcr1 mutants. Moreover and somewhat surprisingly, results indicated a highly synergistic interaction between S. gordonii and the C. albicans ∆efg1 and ∆brg1 mutants, even in the absence of filamentation, which was particularly noticeable when the mixed biofilms were grown in synthetic saliva. In all instances biofilm formation led to increased levels of antimicrobial resistance. Overall these results unravel a high degree of complexity in the reciprocal interactions between C. albicans and S. gordonii in mixed species biofilms.

106

P9A

The Regulatory Subunit of Protein Kinase A (Bcy1) in Candida albicans Plays Critical Roles in Filamentation and White-Opaque Switching but is Not Essential for Cell Growth

Xuefen Ding, Chengjun Cao, Qiushi Zheng, Guanghua Huang Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

The conserved cAMP-dependent protein kinase (PKA) is composed of the regulatory and catalytic subunits and acts as the central component of the cAMP signaling pathway. In the human fungal pathogen Candida albicans, the PKA regulatory subunit Bcy1 plays a critical role in the regulation of cell differentiation and death. It has long been considered that Bcy1 is essential for cell viability in C. albicans. In the current study, surprisingly, we found that Bcy1 is not required for cell growth, and we successfully generated a bcy1/bcy1 null mutant in C. albicans. Deletion of BCY1 leads to multiple cellular morphologies and promotes the development of filaments. Filamentous and smooth colonies are two typical morphological types of the bcy1/bcy1 mutant, which can undergo spontaneous switching between the two types. Cells of filamentous colonies grow better on a number of different culture media and have a higher survival rate than cells of smooth colonies. In addition, deletion of BCY1 significantly increased the frequency of white-to-opaque switching on N-acetylglucosamine-containing medium. The bcy1/bcy1 null mutant generated herein provides the field a new resource to study the biological functions of the cAMP signaling pathway in C. albicans.

107

P10A

Functional genomic analysis of echinocandin resistance in Candida albicans

Tavia Caplan, Elizabeth J. Polvi, Nicole Robbins, Leah E. Cowen Department of Molecular Genetics, University of Toronto, Toronto, Canada

Candida albicans is a leading cause of fungal infections, with mortality rates of ~40%. Effective treatment is hindered by the paucity of antifungal drugs, as well as the ability of fungal pathogens to exploit diverse strategies to tolerate and develop resistance to antifungals. Defining the cellular circuitry governing drug tolerance and resistance is key to identifying novel strategies to block resistance and enhance drug efficacy. To identify novel mechanisms of tolerance to the newest class of antifungals, the echinocandins, we screened two C. albicans mutant libraries collectively covering ~50% of the genome. We identified 13 novel regulators of echinocandin tolerance, most of which were specifically required for cell wall stress responses. Four genes, TSC11, KEX2, RPS5 and CCT8, also enabled clinically-relevant echinocandin resistance. Cct8 is one of eight subunits of the chaperonin containing TCP-1 (CCT) complex, required for the folding of proteins such as actin. By depleting each CCT subunit, we implicated the entire CCT complex in echinocandin tolerance. Chemical inhibition of actin phenocopied depletion of the CCT complex, suggesting that this complex mediates echinocandin tolerance at least in part through actin. Analogous to actin perturbation, depletion of CCT8 activated the cell wall stress response, as demonstrated by activation of the mitogen-activated protein kinase Mkc1. This work uncovers novel regulators of echinocandin tolerance and resistance, and unveils a role for the CCT complex in echinocandin resistance.

108

P11A

Riboswitch Discovery in Saccharomycotina Species

Paul Donovan1, Aisling Coughlan2, Des Higgins2, Geraldine Butler1 1School of Biomedical and Biomolecular Science, Conway Institute , University College Dublin, Belfield, Dublin, Ireland, 2School of Medicine and Medical Science, Conway Institute , University College Dublin, Belfield, Dublin, Ireland

Riboswitches are regulatory elements of mRNA that control gene expression, usually by binding a ligand. Thiamine pyrophosphate (TPP) riboswitches have been described in many bacteria, some plant species, and the filamentous fungi Neurospora crassa and Aspergillus oryzae. Early reports suggested that riboswitches were not present in the Saccharomycotina, including Candida and Saccharomyces species.

We used Infernal with the RFAM covariance model to identify riboswitches in Saccharomycotina species. Riboswitches were identified in thiamine biosynthesis genes (THI4 and THI5), and in a gene of unknown function (DUR31). We confirmed that the thiamine-regulated riboswitch mediates alternative splicing in DUR31 in Candida parapsilosis and Ogataea polymorpha. C. parapsilosis and C. albicans DUR31 deletion strains grow in the presence of pyrithiamine, a toxic thiamine analogue. DUR31 therefore likely encodes a thiamine transporter.

DUR31 has been lost from the genome of Saccharomyces cerevisiae and relatives. However, it is present in most other species in the Saccharomycotina. In S. cerevisiae, thiamine is imported by Thi7 and related proteins. This family is restricted to species closely related to S. cerevisiae. We therefore suggest that Dur31 is a major thiamine transporter in most Saccharomycotina species.

Here, we show that riboswitches are widespread in the Saccharomycotina, including most species in the CTG-Ser clade. Surprisingly, they are lost from DUR31 in some of the best-researched species, including C. albicans and C. tropicalis. We found that all TPP riboswitches are lost from the Saccharomycetaceae.

109

P12A

Spores and hyphae of Aspergillus fumigatus make distinct contributions to epithelial responses and damage

Uju Joy Icheoku1 ,2, Margherita Bertuzzi1, David Moyes2, Elaine Bignell1, Julian Naglik2 1MFIG, Department of Infection, Immunity and Respiratory medicine. The University of Manchester, Manchester, UK, 2Mucosal and Salivary Biology Division, Dental Institute, King’s College, London, London, UK

The human airway continually encounters inhaled airborne spores of the pathogenic fungus A. fumigatus which is globally the most prevalent cause of fungal lung diseases. Moderation of respiratory responses to A. fumigatus spores and hyphae represents a promising therapeutic strategy but understanding of the host-pathogen interaction remains fragmented. This study demonstrated the relevance of A. fumigatus morphotypes and secreted products in orchestration of dynamic host responses and epithelial cell damage, by challenging cultured type II alveolar epithelia (A549) cells with either A. fumigatus spores (LF), or culture filtrate (CF) and monitored for detachment, lactate dehydrogenase release (lytic cell damage), phosphorylation of signalling molecules, transcriptional responses, and cytokine expression. We found that A549 alveolar epithelial cells (ECs) respond differentially to distinct A.fumigatus morphotypes during early and late infections and this response was independent of fungal internalization by epithelial cells. Inhibition of activated epithelial cell signalling pathways rescued cells from damage following both LF and CF challenges but without an observable additive effect. Hyphal products secreted into culture filtrate (CF) are potent effectors of epithelial damage and immunomodulation. Responses to A. fumigatus mutants lacking gliotoxin and /or secreted proteases revealed significant redundancy of anti-epithelial activity amongst the fungal secretome. Our results suggest that A. fumigatus challenge stimulates several mechanistically distinct host responses, via both contact-mediated and diffusible mechanisms, and that secreted fungal products can significantly remodel the host immune response.

110

P13A

Reversible, specific, functional RNA-protein granules triggered by heat shock in budding yeast

Edward Wallace1, Christopher Katanski2, D. Allan Drummond2 1The University of Edinburgh, Edinburgh, Scotland, UK, 2The University of Chicago, Chicago, IL, USA

A pathogen infecting a warm-blooded host encounters a change in temperature, and survival of this heat shock is essential for virulence. Heat shock triggers massive changes in a cell, including to transcription, translation, membrane composition, and reorganization of some RNA and protein into granules. Heat shock granule formation is conserved across eukaryotes, including the ascomycete yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the basidiomycete pathogen Cryptococcus neoformans. However, the function of heat shock granules has remained unclear. We carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in S. cerevisiae, identifying >170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. Stable-isotope labeling revealed that these aggregated proteins are not misfolded and destined for degradation. We further used RNA-seq to show that most mRNA species partially localize to granules upon heat shock, but specific functional groups of mRNAs are excluded. RNA exits granules during recovery from stress, consistent with our protein measurements. Granule-localized mRNA is physically separated from polysomes and specifically protected from stress-triggered degradation. Measurements of RNA-protein association in unstressed cells poorly predict granule localization, implying massive stress-triggered changes in mRNA-protein binding. We have shown that these stress-triggered granules are not a random collection of RNA and proteins, nor are they a sink for terminally damaged molecules. We propose that heat shock granules are part of an adaptive, autoregulatory process that silences and protects specific RNA and protein to aid cellular adaptation to thermal stress.

111

P14A

Different impact of Nrg1 and Tup1 on morphology and gene expression in Candida albicans

Ronny Martin1 ,2, Enrico Garbe3, Joachim Morschhäuser4, Oliver Kurzai1 ,2 1Septomics Research Center, Leibniz Institute for Natural Product Research and Infection Biology –Hans Knoell Institute, Jena, Germany, 2Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany, 3Research Group Host Fungal Interfaces, Friedrich Schiller University, Jena, Germany, 4Institute for Molecular Infection Biology and Research Center for Infectious Diseases (ZINF), Julius Maximilians Universität Würzburg, Würzburg, Germany

The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for fungal virulence. Formation of hyphae correlates with the up-regulation of a core set of genes like ALS3 and ECE1 which were also shown to be involved in pathogenicity processes like invasion, iron acquisition and cell damage. Since their first description several years ago, Tup1 and Nrg1 are thought to be main antagonists of hyphal development in C. albicans. With the help of a split-GFP assay, we were able to show that both repressors interact in living yeast cells. However, this interaction diminished after the initiation of hyphal growth. Consequently, we examined the transcription of the core filamentation response genes in mutants lacking either Tup1 or Nrg1 or both. QRT PCR results revealed that the presence of Tup1, but not Nrg1 was required for high level expression of ALS3 and ECE1 under serum-inducing conditions. In accordance, overexpression of TUP1 supported transcription of these two genes. In contrast, overexpression of NRG1 repressed both genes, but mainly in a Tup1-dependent mechanism. Surprisingly, TET-promoter driven NRG1 overexpression induced the formation of yeast-like structures in a tup1Δ background, indicating that Nrg1 can repress filamentation at least partially without Tup1. Overall, these observations suggest that Nrg1 and Tup1 work together in the repression of hyphal growth, but after this repression is released, Tup1 participates in the activation of ALS3 and ECE1 transcription in hyphae.

112

P15A

Investigating the roles of a chromatin acylation reader in Candida albicans

Nikolina Vidan1, Bella Qi Wang2, Jiyoti Verma-Gaur2, Mary Sopta1, Ana Traven2 1Ruder Boskovic Institute, Zagreb, Croatia, 2Monash University, Biomedicine Discovery Institute, Melbourne, Australia

Chromatin regulation by histone lysine acetylation is important for transcriptional regulation. In Candida albicans, phenotypes of mutants in histone acetyltransferases and deaceytlases have shown that histone acetylation is important for the regulation of pathways that determine virulence, such as filamentation and the susceptibiliy to antifungal drugs and other stressors. In addition to enzymes that append or remove histone acetylations, «reader» proteins interact with the modified histones and recruit chromatin regulators and transcriptional complexes to DNA to enable biological outcomes. We are focusing on understanding the functions of the YEATS domain histone modification readers in C. albicans. The YEATS domain has been shown to interact with acetylated histones, and more recently was identified as the first reader of histone crotonylation, a novel acyl modification. It has been suggested that distinct histone acylations are regulated by cellular metabolism, making this an interesting problem to study in pathogenic yeasts for which metabolic adaptation is crucial for fitness in diverse host niches. The C. albicans genome encodes two YEATS domain proteins, while Saccharomyces cerevisiae contains three family members. We constructed a mutant in one of the C. albicans YEATS protein-encoding genes, and show roles in filamentation under some conditions, and functions in growth in the presence of cell wall damaging agents. We are currently extending our anaysis to understand how the YEATS proteins control gene expression programs in C. albicans, as well as addressing the conservation of functions of YEATS proteins between C. albicans and S. cerevisiae.

113

P16A

Development of a novel antifungal bioadhesive hydrogel formulation against oral candidiasis

Mary Ann Jabra-Rizk, Eric Kong, Christina Tsui, Ahmed Sultan University of Maryland, Baltimore Maryland, USA

Oral candidiasis (OC) caused by Candida albicans is the most common opportunistic infection in HIV+ individuals and other immunocompromised populations. The dramatic increase in resistance to common antifungals has emphasized the importance of identifying unconventional therapeutic options. Antimicrobial peptides have emerged as promising candidates for therapeutic intervention. Histatin-5 (Hst-5) specifically has exhibited potent anti-candidal activity indicating its potential development as an antifungal agent. In this study, a biocompatible bioadhesive hydrogel delivery system for Hst-5 was designed specifically for topical oral application against OC. The new formulation was evaluated in vitro and in vivo in a mouse model of OC. The findings demonstrated a controlled sustained release of Hst-5 from the polymer, rapid killing ability and stability of antimicrobial activity. Based on viable C. albicans counts recovered from tongues of treated and untreated mice, three daily applications of the formulation beginning one day post infection with C. albicans was effective in protection against development of OC. These findings were confirmed by histopathology analysis of tongue tissue. In addition, the formulation was effective in inhibiting adherence of C. albicans to denture acrylic material and rat palatal tissue. A rat model of denture stomatitis was developed and is currently being used to evaluate the efficacy of the formulation in prevention of Candida-associated denture stomatitis. Coupled with its lack of toxicity as well as its anti-inflammatory properties, the findings from this study support the commercial feasibility of the Hst-5 formulation as a novel therapeutic agent.

114

P17A

The establishment of a specific response during paracoccidioidomycosis is dependent of CD11c+ cells

Suelen dos Santos, Sandro de Almeida Universidade de São Paulo, Sao Paulo, SP, Brazil

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America and the most frequent systemic mycosis in Brazil. The dendritic cells (DCs) are antigen presenting cells able to do the link between innate and adaptive immune response that play a pivotal role in important infections caused by airborne pathogens. During the first 12 hours of PCM infection, lung DCs migrate to lymph nodes and induce a mixed pattern of CD4 T cell cytokines, compatible with a Th1/Th2 response. The aim of this work is to characterize the immunological and cellular profile of mice infected with P. brasiliensis after CD11c+ cells depletion and how the lack of this population interferes in the infection outcome. We evaluate the depletion of CD11c+cells in C57BL/6.CD11c-DTR mice, after 24 hour of administration of diphtheria toxin (DTX). With 4ng/g of animal, we get almost 80% CD11c+ cells reduction in the spleen. After this period, we infected the animals intratracheally with P. brasiliensis. After 7 and 15 days we had a CD4+ cells reduction in lymph nodes, and a small proliferation of this cells obtained from spleen in response of P. brasiliensis antigens, in comparison of not depleted animals. In the absence of CD11c+ cells we saw an increase of IL4, IL-6 and IL-10 and a decrease of Il-17 after 7 days of infection. In conclusion, animals without CD11c+ during the infection had a different pattern of cytokines and a small lymphocyte specific proliferation and altogether these results showed the importance of CD11c+ cells during the establishment of PCM infection.

115

P18A

Intracellular zinc transport and dynamics in Candida albicans

Aaron Crawford, Laura Lehtovirta-Morley, Omran Alamir, Duncan Wilson University of Aberdeen, Aberdeen, UK

Zinc is an essential micronutrient for the growth and development of all organisms, and zinc restriction due to host nutritional immunity results in extreme limitation for pathogens during infection. Candida albicans can coexist as a commensal member of the microbiota, cause vulvovaginal candidiasis and life-threatening invasive infections. The presence and bioavailability of zinc in these conditions can vary greatly. We hypothesise that the ability of this pathogen to thrive in these environments relies on its capacity to create and utilise intracellular zinc stores.

We observe that C. albicans generates vacuolar and vesicular (zincosomes) zinc stores when zinc is present in the environment, and these reservoirs can fuel growth under subsequent nutrient restriction. Using bioinformatics, we have identified two plasma membrane and seven intracellular predicted zinc transporters in C. albicans and created deletion mutants of all of them. Our evidence suggests that one plasma membrane and two intracellular transporters are involved in the generation of zincosomes. Moreover, using fluorescence microscopy and flow cytometry, we have found that zincosomal accumulation is rapid, occurring within 10-20 minutes of exposure to zinc. We are generating fluorescent protein fusions to assess the localisation of these transporters. We are also developing a C. albicans-optimised BiFC (Bimolecular fluorescence complementation) split fluorophore system to assess potential homo and hetero dimerization of these proteins. With these approaches, we hope to elucidate the dynamics of zinc storage and utilisation in Candida albicans, and to further understand the role these processes play during infection, within the context of mammalian nutritional immunity.

116

P19A

Mechanistic basis of epithelial damage in response to A. fumigatus infection

Uju icheoku, Margherita Bertuzzi, Norman van Rhijn, Takanori Furukawa, Zorana Carter, Lea Gregson, Sayema Khan, Panagiotis Papastamoulis, Magnus Rattray, Michael Bromley, Elaine Bignell University of Manchester, Manchester, UK

The human lung is continually exposed to spores of the airborne mould Aspergillus fumigatus. Inhaled spores are small enough to bypass mucociliary clearance mechanisms and reach the alveoli of the lung where interaction between host and pathogen cells can lead to fungal clearance, or to development of inflammatory or invasive fungal diseases. In order to find out why A. fumigatus is cytotoxic to human epithelia we have observed the interaction between host and pathogen in laboratory culture, and measured epithelial decay, lytic death of host cells, host cell signalling and cytokine degradation in response to fungal challenge. This research has revealed multiple mechanisms involved in eliciting epithelial damage, occurring at different stages of the host-pathogen interaction and involving different fungal morphotypes. We have used laboratory experimentation to ask the following questions: Why is A. fumigatus cytotoxic to human cells? Does the host or the pathogen drive epithelial damage and how? How do pathogen-derived proteins shape the immune environment? How is damage driven in the whole animal host? Which elicitors of host damage are produced by A. fumigatus, and have they recently evolved? We also introduce a new high throughput study addressing the A. fumigatus regulatory network driving epithelial damage in cultured human lung cells.

117

P20A

The antifungal caspofungin increases fluoroquinolone activity against Staphyloccocus aureus biofilms by inhibiting N-acetylglucosamine transferase activity

Wafi Siala1, Sona Kucharikova2 ,3, Paul M. Tulkens1, Marie-Paule Mingeot-Leclercq1, Patrick Van Dijck2 ,3, Françoise Van Bambeke1 1Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium, 2VIB-KU Leuven Center for Microbiology, Leuven, Belgium, 3Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KULeuven, Leuven, Belgium

Biofilms play a major role in Staphylococcus aureus pathogenicity but poorly respond to antibiotics. We show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against biofilms grown in vitro (96-well plates or catheters) as well as in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates was inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibited the activity of IcaA, which shares homology with β-1-3-glucan synthase (caspofungin pharmacological target in fungi). This inhibition destructured the matrix, reduced the concentration and polymerization of exopolysaccharides in biofilms, and increased fluoroquinolone penetration inside biofilms. This study describes for the first time a bacterial target for caspofungin. In a broader context, it highlights the interest of drugs inhibiting IcaA in biofilm-related infections.

118

P21A

Expansion of PacC gene family in Fusarium oxysporum NRRL 32931 contributes to fungal pathogenesis

He Yang, Matt James, Peter Tao, Dilay Hazal Ayhan, Li-Jun Ma University of Massachusetts Amherst, Amherst, MA, USA

Pathogens have the ability to perceive the fluctuating environment and tailor their biological activities appropriately to survive in the host. In fungi, the PacC/Rim signaling pathway governs pH response through the zinc finger transcription factor PacC, which is critical for survival and invasive growth. In response to neutral to alkaline pH, Rim/Pal signaling molecules relay signals to PacC, ultimately leading to its cleavage and subsequent nuclei translocation. The signaling pathway is well conserved in fungi: only a single gene for each signaling molecule, including PacC. However, this is not the case in Fusarium oxysporum clinical strain NRRL 32931. One PacC ortholog and three pacC homologues are present in its genome, all retaining the intact zinc finger domain, suggesting active roles for each duplicated gene. Gene expression data confirmed that these NRRL 32931 PacC genes were indeed expressed in alkaline but not acidic conditions. Furthermore, PacC_O knockouts displayed normal fungal growth under both acidic and alkaline conditions, suggesting functional overlay or redundancy. Individual PacC-GFP constructs, under the control of its native promoter, displayed gene expression and nuclei translocation when transfer from acidic to alkaline pH. We are currently working on 1) ChiP sequencing to answer the questions of whether PacC genes regulate the expression of different genes, 2) determine whether PacC duplication is a common feature in clinical strains by using comparative genomics approaches to sequence and analyze the genome of two other F. oxysporum clinical strains, isolated from different tissues/organs, and 3) animal models to dissect their role in pathogenesis.

119

P22A

Exploring fungal virulence using C. elegans

Xing Zhang, Jolanta Polanowska, Nathalie Pujol, Jonathan Ewbank Centre d’Immunologie de Marseille - Luminy (CIML), Marseille, France

We are using the interaction between Drechmeria coniospora and the nematode Caenorhabditis elegans as a model system to investigate fungal pathogenesis and the host response to infection. D. coniospora, an obligate nematophagous fungi, was first reported as Meria coniospora by Drechsler 70 years ago. In the 1980s and 90s, Jansson and Dijksterhuis described in detail how it infects nematodes. The infection process starts with the attachment of spores to the surface of the nematode. Following the formation of appressoria, the fungus penetrates the cuticle, then hyphae grow inside the nematode. After infection, worms die in 2-3 days. Hyphae grow out from the worm and generate new spore clusters.

Given the long co-evolution between D. coniospora and its nematode hosts, we predict that it has developed mechanisms to interfere with host defence. As a first step to investigate this possibility, we sequenced the D. coniospora genome and, combined with RNAseq data, used this to predict a first complete gene set for D. coniospora. Using transcriptome data for different stages of D. coniospora’s life-cycle, we identified genes that are preferentially expressed during infection. We are focusing on genes that are predicted to be secreted into the host. We have characterized a novel fungal virulence factor, an inhibitory saposin A-domain protein that can bind to the nematode antimicrobial saposin B protein (Lebrigand, He et al., PLoS Genetics, 2016). We will use co-immunoprecipitation and Y2H screens to identify host proteins that interact specifically with selected fungal effectors.

120

P23A

PNEUMOCYSTIS: WHAT ARE THE HOST INNATE IMMUNE MECHANISMS CONTROLLING INFECTION?

Patricia Otieno-Odhiambo1, Nontobeko Mthembu1, Suraj P. Parihar1 ,2, Frank Brombacher1 ,2, Jay Kolls4, Gordon D. Brown3, J. Claire Hoving1 1Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa, 2International Centre for Genetic Engineering and Biotechnology, Cape Town, South Africa, 3Aberdeen Fungal Group, Institute of Medical Sciences,University of Aberdeen, Aberdeen, UK, 4The Richard King Mellon Foundation,Institute of Paediatric Research, University of Pittsburgh,School of Medicine, Pittsburgh, USA

The high prevalence of diseases such as HIV/AIDS on the African continent has rendered patients immunocompromised. This is strongly associated with the prevalence of life threatening opportunistic fungal pathogens. Even so, fungal infections are an often overlooked clinical and public health issue. Pneumocystis jirovecii, causes pneumonia (PCP) in HIV/AIDS patients, with an estimated 400,000 cases and mortality of 150,000 per annum. The introduction of highly active antiretroviral therapy has certainly improved the HIV pandemic outlook but PCP related morbidity and mortality remain high. Innate immunity drives pathogen recognition with significant downstream effects on adaptive responses. Both Toll like receptors (TLRs) and C-type lectin receptors (CLRs) are key pattern recognition receptors (PRRs) involved in antifungal immunity. Evidence suggests that Pneumocystis recognition involves PRRs as well. By using a Pneumocystis mouse model and mice deficient in CLRs including Dectin-1, Clecsf8 (Cle4d, Mcl, Dectin 3) or the downstream signalling molecule PKCδ (protein kinase c delta), we hope to dissect the innate immune response in Pneumocystis infection. Preliminary data using PKCδ-/- mice that has been shown to engage Syk Kinase-coupled C-type lectin receptors, shows higher lung burden compared to the wild type mice suggesting Pkcδ plays a role in the immunity against Pneumocystis infection. With the indicative preliminary results, and continual investigation, we hope to discover new insights into underlying host mechanisms involved in protective immunity to Pneumocystis which may lead to improved treatments for immunocompromised hosts.

121

P24AOrchestration of chitin synthesis in Candida albicans

Maria Spyrou, Megan Lenardon, Alistair Brown, Neil Gow University of Aberdeen, Aberdeen, UK

Septation is a highly regulated process, fundamental for cell viability, taking place during cell division. Understanding the biochemistry and cell biology of fungal septation may lead to the identification of tractable drug targets for future antifungal chemotherapeutic strategies. In the pathogenic fungus C. albicans, a primary septum which is made out of chitin is formed between two dividing cells. In C. albicans, chitin is synthesised by four chitin synthases: Chs1, Chs3, Chs2 and Chs8 and all four enzymes localise to sites of septation. Strains expressing pairs of fluorescently tagged chitin synthases were generated to examine timing and position of recruitment in relation to each other. Based on data obtained by live-cell fluorescence microscopy, a model is proposed demonstrating the spatial and temporal distribution of all four chitin synthases during septation in C. albicans hyphae. We demonstrate for the first time that Chs3 arrives at the septation site first prior to the other three chitin synthases and that Chs1 Chs2 and Chs8 form contractile rings that either disappear or are retained as spots upon completion of septation. Proteomic analyses of GFP pull down assays identified further proteins that may be part of the septation complex. These data are sued to present a new model of cell wall synthesis during septation in this fungus.

122

P25A

The chromatin state of DNA repeats controls genome plasticity in Candida albicans

Veronica Freire Beneitez1, Jordan Robert Price1, Daniel Tarrant1, Judith Berman2, Alessia Buscaino1 1University of Kent, Canterbury, UK, 2Tel Aviv University, Tel Aviv, Israel

DNA repeats, found at the ribosomal DNA locus, telomeres and subtelomeric regions, are unstable sites of eukaryotic genomes that are usually assembled into transcriptionally silent heterochromatin. A fine balance between genetic variability and genomic stability tunes plasticity of these chromosomal regions. This tuning mechanism is particularly important for organisms such as microbial pathogens that utilise genome plasticity as a strategy for adaptation. We have recently analysed the chromatin state associated with DNA repeats in the most common human fungal pathogen Candida albicans (1, 2). Our data demonstrate that, in C. albicans, different types of DNA repeats are associated with distinct chromatin states. We have shown that the chromatin state associated with C. albicans subtelomeres controls genomic plasticity via a novel DNA element (3). We are currently investigating the contribution of different chromatin factors to C. albicans epigenetic state and genome stability. These published and unpublished data highlight how mechanisms regulating genome stability are rewired in C. albicans.

1. Freire-Benéitez,V., et al (2016) Candida albicans repetitive elements display epigenetic diversity and plasticity. Sci. Rep., 6, 22989.

2. Freire-Benéitez,V., Price,R.J. and Buscaino,A. (2016) The Chromatin of Candida albicans Pericentromeres Bears Features of Both Euchromatin and Heterochromatin. Front. Microbiol., 7, 759.

3. Freire-Benéitez,V., et al (2016) Sir2 regulates stability of repetitive domains differentially in the human fungal pathogen Candida albicans. Nucleic Acids Res., 10.1093/nar/gkw594.

123

P26A

Attenuation of neutrophil recruitment contributes for development of A. fumigatus induced allergic airway inflammation

Natalia Troyanova, Alexander Sapozhnikov, Marina Shevchenko Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia

A. fumigatus fungi can induce neutrophil-mediated inflammation when inhaled in a high dose or proallergic eosinophil-mediated response after multiple inhalations of low doses of fungal extract or conidia. Here we prove the hypothesis that allergic airway response development is a result of insufficiency of neutrophil recruitment. High (150 µg/mouse/injection) and low (10 µg/mouse/injection) doses of A. fumigatus extract were administrated to the oropharyngeal cavity of BALB/c mice twice a week. Bronchoalveolar lavage (BAL) and bone marrow (BM) cells were analyzed at different time points (4, 8, 12, 48, 72 hours) after distinct numbers of extract applications (up to 6). Morphological analysis of BAL and BM cells was performed using Diff Quick staining. Neutrophils were obtained by negative separation of BM cells. CXCR2 and CXCR4 expression on BM neutrophils was detected by flow cytometry. Inflammatory leukocytes recruited to the airways in an allergen dose dependent manner, however at acute phase (12 hours after single extract inhalation) BAL cells of mice that received both high and low allergen doses were represented primarily with neutrophils. In mice that received low doses of A. fumigatus extract further challenges leaded to replacement of BAL neutrophils with eosinophils. Each next application of high dose of extract induced repeated recruitment of neutrophils and decreased the population of immature CXCR4+ CXCR2- BM neutrophils. Applications of low allergen doses did not significantly affect immature BM neutrophils. Thus, A. fumigatus extract induces allergic airway inflammation manifests in the absence of de novo neutrophil recruitment from bone marrow to the airways.

124

P27A

Development of a plasmid based system for CRISPR-Cas9 mediated mutation in Candida parapsilosis

Lisa Lombardi, Siobhán Turner, Fang Zhao, Geraldine Butler School of Biomedical and Biomolecular Science, Conway Institute, University College Dublin, Dublin, Ireland

The RNA-guided CRISPR-Cas9 endonuclease system has revolutionized genome editing in eukaryotes, and its popularity exponentially increases, with almost 1500 papers published in 2016. The CRISPR-Cas9 elements (Cas9 and guide RNA, gRNA) can be used in the fungal pathogen Candida albicans to knockout gene function (Vyas et al., 2015; Mitchell et al., 2016). However, the published methods require either integration of CAS9 into the genome of the host organism, or its transient expression and integration of a selectable marker. Here we describe the first plasmid-based CRISPR-Cas9 system in Candida species. We have developed a method for gene editing in the diploid yeast Candida parapsilosis, which is frequently responsible for infections in infants. The autonomously replicating plasmid confers Nourseothricin resistance, thus avoiding the need for integrating a selectable marker. Both CAS9 and the gRNA are carried on the same plasmid, and ribozymes flank the gRNA to allow its expression from a PolII promoter (Gao et al., 2014). The system successfully mutated ADE2 in a panel of C. parapsilosis strains, including clinical isolates, with an efficiency ranging from 20 to 80%. The plasmid is lost in absence of selection, so that detrimental effects of prolonged Cas9 expression can be avoided. Overall, this system may allow high throughput generation of mutations in relevant clinical isolates, as well as simultaneously target several genes, which would facilitate the characterisation of gene families in C. parapsilosis.

125

P28A

Potential of intraepithelial airway mucosal dendritic cells to maintain the antifungal defense in immunosuppression

Marina Shevchenko1, Andrey Bogorodskiy2, Valentin Borshchevskiy2, Alexander Sapozhnikov1 1Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia, 2Moscow Institute of Physics and Technology, Dolgoprudny, Moscow region, Russia

Intraepithelial dendritic cells (i.e.DCs) of the airway mucosa locate beneath the epithelial barrier in a close proximity to the epithelial cells and form trans-epithelial processes to the airway lumen. They can sense airborne pathogens, including Aspergillus fumigatus conidia. Together with neutrophils i.e.DCs can internalize conidia. Susceptibility to fungal infection is commonly associated with neutropenia. The aim of the study is to investigate the potential of i.e.DCs to maintain the sufficient antifungal defense in the absence of neutrophils.

Oropharyngeal application of A. fumigatus conidia was performed 24 hours after either immunosuppression with cyclophosphamide and cortisone acetate or neutrophil depletion. 8 hours after lungs were perfused, fixed, airways were dissected and subjected to immunochemistry as whole-mounts. Images were acquired using Zeiss LSM 780 as Z-stacks and processed with ZEN and FIJI software. i.e.DCs were identified according to MHCII expression, morphology and location.

Injection of cyclophosphamide and cortisone acetate significantly decreased the number of MHCII-positive dendritic cells including i.e.DCs compare to that in intact mice. Neutrophil depletion by both anti Gr-1 and anti Ly-6G did not alter i.e.DC number. The number of i.e. DCs was also uneffected after A. fumigatus conidia application.

Thus neutropenia does not affect morphology and number of i.e.DCs in the airway mucosa. Therefore i.e.DCs can be considered as potential therapeutic targets that are able to control fungal dissemination and recover antifungal defense.

The work was supported by Russian Foundation for Basic Research project No. 16-04-00989 А

126

P29A

Complete X-ray structure at 3,7 Å resolution of the Candida albicans 80S ribosome

David Bruchlen1 ,2, Simone Pellegrino1, Yacine Dahman3, Marcela Sabou2, Mélanie Meyer1, Fabrice Jossinet3, Julie Denis2, Marat Yusupov1, Ermanno Candolfi2 1Biologie Structurale Intégrative, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg; CNRS, UMR7104; INSERM U964, Illkirch, France, 2Institut de Parasitologie et de Pathologie Tropicale de Strasbourg, Université de Strasbourg, Strasbourg, France, 3Institut de Biologie Moléculaire et Cellulaire, Architecture et Réactivité de l’arN, UPR9002 CNRS, Strasbourg, France

Candida albicans is an opportunistic pathogenic fungus which causes mostly oral and genital infections in humans. It has become a very important subject since C. albicans can ultimately lead to death of immunocompromised patients. Counteracting its growth is therefore needed to avoid such health threat.

In our laboratory we developed the technology for obtaining crystals of 80S ribosome from S. cerevisiae diffracting at 3 Å resolution (Ben-shem et al. (Science), 2011), paving the way for the understanding of the mechanism of translation in eukaryotes. Thanks to this knowledge we solved the structure of the vacant 80S ribosome from C. albicans at high resolution (3,7 Å), by X-ray crystallography. The structure shows a high degree of identity with the previous solved 80S ribosome structure of S. cerevisiae. Nevertheless, differences in important functional sites are present and can be unravelled.

These new crystallization conditions can be used to solve the structure of the 80S ribosome in complex with functional ligands, ultimately leading us to gain new insights about the regulation of protein biosynthesis in C. albicans. The vacant 80S structure will also serve as a platform to perform drug design and screening in order to identify new molecules that could specifically target this pathogen.

127

P30A

Extracellular vesicles from stressed Candida albicans cultures: differential transcriptome and effect in fungal cell communication

Natanael P. Leitão Jr.1, Maria G. Amorim2, Ayrton Carvalhedo1, Diana N. Nunes2, Emmanuael Dias-Neto2, Rosana Puccia1 1EPM-UNIFESP, São Paulo, SP, Brazil, 2A. C. Camargo Cancer Center, São Paulo, SP, Brazil

Extracellular vesicles (EVs) are membranous closed structures that carry a wide variety of cell components outside prokaryotic and eukaryotic organisms. In pathogenic fungi, such as Paracoccidioides brasiliensis and Candida albicans, EVs carry components that might be relevant to host/pathogen interaction and immune modulation. We have previously characterized P. brasiliensis EVs and described their protein, RNA, lipid, and carbohydrate composition. Current projects aim at characterizing the differential protein and RNA contents of EVs from C. albicans and P. brasiliensis under oxidative/nitrosative stress and farnesol/tyrosol stimulus, and investigating the role of EVs in fungal/fungal communication. For the present work, EVs were isolated from cell-free culture supernatants by standard differential centrifugation procedures and recovered after a final 1-h ultracentrifugation (100,000×g). Here we show: a) that when C. albicans was previously incubated with EVs, the association of fungal cells with RAW 264.7 was inhibited by 15-20% in 2-to-4-h co-cultures. The result was similar for control EVs and EVs isolated from cultures stimulated with tirosol (EVtir) or H2O2; b) that EVtir stimulated C. albicans hyphal formation within specific culture conditions; c) differential EV transcriptome, as analysed by eletrophoregrams and RNAseq, from C. albicans grown under oxidative stress or in the presence of farnesol and tirosol. Comparison between EV and total C. albicans transcriptome suggested that the EV population does not reflect the cell RNA population. Financial support: FAPESP, CNPq, CAPES.

128

P31A

Host response to infection with Candida species

Elise Iracane, João Pacheco, Geraldine Butler University College Dublin, Dublin, Ireland

Candida parapsilosis and Candida tropicalis are human commensal yeasts but are also responsible for fungal infection, including new born or immunocompromised patients. Understand the mechanisms of infection of these emerging pathogens and the host response during the infection is important to fight and prevent fungal infection. We are therefore studying the interaction of both species with the human host, using oral and vaginal epithelial cell lines as ex vivo infection models. (TR-146, A-431 and HMV-II) We are assessing adhesion, invasion and damage of each cell line by both Candida species. The adhesion and invasion assay are measured by double staining of fungal and human cells with Calcofluor white and concanavaline A. Damage of the host cell is evaluated after 24h and 48h by measuring the levels of LDH released. C. parapsilosis and C. tropicalis weakly invade epithelial tissue, compared to C. albicans. We have assessed the damage inflicted on A-431 cells by 74 isolates of C. tropicalis and 27 isolates of C. parapsilosis. Both species confer damage after 48h of infection. The amount of damage varies between isolates, but C. tropicalis and C. parapsilosis damage is on average 1.5-fold and 20-fold lower than C. albicans, respectively. The next stage is to use RNA-seq to determine the transcription profile of host and pathogen during different stages of infection with both Candida species. This data will be compared to identical experiments evaluating infection by C. albicans and C. glabrata, and will be used to discover potential infection markers, and ultimately for development of diagnostic tools.

129

P32A

The Candidalysins are a functionally conserved family of fungal toxins

Jonathan Richardson, Shir-Lynn Tan, Giulia Carrano, David Moyes, Julian Naglik King’s College London, London, UK

Candida albicans is a commonly encountered opportunistic fungal pathogen of humans responsible for mucosal and life threatening systemic ailments that contribute to high morbidity and mortality worldwide. During mucosal infection, C. albicans grows as infiltrating pathogenic hyphae that secrete Candidalysin, a 31 amino acid peptide toxin derived from its parent protein, Ece1p. Candidalysin destabilises the structural integrity of epithelial cell plasma membranes, facilitating infection. Epithelial cells respond to Candidalysin through a bi-phasic p38/c-Fos signalling pathway that culminates in a strong inflammatory response at mucosal surfaces. Analysis of Ece1p amino acid sequences from different Candida species revealed the presence of additional putative Candidalysin toxins in C. dubliniensis, C. tropicalis, C. albicans 529L and C. maltosa. We used solid phase peptide synthesis to generate these peptide sequences and quantified their ability to cause damage, activate mucosal inflammatory responses and the p38/c-Fos response pathway in oral (TR146) and vaginal (A431) epithelial cells. All putative toxins caused epithelial damage as evidenced by quantification of lactate dehydrogenase activity and induced epithelial signalling (p38/c-Fos) and cytokine secretion. Notably, the putative toxins of C. tropicalis and C. dubliniensis exhibited greater potency in vitro when compared with the Candidalysin of C. albicans. These data suggest conservation of function between the different Candidalysins despite differences in amino acid sequence and identify the Candidalysins as a family of functionally related fungal toxins.

130

P33A

Microbiological characterization of the colonization by Candida spp. in patients with orthodontic fixed appliances and evaluation of immunological parameters in saliva.

Christian R Batarce1 ,2, Jose Amaro1, German Hermosilla1, Paula I Rodas3, Ivan Hidalgo4, Catalina Batarce1 ,2, Cecilia V Tapia1 ,5 1Universidad de Chile, Santiago, Chile, 2Centro Medico y Dental Animet, Linares, Chile, 3Universidad Andres Bello, Santiago, Chile, 4Instituto de Ortodoncia Santiago, Santiago, Chile, 5Clinica Davila, Santiago, Chile

The use of orthodontic fixed appliances has increased significantly in our country especially by esthetic reasons. Fixed appliances have an effect in the local microbiota facilitating the colonization by Candida spp. We characterized microbiologic and immunologically the colonization by Candida spp. in patients with fixed appliances Methods: Seventy-five patients (51 females and 24 males) were studied. Fungal burden of Candida spp, susceptibility to fluconazole (by Yeast OneR test) and genetic diversity (by RAPD) were determined. Interleukin-1ß and IL-10 were measured in saliva by ELISA and the gene expression of human beta defensin 3 (HBD3) by RT-PCR. Candida isolates were identified by MALDI-TOF. Results: Only C. albicans was isolated and the colonization rate was 6.7% (5/75). The fungal burden was 81 + 49.3 ufc/cm2 of oral mucosa. Five genetic clusters were identified, being the colonies highly related by patient (similarity coefficient or SAB>0.9), and very similar between patients. Treatment time was higher in colonized patients (p<0.05). The fluconazole MIC90/susceptibility range was 1/0.25-1 µg/mL. A negative correlation between the fungal burden and IL-1ß levels was found in colonized patients and the opposite for IL10. Significantly, non-colonized patients expressed higher expression levels of HBD3 compared to colonized (p<0.05). Conclusions: Candida albicans was the only colonizing agent. Patients had their own C. albicans cluster, but clusters between patients were highly related probably due to the same geographic location. Colonized patients coursed longer treatment times than non-colonized, being a risk factor. Higher levels of IL-1ß and HBD3 in saliva seem protective against colonization.

131

P34A

The Rim pathway is involved in antifungal tolerance in C. albicans

Cécile Garnaud1 ,2, Encarnacion García-Oliver3 ,4, Danièle Maubon1

,2, Jérôme Govin3 ,4, Muriel Cornet1 ,2 1CHU Grenoble Alpes, Grenoble, France, 2Laboratoire TIMC-IMAG TheREx, UMR5525 CNRS-UGA, Université Grenoble Alpes, Grenoble, France, 3U1038, Université Grenoble Alpes, Institut de Biosciences et Biotechnologies de Grenoble (BIG), Institut national de la santé et de la recherche médicale (Inserm), Grenoble, France, 4Laboratoire Biologie à Grande Echelle (BGE), Commissariat à l’énergie atomique et aux énergies alternatives (CEA), Institut de Biosciences et Biotechnologies de Grenoble (BIG), Grenoble, France

Invasive candidiasis (IC) is a major cause of morbidity and mortality, despite antifungal treatment. Azoles and echinocandins are used as first-line antifungals for the treatment of IC. However, their efficacy is limited by both tolerance and the emergence of acquired resistance. The pH-responsive pathway, named rim pathway in yeasts, is fungal-specific and conserved among the fungal kingdom. It is involved in both C. albicans pathogenesis and virulence. In addition, its terminal transcription factor, rim101p, was shown to be involved in tolerance to azoles and echinocandins in this species. This study aimed at investigating the implication of the other Rim proteins in antifungal tolerance in C. albicans, as well as the mechanisms underlying it. Time-kill curves experiments and colony formation tests showed that all the Rim proteins were involved in azoles and echinocandins tolerance. Transcriptomic analysis of a rim101-/- mutant, a strain constitutively-overexpressing RIM101 and a wild-type strain allowed to identify several genes involved in antifungal tolerance as Rim-dependent genes, among which HSP90. Rim mutants were also hypersensitive to pharmacological inhibition of Hsp90, suggesting that RIM101 acts upstream HSP90. Targeting the Rim pathway could therefore represent a promising antifungal strategy, in combination with existing antifungal drugs, to indirectly target HSP90.

132

P35A

From Bug to Drug: In-house production of bespoke antifungal peptide aptamers

Bethany McCann, Christopher Grice, Darren Thomson, Margherita Bertuzzi, Elaine Bignell University of Manchester, Manchester, Greater Manchester, UK

Of around 250 Aspergillus species, Aspergillus fumigatus is the most pathogenic to humans causing invasive fungal infections, and killing upwards of 200,000 people annually. However the number of available antifungal drugs is limited and occurrence of antifungal resistance is increasing.

Peptide aptamers (PAs) can be defined as short; 8-20 amino acids in length, combinatorial peptide fragments doubly constrained within inert protein scaffolds which exhibit functionality much like antibodies. High specificity and affinity for target proteins permits PAs the ability to modulate functionality of targets. Such ligand based perturbation of function paves the way for the elucidation of novel drug targets and the design of potential inhibitory agents against virulence-causing mechanisms.

A. fumigatus mutants lacking the pH-dependent transcription factor PacC, or an upstream pH-responsive receptor protein PalH, display highly attenuated virulence resulting in non-invasive phenotypes. Inhibition of PalH mediated pH signalling is therefore an attractive novel antifungal strategy.

In an initial screen for selection of anti-PalH PAs, we used a yeast membrane two hybrid approach expressing the PalH receptor as bait, and a library of thioredoxin constrained peptide aptamers as preys. The approach successfully identified PalH-binding PAs, however in all instances expression in A. fumigatus delivered gain of pH function phenotypes. We describe the development of two critical new tools, of PAs causing loss of pH signalling, using direct selection in A. fumigatus: a randomised library of Peptide Aptamers (PAs) expressed, under regulatable control, in A. fumigatus, additionally a split-fluorescence approach for direct detection of protein-protein interactions, and their subsequent inhibition.

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P36A

Insights into host-pathogen interactions by dual RNAseq.

Jiyoti Verma1, Timothy Tucey1, Paul Harrison2, Traude Beilharz1, Thomas Naderer1, Ana Traven1 1Infection and Immunity Program and the Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Australia, 2Victorian Bioinformatics Consortium, Monash University, Melbourne, Australia

Candida albicans is the most prevalent fungal species causing human infections. Macrophages are the first line of immune defence against fungal infections. We have previously established that Candida co-culture with macrophages causes host cell death in two distinct phases termed Phase I and II. Phase I death lasts for the first 6-8 hours and results from NLRP3/caspase 1 inflammasome-dependent pyroptosis. Phase II death starts later, it is caspase 1-independent, and it kills the majority (60-70%) of the macrophage population under our experimental conditions, showing it is dominant over pyroptosis. In order to understand host and fungal factors that coordinate these distinct macrophage death pathways, we have performed dual RNAseq of Candida infecting murine bone-marrow-derived macrophages at time points corresponding to early and mid Phase I death, and early Phase II death. We observed changes to the transcriptional programs linked to host antimicrobial responses, host cell death pathways and host and pathogen metabolism as the infection progressed through the Phase I and Phase II stages of host cell death. Our data shows that the transcriptional activators Tye7 and Gal4 play a role in enabling C. albicans to adapt after it escapes from macrophages following pyroptosis, thereby controlling the ability of the pathogen to rapidly elicit Phase II death. Collectively, our study addresses how, by adapting to intracellular and then extracellular infection environments and usurping changes in host physiology during infection, C. albicans is able to thrive and trigger macrophage death.

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P37A

Functional characterization of pathogenesis-related proteins in Candida albicans

Yannick Bantel1, Rabih Darwiche3, Nick K. Olrichs4, Steffen Rupp1 ,2, J. Bernd Helms4, Kai Sohn1 ,2 1University of Stuttgart, Stuttgart, Germany, 2Fraunhofer IGB, Stuttgart, Germany, 3University of Fribourg, Fribourg, Switzerland, 4Utrecht University, Utrecht, The Netherlands

Members of the CAP protein superfamily are found among all kingdoms of life and their biological function seems to be very diverse. Despite their broad evolutionary distribution and intensive research efforts the molecular function of these proteins is still controversial. Currently binding and export of sterols and the formation of amyloid fibrils is discussed as a potential function. In pathogenic fungi like Candida albicans and Fusarium oxysporum, CAP proteins have recently emerged as novel virulence factors. Deletion of RBE1 and RBT4, two members of the CAP protein superfamily in C. albicans, leads to reduced virulence in a systemic mouse infection model. Aiming to dissect the role of Rbe1p and Rbt4p in pathogenesis, we found that in contrast to the CAP proteins Pry1p and Pry2p in S. cerevisiae, in vitro sterol binding could not be observed for Rbe1p and Rbt4p, even though the sterol export defect of a ∆pry1∆pry2 mutant could be rescued by expression of RBE1 and RBT4. Furthermore no amyloid fibril formation of Rbe1p or Rbt4p was induced in the presence of liposomes in vitro. Surprisingly, immunofluorescence showed that Rbe1p was enriched at budding sites and this localization was strongly dependent on cell morphology. Using a chemical crosslinker, Rbe1p could be linked to an SDS-insoluble component of the cell wall. The collected data suggests a different molecular function for Rbe1p and Rbt4p in comparison to other CAP family members, especially Rbe1p seems to play a role in cell wall associated processes.

135

P38A

IL-10 overexpression predisposes to invasive aspergillosis by suppressing innate and adaptive pulmonary antifungal immunity

Cristina Cunha1 ,2, Samuel Gonçalves1 ,2, Cláudio Duarte-Oliveira1 ,2, Luís Leite3, Katrien Lagrou4 ,5, Carmen Lupiañez6 ,7, Inês Mesquita1

,2, Joana Gaifem1 ,2, Ana Margarida Barbosa1 ,2, Oliver Kurzai8, Fernando Rodrigues1 ,2, António Gil Castro1 ,2, Ricardo Silvestre1 ,2, Juan Sainz6 ,7, Johan Maertens9, Egídio Torrado1 ,2, Ilse Jacobsen10, João Lacerda11 ,12, António Campos Jr.3, Agostinho Carvalho1 ,2 1Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal, 2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal, 3Serviço de Transplantação de Medula Óssea (STMO), Instituto Português de Oncologia do Porto, Porto, Portugal, 4Department of Microbiology and Immunology, KU Leuven - University of Leuven, Leuven, Belgium, 5Department of Laboratory Medicine and National Reference Center for Medical Mycology, University Hospitals Leuven, Leuven, Belgium, 6Genomic Oncology Area, GENYO, Center for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Granada, Spain, 7Hematology Department, Virgen de las Nieves University Hospital, Granada, Spain, 8Septomics Research Centre, Friedrich Schiller University and Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany, 9Department of Hematology, University Hospitals Leuven, Leuven, Belgium, 10Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany, 11Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisboa, Portugal, 12Serviço de Hematologia e Transplantação de Medula, Hospital de Santa Maria, Lisboa, Portugal

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Aspergillus fumigatus causes superficial and invasive infections, and allergic reactions. The risk of disease varies significantly among patients with similar predisposing conditions, but the genetic mechanisms that influence the efficiency of individual antifungal immune responses remain largely undefined. To dissect the contribution of genetic variation within the immunoregulatory cytokine IL-10 to the development of invasive aspergillosis (IA), we analyzed variants at the IL10 locus modifying the risk of IA in 413 recipients of allogeneic hematopoietic stem-cell transplantation and their donors. The functional consequences of genetic variation were investigated in vitro, in patient specimens, and using IL-10-overexpressing mice. We report that the donor rs1800896 variant in IL10 increased the risk of IA after transplantation. The risk genotype was correlated with an enhanced production of IL-10, associated with an impaired proinflammatory profile of cytokines in infected patients. Human macrophages carrying the risk genotype secreted high amounts of IL-10 in response to A. fumigatus infection, which suppressed innate antifungal effector function and inflammatory cytokine production. Furthermore, IL-10-overexpressing mice displayed increased susceptibility to pulmonary aspergillosis, as revealed by the higher fungal burden throughout the course of infection. The elevated levels of IL-10 were associated with a defective inflammatory response to the fungal challenge, supported by an impaired leukocyte accumulation and T-cell expansion in the lung. Our findings highlight a pivotal IL-10-mediated mechanism that regulates the balance between anti- and proinflammatory immune responses to A. fumigatus and plays an essential role in protective antifungal immunity in mice and humans.

137

P39A

The Candida albicans HIR histone chaperone guards morphogenetic yeast-to-hyphae transition by adjusting transcriptional amplitudes

Sabrina Jenull1, Michael Tscherner1, Megha Gulati2, Clarissa J. Nobile2, Karl Kuchler1 1Medical University of Vienna, Department of Medical Biochemistry, Max F. Perutz Laboratories, Campus Vienna Biocenter, Vienna, Austria, 2Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA, USA

Morphological plasticity such as the yeast-to-hyphae transition is a key virulence factor of the human fungal pathogen Candida albicans. Hyphal formation requires a multi-layer signaling network composed of environmental sensing, signal transduction, dedicated transcriptional modulators and chromatin modifications. Here, we demonstrate a novel role for the replication-independent HIR histone chaperone complex in morphogenesis, as HIR acts as a crucial activator of hyphal development. Genetic removal of the HIR complex subunit Hir1 decreases sensitivity to morphogenetic stimuli. Strikingly, HIR1-deficient cells display altered transcriptional amplitudes upon hyphal initiation, suggesting that Hir1 fine-tunes gene expression to establish the transcriptional threshold required for morphogenetic fate decisions. Furthermore, ectopic expression of the transcription factor UME6, which facilitates hyphal maintenance, rescues the filamentation defects of hir1Δ/Δ cells, implying that Hir1 regulates the early transcriptional phase of hyphal initiation. Hence, chromatin-mediated fine-tuning of transcriptional loads is crucial for driving morphogenetic conversions in the fungal pathogen C. albicans.

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P40A

Role of Candida albicans-derived prostaglandin E2 in fungal colonization of the host

Tze Guan Tan, Norman Pavelka Singapore Immunology Network, Singapore, Singapore

Production of prostaglandin E2 (PGE2) by Candida albicans has been described to influence fungal growth and hyphal formation in vitro, as well as promote allergic responses in mice upon fungal overgrowth in the intestines. However, the physiological role of fungus-derived PGE2 during steady-state colonization of the gut by C. albicans remains undefined. In mice, PGE2 augments intestinal barrier function and suppresses inflammatory responses from a variety of immunocyte populations. We hypothesize that C. albicans-derived PGE2 promotes the asymptomatic colonization of the intestines by the fungus, thereby maintaining host-fungus symbiosis. To test this hypothesis, we are examining the impact of boosting or inhibiting PGE2 signalling on intestinal C. albicans titres and dissemination in mice. Additionally, we will generate C. albicans mutants deficient in PGE2 production and assess their capacity to colonize the murine gut relative to that of the wildtype strain. Understanding the role of PGE2 in mediating C. albicans commensalism versus pathogenicity in the host will yield insight into the means by which C. albicans colonization and virulence in people can be controlled.

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P41A

Are Candida glabrata drug efflux pumps from the MFS clinically relevant?

Catarina Costa1 ,2, Jonathan Ribeiro1 ,2, Isabel M. Miranda3 ,4, Ana Silva-Dias3 ,4, Mafalda Cavalheiro1 ,2, Sofia Costa-de-Oliveira3 ,4, Acácio R. Gonçalves3 ,4, Miguel C. Teixeira1 ,2 1Department of Bioengineering, Instituto Superior Técnico, University of Lisbon, Lisbon, Portugal, 2iBB - Institute for Bioengineering and Biosciences, Biological Sciences Research Group, Lisbon, Portugal, 3Department of Pathology, Division of Microbiology, Faculty of Medicine, University of Porto, Porto, Portugal, 4CINTESIS - Center for Health Technology and Services Research, Faculty of Medicine of the University of Porto, Porto, Portugal

Azole drug resistance in Candida species has long been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters. Recently, drug efflux pumps from the Candida glabrata Drug:H+ Antiporter (DHA) family were found to play a role in this process. CgAqr1 [1] and CgQdr2 [2] confer resistance to imidazole antifungals, CgQdr2 expression being controlled by CgPdr1. CgTpo3 [3], CgTpo1_1 and CgTpo1_2 [4] confer resistance to imidazoles, but also triazole antifungals. These transporters were shown to catalyze azole extrusion across the plasma membrane [1-4]. A link between DHA gene expression and the clinical acquisition of antifungal drug resistance was recently demonstrated [5]. After screening 138 Candida glabrata clinical isolates for azole drug resistance, harvested from patients attending two major Hospitals in Portugal, ten susceptible and ten resistant to clotrimazole were selected for further analysis. The transcript levels of CgAQR1, CgQDR2, CgTPO1_1 and CgTPO3 were found to be significantly upregulated in resistant isolates when compared to the susceptible ones, with a level of correlation similar to that observed for the ABC encoding gene CgCDR2. Altogether, our results suggest that azole resistance is not the result of the action of a single effector, but rather the combined action of several drug efflux pumps from the ABC and MF Superfamilies.

[1] Costa et al, Front Microbiol, 4:170, 2013; [2] Costa et al, AAC, 57:3159, 2013; [3] Costa et al, JAC, 69:1767, 2014; [4] Pais et al, MCP, 15:57, 2016; [5] Costa et al, Front Microbiol, 7:526, 2016.

140

P42A

Bacterial secreted factors regulate mucormycete germination

Courtney Kousser, Kerstin Voelz, Rebecca A Hall Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK

Within the human body, microorganisms reside as part of a complex and varied ecosystem, where they rarely exist in isolation. Therefore, bacteria and fungi have co-evolved to develop elaborate and intricate relationships, utilising both physical and chemical communication mechanisms. Mucormycetes are spore-forming fungi belonging to the order Mucorales and are the causative agent of potentially fatal mucormycosis in immunocompromised individuals. Key to the pathogenesis of mucormycetes is the ability to swell and germinate leading to penetration of the surrounding tissues, angioinvasion, vessel thrombosis, and tissue necrosis. Mycomycete spores are found ubiquitously in the environment and in wounds, where they encounter a myriad of bacterial and fungal species including Pseudomonas aeruginosa. Here we show that culture supernatants from P. aeruginosa inhibit the germination of Rhizopus microsporus spores, without affecting spore swelling. This inhibition of germination was not due to the presence of quorum sensing molecules, but was instead due to the iron restriction, as addition of exogenous iron restored germination. We hypothesise that P. aeruginosa secreted iron siderophores, which sequester the available iron inhibiting fungal germination. Therefore, treatment of P. aeruginosa in trauma wounds could result in the release of this inhibition of germination, leaving the patient prone to an underlying fungal infection.

141

P43A

Molecular tools for studying Candida parapsilosis virulence

Tibor Mihaly Nemeth1, Sara Pal1, Leszek Pryszcz2, Toni Gabaldon2, Attila Gacser1 1Department of Microbiology, University of Szeged, Szeged, Hungary, 2Bioinformatics and genomics, Centre for Genomic Regulation, Barcelona, Spain

Candida parapsilosis is a diploid yeast belonging to the CUG clade of Ascomycetes that gained importance since the mid-1980s as a common fungal pathogen of low birth weight neonates. To characterise its pathogenesis related properties and interaction with the host, several genetic tools were developed, applied and published in the literature during the last decade. Gene deletion mutants provided essential data on this field, however opposite approach namely overexpressing selected genes has never been performed in this species. To identify virulence related genes, we set up an in vitro infection model utilising THP-1 human monocytes and a clinical isolate of the yeast in question. RNAseq analysis of the fungus revealed several transcription factors and kinases representing altered expression upon infection. Additionally this set of genes was supplemented with C. albicans orthologs encoding proteins related to resistance, cell-wall synthesis or adhesion and served as candidates for overexpression. For such an approach the Invitrogen Gateway system was adapted and framed into C. parapsilosis. Interestingly target region must have been modified to gain homogeneous expression of reporter genes GFP and mCherry. Overexpression mutants of selected genes were characterised under various conditions ranging from regular vitality capabilities to stress circumstances related to virulence. Further modification of the system led us to create reintegrant derivatives of deletion mutants, moreover it is also planned to employ this system to regulate expression of desired genes by using TETON/TETOFF system.

142

P44A

Next generation sequencing (NGS) based diagnostics of fungal blood stream infections

Silke Grumaz1, Philip Stevens1 ,4, Christian Grumaz1, Sebastian Decker2, Markus Weigand2, Stefan Hofer2, Thorsten Brenner2, Arndt von Haeseler3, Kai Sohn1 1Fraunhofer IGB, Stuttgart, Germany, 2University clinic, Heidelberg, Germany, 3CIBIV, Vienna, Austria, 4IGVP, Stuttgart, Germany

Rapid diagnosis of infecting agents is the most critical step in order to effectively treat critically ill patients suffering from fungal blood stream infections. Generally, culture based diagnostic approaches are time-consuming and are limited with respect to sensitivity and specificity. Similarly, targeted molecular tests in many cases are biased and in many cases provide ambiguous results. Levels of circulating cell-free DNA from microbial origin in plasma are elevated in septic patients thus providing a promising class of biomolecules for the detection of pathogens. In fact, by high throughput sequencing of isolated cell-free DNA from septic patients suffering from suspected fungal infections we could unambiguously identify significant levels of fungal DNA. In this context, we established a Sepsis Indicating Quantifier (SIQ) score to discriminate relevant infecting agents from contamination. Accordingly, we were able to diagnose systemic blood stream infections by different Candida species including C. albicans and C. glabrata. In order to compare sequencing based SIQ scores with state of the art microbiological diagnostics, matched blood for every plasma sample at identical time points were drawn for blood culture analyses. Time from sample to diagnosis generally took approximately 24 hours providing results in a reliable and competitive time frame. Diagnostics by SIQ score analyses revealed a higher detection rate for the identification of infecting agents not only for fungi, but also for bacteria and viruses than standard blood culture analyses, indicating that sequencing based diagnostics provides a more sensitive and specific approach for septic blood samples.

143

P45A

The immunogenic Sporothrix brasiliensis peptides promoted proliferation in CD3+CD4+ cells, being vaccine candidates in experimental sporotrichosis.

José Roberto Fogaça de Almeida1, Grasielle Pereira Jannuzzi1, Gilberto Hideo Kaihami1, Sandro Rogério de Almeida1 1Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo - SP, Brazil, 2Institute of Chemistry, University of São Paulo., São Paulo - SP, Brazil

Sporotrichosis is a mycosis caused by S.brasiliensis, which was responsible for the sporotrichosis outbreak in Rio de Janeiro. The 70 kDa glycoprotein, is the major antigen of the S.brasiliensis, that is higher virulent specie, being lethal in experimental esporotrichosis. Our work is developing of novel therapeutic approaches against sporotrichosis. We characterized the immunogenic peptides of S.brasiliensis. We found, 16 spots in the western blot, using Mab P6E7 and the serum of mice infected with S.brasiliensis, this spots were excised from the 2D gel and analysed by mass spectrometry. Through the S. brasiliensis database, 34 proteins were identified, which were analysed peptides with higher degree of engagement with MHC class II. Through the predictions programs, were selected 7 peptides for synthesis. Lymphoproliferation assay demonstrated that 4 peptide promoted proliferation in CD3+CD4+ and CD3+CD8+ cells from mice with sporothrichosis. One of the positive peptides is a peptide of the gp70 protein (ZR8), which in in vivo treatments was able to decrease the lesions size of the mice and to increase the production of IFN-gama and IL-17 in the lesions sites. This approach can aid the developing of new treatment for Sporothrix brasiliensis, that increasing incidence of resistance to antifungal drugs and causes severe disease in humans.

144

P1B

Classification, Prediction and Analysis of Type VI Secreted Effector Proteins

Rishika Sen, Losiana Nayak, Rajat Kr. De Indian Statistical Institute, Kolkata, West Bengal, India

Pathogens (virus, bacteria, prion, fungus, viroid and parasites) invade their host(s) and cause pathogenic conditions via several substrates and pathways. One of the modus operandi by which pathogens ambush the host is via protein secretion. All secretion systems discharge proteins, called effector proteins, into the host body when they come in contact with it. These effector proteins play important role in bacterial pathogenesis. Pathogens, particularly the gram-negative bacteria, have six different, i.e., T1, T2, T3, T4, T5 and T6 secretion systems. Among them, T6 Secretion System (T6SS) is identified in 2006. Effector proteins of the T6SS of many species have not yet been discovered. In this respect, we have considered the known effector proteins of a few species from an established T6 effector protein database and designed a Machine Learning based T6 effector protein prediction method. Using this method, a set of 125 T6 effector proteins in Vibrio cholerae and 180 T6 effector proteins in Yersinia pestis were predicted as effector proteins. Biological validation by literature survey and gene ontology-based information confirmed many of them have the potential for being an effector protein. In addition, the 3D structures of the T6 effector proteins were analyzed by convex hull theory. This theory considers 3D composition and core structures of a given molecule. On analyzing the features, we have found that there is a high correlation of 0.897 between the layers of convex hull and the radius of gyration of the T6 effector proteins.

145

P2B

Candida albicans takes a licking during infection

Alex Hopke1, Nadine Nicke1, Erica Hidu1, Genny Degani2, Laura Popolo2, Robert Wheeler1 1University of Maine, Orono, ME, USA, 2University of Milan, Milan, Italy

Pathogens hide immunogenic epitopes from the host to evade immunity, persist and cause infection. Candida albicans, which can cause fatal disease in immunocompromised patient populations, masks the inflammatory epitope β-glucan in its cell wall from host recognition. While β-glucan becomes exposed during infection in vivo, the mechanism behind this exposure was unknown. Here, we show that this unmasking involves neutrophil extracellular trap (NET) mediated attack, which triggers changes in fungal cell wall architecture that enhance recognition by the Dectin-1 β-glucan receptor in vitro. Furthermore, using a mouse model of disseminated candidiasis, we demonstrate the requirement for neutrophils in triggering these fungal cell wall changes in vivo. Importantly, we found that fungal epitope unmasking requires an active fungal response in addition to the stimulus provided by neutrophil attack. NET-mediated damage initiates fungal MAP kinase-driven responses, particularly by Hog1, that dynamically relocalize cell wall remodeling machinery including Chs3, Phr1 and Sur7. Neutrophil-initiated cell wall disruptions augment some macrophage cytokine responses to attacked fungi. This work provides insight into host-pathogen interactions during disseminated candidiasis, including valuable information about how the C. albicans cell wall responds to the biotic stress of immune attack. Our results highlight the important but underappreciated concept that pattern recognition during infection is dynamic and depends on the host-pathogen dialog.

146

P3B

The Dark Side of the Wall: Atomic Force Microscopy Revelations on Drug Resistance and Adhesion.

Helene Martin-Yken1, Cécile Formosa-Dague1, Marion Schiavone1, Etienne Dague2 1LISBP INRA Universite de Toulouse, Toulouse, France, 2LAAS CNRS, Toulouse, France

The cell wall of yeast and fungi plays a crucial role in the way these cells sense, respond and adapt to environmental perturbations. Using recent Atomic Force Microscopy technological developments, the biophysical consequences of different stresses on the major human fungal pathogen, Candida albicans, were imaged and measured. Morphological changes were characterized at the nanoscale, including surface roughness, elasticity and adhesive properties. Exposure to the antifungal Caspofungin was shown to cause a deep cell wall remodeling with major modifications of chitin and beta-glucan content. Remarkably, a low dose of Caspofungin (i.e., 0.5 × MIC) provoked a strong expression of adhesive proteins on the cell surface of C. albicans, a side effect highly relevant considering its wide spread medical use. Moreover, Single Molecule Force Spectroscopy (SMFS) experiments by AFM allowed us to visualize the organization of these adhesins, to map them on the cell surface and to quantify the adhesion forces, including on cells undergoing morphogenetic differentiation. Combined with molecular biology tools, this approach enabled us to unravel the particular contribution of previously uncharacterized proteins (PGA22 and PGA59) to C. albicans adhesion mechanism. In addition, functionalizing the AFM tip with antibodies allows following the appearance of specific proteins, while precisely mapping them at the cell surface, and even measuring the time scale of their progression through the cell wall. The example of Hwp1 appearance on geminating hyphal tubes will be illustrated.

147

P4B

Role of RSC chromatin remodeling complex in genome stability and morphogenesis in Candida albicans.

Priya Prasad, Santanu Kumar Ghosh Indian Institute of Technology, Bombay, Mumbai, Maharashtra, India

Candida albicans is a commensal organism of human gastrointestinal tract and a prevalent opportunistic pathogen exhibiting different morphological forms to survive in different host niches. A high frequency of morphological switching argues for an epigenetic rather than a genetic control on these events where chromatin architecture is involved. C. albicans have regional centromeres, and its genome stability appears to be influenced by epigenetic determinants like histone modifier and chromatin remodeler some of which have the roles in morphological changes. We hypothesize the role of ATP-dependent chromatin remodeler RSC in genome stability, morphogenesis and myriad stress tolerance in C. albicans, given its role on chromosome segregation and stress tolerance in the distant yeast Saccharomyces cerevisiae. In this study, to decipher the functions of RSC complex in C. albicans, Sth1, an ATPase and key component of RSC complex is being characterized. We found CaSTH1 an essential gene and depletion of Sth1p showed an elongated pseudohyphal phenotype. sth1 mutants showed abnormal spindle and sensitivity towards anti mitotic drugs thiabendazole suggesting a role of CaSth1 in kinetochore-microtubule mediated processes. The mutant affects global cohesin-chromatin association with a consequent premature separation of kinetochores. Sth1 appears to be also involved in DNA metabolism as the mutant showed sensitivity towards hydroxy urea, but not to MMS and the double mutants sth1 rad9 and sth1 mec1 showed aggravated phenotypes. Justifying the chromatin remodeling activity of RSC with the above phenotypes, a gross change in the chromatin architecture has been observed in the cells with altered level of Sth1.

148

P5B

Pathogenic yeasts express virulence determinants in a new natural model system to defend against amoeba predation

Silvia Novohradská1, Renáta Tóth2, Sascha Brunke1, Attila Gácser2, Jörg Linde1, Falk Hillmann1 1Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena, Germany, 2Department of Microbiology, University of Szeged, Szeged, Hungary

Candida, Aspergillus and Cryptococcus are the leading causative agents of systemic mycoses. Many of their representatives have also been isolated from non-human sources, suggesting that their virulence potential might have been partially shaped in their natural niche as a strategy to counteract environmental predators. Previously we demonstrated that phagocytic interactions of filamentous fungi are conserved between soil amoeba and macrophages. We have now established a new natural amoeba model system to study evolutionary forces that could have led to the generation and maintenance of virulence traits in human pathogenic yeasts. Protostelium fungivorum is an exclusively mycophagous amoeba, widely spread in nature, which we have successfully isolated and completely sequenced its genome. Large-scale feeding experiments revealed a broad prey spectrum within yeasts and filamentous fungi. With an extreme efficiency, the yeasts were recognized, phagocytosed and killed in a range of few minutes. As C. parapsilosis was found to be a preferential prey, we used this pathogen in a dual-transcriptome approach to identify targets of this predatory-prey interaction. Amoeba-responsive genes included those involved in the elevated metal efflux, oxidative stress response, filamentous growth and secreted lipase, suggesting predatory selection pressure on these important virulence determinants. As a killing mechanism, we propose mobilization of internal copper resources leading to an impaired oxidative stress response and intoxication inside of the acidic phagolysosome. Constructed deletion mutants for the most promising targets will further reveal if traits that have originated to counteract with natural predators could also have supported the resistance against innate immune cells.

149

P6B

Anti-fungal drug susceptibility testing in Malassezia spp.

Cheryl Kit Mun Leong, Antonino Buttafuoco, Martin Glatz, Philipp Bosshard University and University Hospital Zurich, Zurich, Switzerland

Malassezia spp, are a lipid dependent genus of commensal yeast which have been implicated in a host of dermatological conditions such as atopic dermatitis and seborrheic dermatitis. They may sometimes also be associated with severe systemic infection, particularly in infants or the immune-compromised. Severe skin diseases or systemic infections require topical or systemic anti-mycotic treatment. Anti-fungal drug resistance is an emerging concern in the treatment of such conditions, where it is associated with poor clinical outcomes and higher healthcare costs. However, the susceptibility of Malassezia spp. to anti-mycotics is not well established due to the lack of standardized testing assays available.

Using our optimized broth dilution method adapted from NCCLS/EUCAST guidelines, we established the resistance profiles of 13 species of Malassezia spp. alongside 39 clinical strains with 11 commonly used anti-mycotics. All strains showed intrinsic resistance to the echinocandin drug class as well as to griseofulvin. The triazoles itraconazole, posaconazole and voriconazole were the most effective anti-mycotics. Fluconazole and ketoconazole had the widest MIC ranges and was the least effective drug for many strains, in particular for strains of animal-host associated species. M. furfur had the widest range of MIC values for most drugs. These results were consistent with known findings on Malassezia drug susceptibility and most strains tested were within published MIC ranges of the tested drugs. The feasibility of anti-fungal drug susceptibility testing provides valuable information on suitable anti-mycotics for treatment of Malassezia associated conditions and enhances our understanding of the role of Malassezia in skin disease.

150

P7B

Contribution of a Candida albicans secreted cysteine-rich protein in fungi-host interaction

Wenjuan Wang, Wencheng Zhu, Hongyu Wu, Qun Zhao, Jiangye Chen Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

Candida albicans is the most frequently isolated opportunistic pathogen of humans. It is a prevalent fungal pathogen but also a normal resident in human niches. It can colonize and infect many host niches, such as the gastrointestinal tract, oral cavity, bloodstream, and the vagina, give rise to mucosal infections and diverse symptoms in immunocompromised patients, including serious disseminated infections. To identify the secreted proteins required for C. albicans commensalism and pathogenesis in human, we characterized the small secreted cysteine-rich proteins (SCPs) in C. albicans by bioinformatic and functional analyses. Our results showed that among the SCPs, Scp1 acts as an effector to mediate the fungi-host interaction. The expression and secretion of C. albicans Scp1 is highly increased when C. albicans co-cultured with host cells. The Scp1 secreted from C. albicans could trigger the activation of MAPK and NF-kB pathways and increase the production of cytokines and chemokines in macrophages. In systemic mouse infection model, the scp1 deletion mutant cells are more virulent, whereas the purified Scp1 proteins could protect mice against C. albicans infection. Our data suggest that the secreted Scp1 functions as an alertor to trigger host immune responses during C. albicans infection and host defence.

151

P8B

Drug resistance via RNAi-dependent epimutations in Mucor circinelloides

Zanetta Chang1, Blake Billmyre1, Soo Chan Lee2, Joseph Heitman1 1Duke University, Durham, North Carolina, USA, 2University of Texas at San Antonio, San Antonio, Texas, USA

The opportunistic fungal infection mucormycosis is notable for high mortality as well as increasing incidence. Treatment is complicated by the fact that Mucor circinelloides, a major cause of mucormycosis, demonstrates high intrinsic resistance to most antifungal agents. However, the mechanisms driving this extensive resistance remain poorly understood. Previous work demonstrated that Mucor is capable of developing transient resistance to the antifungal FK506 through a novel, RNA interference-dependent mechanism known as epimutation. Epimutants silence the drug target gene and can be selected by exposure to FK506; the target gene is re-expressed in these strains following passage without selective pressure. This silencing process involves the generation of small RNAs (sRNA) against the target gene via core RNAi pathway proteins. To further investigate the role of epimutation in Mucor’s intrinsic antifungal resistance, we studied the development of resistance to a second drug, 5-fluoroorotic acid (5-FOA). We have identified epimutants that exhibit resistance to 5-FOA without mutations in either of the target genes, pyrF or pyrG. We conducted sRNA hybridization analysis to document the presence of sRNA against pyrF or pyrG in these epimutants, and that this sRNA is lost after reversion to drug sensitivity. Analysis of sRNA libraries generated from these epimutants demonstrated expression of sRNA against the pyrF and pyrG loci, respectively. From this, we conclude that epimutation is a general mechanism through which Mucor can develop resistance to multiple antifungal agents.

152

P9B

Unveiling transcriptional regulation in Candida species: using the PathoYeastract database

Pedro T. Monteiro3 ,4, Pedro Pais1 ,2, Catarina Costa1 ,2, Isabel Sá-Correia1 ,2, Miguel C. Teixeira1 ,2 1iBB - Institute for Bioengineering and Biosciences, Lisbon, Portugal, 2Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal, 3Department of Computer Science and Engineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal, 4INESC-ID, Lisbon, PortugalIt is essential to understand the structure and functioning of the transcription networks regulating the early response to clinically relevant environmental changes, in Candida species, to be able to understand and circumvent the long term acquisition of virulence and drug resistance-related phenotypes. In that sense, the PathoYeastract (http://pathoyeastract.org) database was developed for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in the pathogenic yeasts Candida albicans and C. glabrata [1]. This freely available information system currently includes 28,000 unique documented regulatory associations between transcription factors (TF) and target genes and 107 DNA binding sites, considering 134 TFs in both species. Being a twin of the YEASTRACT database [2], PathoYeastract makes available bioinformatics tools to predict the TFs involved in the regulation of a gene or genome-wide transcriptional response. Each search can be filtered to selected only specific environmental conditions or experimental evidence. Promoter analysis tools and visualization tools for the representation of TF regulatory networks are also provided. A preliminary comparative genomics setup for the study of cross-species evolution of regulatory networks is further offered, aiming the prediction of gene and genomic regulation based on orthologous regulatory associations described for other yeast species.

[1] Monteiro et al, Nucleic Acids Res, 45:D597, 2017; [2] Teixeira et al, Nucleic

153

Acids Research, 42: D161, 2014.

P10BCharacterization of the fungal phosphatidylserine synthase as a drug target

Chelsi Cassilly, Todd Reynolds University of Tennessee, Knoxville, Knoxville, TN, USA

Treatments for Candida albicans systemic infections are limited to three classes of antifungals, some of which have harmful side effects or increasing instances of antifungal resistance. This illustrates a need for new antifungals. The phosphatidylserine (PS) synthase, Cho1p, represents a potential drug target that can address this need. Cho1p is involved in a major phospholipid biosynthesis pathway and represents a novel drug target for two key reasons: 1) It is required for virulence, indicating that inhibitors of Cho1p would render the organism incapable of causing infection; and 2) it is absent in mammals, so inhibitors potentially have no toxic side effects. In order to take full advantage of Cho1p as a drug target, we have begun detailed characterization of the biochemistry of this protein. Cho1p has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. We have found that the Km for serine is 1.3 mM and the Km for CDPDAG is 0.069 mM. Further, western blot analysis indicates that the activity of this protein is likely regulated via phosphorylation. We have confirmed via site-directed mutagenesis that a conserved CDP-binding motif is present in the sequence of Cho1p and is involved in PS synthesis. Work is currently underway using homology modeling and site-directed mutagenesis to identify the serine binding region. The results of his project will lead to more direct approaches for finding Cho1p inhibitors, leading ultimately to more effective antifungals, as well as a greater understanding of enzymes involved in phospholipid biosynthesis, which is necessary for virulence in this pathogen.

154

P11B

Role of Arf GTPases in Candida albicans filamentous growth and virulence

Hayet Labbaoui1, Stéphanie Bogliolo1, Scott G Filler2, Norma V Sollis2, Robert A Arkowitz1, Martine Bassilana1 1Université Côte d’Azur, CNRS, Inserm, iBV, Nice, France, 2Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, USA

Virulence of the human fungal pathogen Candida albicans is associated with the switch from budding to hyphal growth. Sustained membrane traffic is critical for such polarized growth and the secretion of virulence factors. The Arf (ADP-ribosylation factor) small GTPases are critical for membrane traffic in a number of organisms. To investigate the importance of this class of proteins during C. albicans hyphal growth, we generated loss of function mutants in all 5 Arf/Arl (Arf like) proteins. Our results reveal that only Arf2 is required for viability and sensitivity to antifungal drugs and that both Arf2 and Arl1 are required for hyphal growth, with arl1 hyphal filaments being 2-fold shorter than that of the wild-type. Furthermore, both Δ/pTetARF2 and arl1 mutants have drastically reduced virulence, with ARL1 particularly critical for oropharyngeal candidiasis. We show that while the defects in Δ/pTetARF2 appear to be due to alteration of the Golgi, the defects in arl1 are likely to result from the inability of this mutant to restrict growth to a single axis. Further analyses of the arl1 mutant revealed that this defect does not result from a misregulation of the GRIP-domain golgin coiled-coil tethering protein Imh1 nor of the phosphatidylserine flippase Drs2. Rather, our results suggest that the arl1 hyphal growth defect results from increased secretion. Together our work identifies Arf2 and Arl1 as key regulators of membrane traffic, critical for hyphal growth and virulence.

155

P12B

Candida albicans genes associated with translocation through intestinal epithelial barriers

Stefanie Allert1, Toni Foerster1, Marc Juraschitz1, Daniela Schulz1, Betty Hebecker2 ,4, Tony Pawlik2, Ilse D. Jacobsen2 ,3, Selene Mogavero1, Lydia Kasper1, Bernhard Hube1 ,3 1Department of Microbial Pathogenicity Mechanisms, Hans-Knöll-Institute, Jena, Germany, 2Research Group Microbial Immunology, Hans-Knöll-Institute, Jena, Germany, 3Friedrich-Schiller-University, Jena, Germany, 4Aberdeen fungal group, University of Aberdeen, Aberdeen, UK

The opportunistic pathogen Candida albicans can cause life-threatening systemic infections, which predominantly originate from the commensal C. albicans population of the intestinal tract. However, C. albicans translocation from the gut into the bloodstream is not well characterized. The aim of this project is to elucidate the molecular mechanisms associated with damage of and translocation through intestinal epithelia and to define potential pathogenicity factors required for intestinal tissue invasion. Characterization of processes associated with epithelial damage and fungal translocation using an in vitro translocation model showed that C. albicans translocation mainly occurs via a transcellular route, which is associated with fungal-induced epithelial damage. Hence, translocation correlates with necrosis, but not apoptosis of intestinal cells. However, translocation can also occur to some extend without any damage, indicating a tight junction-associated paracellular translocation mechanism. Screening of C. albicans libraries identified deletion mutants with reduced or increased damage potential. Selected damage-defective mutants were analyzed for infection-associated attributes, invasive potential and impact on epithelial barrier function in the translocation model. Almost every mutant showed a unique pattern of defects indicating that several biological properties are required for full damage potential. One of the most interesting mutants, however, was the mutant lacking ECE1. The polypeptide Ece1 and the Ece1-derived lesion-forming peptide toxin Candidalysin are dispensable for the ability to produce hyphae, to adhere to or invade into intestinal cells, but are essential for causing intestinal epithelial damage. Our data show that translocation of C. albicans through intestinal layers is associated with Ece1-mediated necrosis and epithelial barrier breakdown.

156

P13B

The in vivo efficiency of anidulafungin and tigecycline against polymicrobial catheter-associated peritonitis

Ona Rogiers, Patrick Van Dijck, Sona Kucharikova VIB-KU Leuven Center for Microbiology, KU Leuven Laboratory of Molecular Cell Biology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium

The emerging use of medical devices encounters an increased occurrence of biofilms-related infections. Both the bacterium Staphylococcus aureus and the yeast Candida albicans are key players in the cause of hospital-acquired infections with an extreme ability to inhabit diverse host niches especially in immunocompromised individuals. It is crucial to study and understand the behavior of these pathogens when coexisting together and to discover a viable option for treatment of not only single species but also mixed species biofilms.

In the present study we focused for the first time on C. albicans - S. aureus device- associated intraperitoneal biofilm infections. With the development of a new in vivo model to study catheter-associated peritonitis caused by C. albicans and S. aureus we provided an insight into the pathogenesis of this dual-species biofilm-associated infection and elucidated possible options for treatment. Both anidulafungin and tigecycline showed promising results against intraperitoneal catheter-associated C. albicans – S. aureus biofilm infections when used in appropriate doses (1mg/kg of body weight/day vs 10mg/kg of body weight/day). Further we examined the efficacy of combination treatment consisting of anidulafungin and tigecycline against mature C. albicans – S. aureus intraperitoneal device-related biofilm infections. All together, these results are promising and suggest that in combination anidulafungin drives tigecycline to bactericidal activity. Currently the in vivo studies are conducted and the results will be presented in the poster.

157

P14B

Novel approaches for understanding and fighting trauma-associated fungal infections

Wioleta Trzaska1, Helen Wrigley1, Joanne Thwaite2, Robin May1 1University of Birmingham, Birmingham, UK, 2DSTL, Salisbury, UK

Trauma-associated fungal infections represent a significant threat to immunocompetent and immunocompromised patients. Consequently, strategies to remove fungal pathogens from hospital surfaces or patient skin offer the potential for significant therapeutic benefit, in particular for patients suffering from traumatic injury.

Fungi such as Mucormycete, Fusarium and Scedosporium species are typically introduced into the wound as spores from environmental contamination, which then germinate and spread causing fatal invasive fungal infections. Many of these species are highly resistant to antifungal treatment and, in addition, drug accessibility to wounded tissues is difficult. Thus, disseminating wound infections are very hard to treat and consequently result in very high mortality.

To try and address this problem, we have been taking a dual approach of a) screening for novel treatments and b) investigating the underlying immune reaction to these organisms.

Using this strategy we have recently demonstrated that very dilute acetic acid represents a powerful, low cost strategy to prevent and treat skin fungal diseases. Also, we explore the antifungal potential of blue light, which appears to be effective against bacterial pathogens without the need to introduce exogenous photosensitizers into the pathogen. By testing a wide range of fungal pathogens, we show that blue light is effective against some but not all species.

In parallel, we have also been investigating host pathogen interaction in order to probe the early immune response to invading fungal pathogens. We will discuss findings from all of these systems at this meeting.

158

P15B

Characterization of the Candida albicans ECE1 promoter

Enrico Garbe1 ,2, Ronny Martin1 ,3, Oliver Kurzai1 ,3, Slavena Vylkova2 1Septomics Research Center, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany, 2Research Group Host Fungal Interfaces, Friedrich Schiller University, Jena, Germany, 3Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany

ECE1 is one of the most abundant genes in C. albicans hyphae. It encodes a cytolytic peptide causing damage to host cells membranes. Like other core filamentation response genes as ALS3 and HWP1, ECE1 has a very long 5’ intergenic region of 3197 bp. In this study we examined which parts of this intergenic region are required for ECE1 regulation during yeast and hyphal growth. 5’ RACE-PCR identified a notably short 5’ UTR of 50 bp, suggesting a TATA box within the first 110 bp upstream of the start codon. Next, various fragments of the intergenic region consisting of the first 500 bp up to the whole length region were fused to GFP and ectopically integrated into the NEUT5L locus. The resulting mutants were screened microscopically for GFP signals and mRNA levels were determined via qRT-PCR. 1200 bp were determined as the minimum size required for a weak fluorescence signal, while 1500 bp and larger fragments induce bright signals, an observation which correlated with detected mRNA amounts. However, a deletion of the first 1000 bp led to the absence of detectable GFP signals, indicating their importance for full activation of ECE1. We further analysed mutants lacking transcription factors potentially binding to the promoter region. Amongst this set Ahr1 turned out as a novel regulator required for high-level transcription of ECE1. Taken together, these findings allow further experiments to define the possible interaction pattern and binding behaviour of transcription factors at this promoter.

159

P16B

Temporal and spatial control of fungal filamentous growth

Patrícia M. Silva, Martine Bassilana, Robert A. Arkowitz Université Côte d’Azur, CNRS, Inserm, iBV, Nice, France

Candida albicans is a human pathogen that can cause life-threatening infections in immunocompromised patients, in part, due to its ability to switch between an oval budding form and a hyphal form. The small-Rho GTPase Cdc42 plays a crucial role in hyphal growth and, in its active state, localizes as a persistent tight cluster at the tips of growing hyphal filaments (1). Our goal is to investigate the importance of this site-specific location and whether it is crucial for initiation and maintenance of hyphal growth. Using a light-activated membrane recruitment system, we are able to control the recruitment of constitutively active Cdc42 to the plasma membrane. We are investigating how such a photo-recruitment disrupts hyphal growth and where, when and how is new growth subsequently initiated. Our results demonstrate that, upon photo-recruitment of active Cdc42, filament extension is abrogated and new growth can occur throughout the cell. Location of new growth correlates with the length of the filament, which may reflect how well- established the growth site is. Before a new growth site appears, the initial filament stops extending, concomitant with the disruption of the cluster of endogenous active Cdc42. We are currently investigating the molecular mechanisms that underlie this disruption at the initial hyphal growth site and the subsequent site- specific initiation of new hyphal growth.

(1) Corvest, Bogliolo, Follette, Arkowitz & Bassilana (2013). Mol Microbiol. 89: 626-48.

160

P17B

The role of APSES transcription factors in Histoplasma capsulatum virulence and adaptation to stress conditions

Larissa V G Longo1 ,2, Rosana Puccia1, Chad A Rappleye2 1Escola Paulista de Medicina - Universidade Federal de São Paulo - UNIFESP, São Paulo, SP, Brazil, 2The Ohio State University - OSU, Columbus, OH, USA

The APSES proteins belong to a fungal-specific family of transcriptional factors of the basic helix-loop-helix (bHLH) class. They function as key regulators of development and other biological processes, such as stress response and control of virulence traits in various fungi. Our work investigates the role of the APSES transcription factors in the biology and virulence of the thermodimorphic pathogenic fungus Histoplasma. capsulatum. The H. capsulatum encodes four APSES proteins: Stu1, Aps2, Mbp1, and Swi6. None of these factors showed increased expression under nitrosative or oxidative stress or during macrophage infection. However incubation at pH 4 caused a 2-fold down-regulation of Swi6 and Mbp1 and a 4-fold up-regulation of Stu1 compared to yeasts at pH 7, suggesting roles in pH-dependent responses. To determine their functional roles in Histoplasma biology, RNA-interference (RNAi) was used to knocked-down APSES protein levels. Lines were generated that had more than 90% silencing of Stu1, Aps2, and Swi6 as determined by co-silencing of the GFP sentinel. However, only 50% knock down was achieved for Mbp1, suggesting some essentiality of Mbp1. All knocked-down lines showed normal growth kinetics of yeasts and virulence in P388D1 macrophages. In a mouse model of histoplasmosis, Stu1- and Aps2-deficient strains were strongly attenuated. In addition, the fungal transition from yeast to mycelial phase was impaired in APSES-deficient cells. These data indicate Histoplasma virulence in vivo requires APSES-family transcription factors and further experiments to assess what aspect of infection is defective are being performed.

161

P18B

Using pathoproteomics to unveil new players in antifungal drug resistance in the human pathogenic yeast Candida glabrata

Pedro Pais1 ,2, Catarina Costa1 ,2, Carla Pires1 ,2, Mafalda Cavalheiro1 ,2, Miguel Teixeira1 ,2 1Institute for Bioengineering and Biosciences, Lisbon, Portugal, 2Instituto Superior Técnico, Lisbon, Portugal

The pathogenic yeast Candida glabrata presents intrinsic and acquired resistance to antifungals, which limits their clinical effectiveness. The understanding of the resistance mechanisms applied by this pathogen is of great importance to develop better therapeutic approaches. This study presents a membrane proteome-wide response of Candida glabrata to the antifungals clotrimazole [1] and 5-flucytosine [2]. Clotrimazole belongs to a family extensively used to treat fungal infections, while 5-FC is used in combination therapy. Using iTRAQ-MS, 37 proteins were involved in clotrimazole response, including translation machinery, mitochondrial function, multidrug resistance transporters and cell wall remodeling. The multidrug resistance transporters CgTpo1_1 and CgTpo1_2 were found to confer clotrimazole resistance by decreasing intracellular drug accumulation. Additionally, clotrimazole was found to exert a deleterious effect over the cell wall, counteracted by cell wall remodeling. Moreover, 32 proteins responsive to 5-FC were identified. Over-represented groups comprise aminoacid metabolism, cell wall remodeling and multidrug resistance transporters. The multidrug resistance transporters CgFlr1 and CgFlr2 were found to contribute to 5-FC resistance by decreasing drug accumulation in Candida glabrata cells. In both clotrimazole and 5-FC exposure, approximately 50% of the responsive proteins were found to be dependent on the transcription factor CgPdr1, highlighting the existence of additional targets of this major regulator of multidrug resistance in clinical isolates.

[1] Pais et al, Mol Cell Proteomics, 15:57, 2016; [2] Pais et al, Front Microbiol, 7:2045, 2016.

162

P19B

Therapeutic implications of Candida albicans and Staphylococcus aureus mixed biofilms

Eric Kong1, Christina Tsui1, Sona Kucharicova2, Patrick Van Dijck2, Mary Ann Jabra-Rizk1 1University of Maryland, Baltimore MD, USA, 2KU Leuven, Leuven, Belgium

Biofilm associated fungal-bacterial mixed infections are responsible for significant morbidity and tend to be challenging to treat. Candida albicans and Staphylococcus aureus are leading microbial pathogens mainly due to their ability to form biofilms on implanted medical devices. However, the impact of mixed species biofilm growth on therapy remains largely understudied. We recently demonstrated in vitro that in mixed biofilms, C. albicans conferred S. aureus with significantly enhanced tolerance to antibacterials. As these findings may be indicative of therapeutic outcomes in a co-infected host, we developed a clinically relevant subcutaneous catheter mouse model to explore the phenomenon of enhanced antimicrobial tolerance in mixed infections under in vivo conditions. In this model, catheters infected with S. aureus, C. albicans or in combination were implanted subcutaneously in mice and biofilms were allowed to form for 48 hours prior to initiation of vancomycin therapy, which was administered for 6 days. Harvested catheters were assessed for microbial recovery and scanning electron microscopy analysis. Results demonstrated that where vancomycin significantly reduced S. aureus recovery in mice infected only with S. aureus, in co-infected animals, vancomycin had no impact on S. aureus recovery. Microscopic images revealed a thick biofilm matrix formed within catheter lumen composed of C. albicans hyphae and embedded S. aureus. These findings underscore the clinical and therapeutic implications of mixed biofilm-associated infections in a host. Understanding microbial interactions central to the persistence and resistance of polymicrobial infections will greatly aid in overcoming limitations of current therapies and in defining potential new drug targets.

163

P20B

Systematic interrogation of Aspergillus fumigatus cyp51A polymorphisms for azole drug resistance

Christoph Sasse2, Elisa Kolander1 ,2, Anna Dudakova1, Gerhard Braus2, Uwe Gross1, Oliver Bader1 1Institute for Medical Microbiology, University Medical Center Goettingen, Goettingen, Germany, 2Institut für Mikrobiologie und Genetik, Georg-August-University, Goettingen, Germany

Azole antifungal drug resistance in Aspergillus fumigatus is an emerging public health problem concerning critically ill patients, such as stem cell transplant recipients, cancer patients or patients under intensive care. We have previously surveyed the presence of known cyp51A polymorphisms in isolates obtained from patients as well as the environment and found both, highly prevalent TR34/L98H rand TR46/Y121F/T289A polymorphisms as well as well as rarer substitutions whose functional relevance was unclear (e.g. F219C), despite correlation with drug resistance. Compilation of MIC values from the literature associated with individual substitutions shows heterogeneous distribution between intermediate and resistance categories in several cases, indicating the necessity of molecular studies to further elucidate this. To assess the relevance of individual polymorphisms known, we chose to express them each in a common genetic background and measure the resulting MIC values according to the EUCAST protocol. A cyp51A deletion mutant and a respective reconstituted strain were created in strain AfS35. The deletion of cyp51A resulted in a 4-fold reduction in MIC, which was alleviated in the reconstituted strain, where cyp51A was reintroduced under control of its native promotor. Introduction of cyp51A alleles known to cause resistance confirmed the phenotype in our system. Testing of other polymorphisms present in the population ruled out their contribution to resistance. We have developed a system, within which we can test the cyp51A polymorphisms present in the A. fumigatus population for their relevance. Results for the most prevalent alleles confirm the resistance or susceptibility profiles observed in clinical or environmental isolates.

164

P21B

Interactions between Candida albicans and sepsis-relevant intestinal bacteria

Mario Kapitan1, Joanna Niemiec1, Steffen Höring2, Tor Monsen3, Bettina Löffler2, Ilse Jacobsen1 1Hans-Knöll-Institute, Jena, Germany, 2University Hospital Jena, Jena, Germany, 3University Hospital Norrland, Umeå, Sweden

As a commensal on mucosal surfaces, Candida albicans not only interacts with the host but also with other members of the microbiota. Furthermore, disseminated infections frequently originate from intestinal microorganisms and coinfections with C. albicans and sepsis-relevant bacteria are not uncommon. We hypothesized that interactions between C. albicans and intestinal bacteria influence not only colonization rates but also initiation and progression of systemic infections. One genus of bacteria associated with mixed bacterial-fungal infections are Enterococci, especially Enterococcus faecalis and E. faecium. To analyse the interactions between C. albicans and Enterococci, we developed co-cultivation and coinfection assays with human enterocytes. Interestingly, the quality of interactions was not only species- but also highly strain-dependent, ranging from antagonistic to synergistic effects, and was influenced by the presence of host cells. In the absence of host cells, most E. faecalis strains showed enhanced growth in presence of C. albicans or impaired fungal long-term survival. In contrast, coinfection of enterocytes commonly led to increased damage if C. albicans was combined with E. faecalis, whereas most tested E. faecium strains reduced overall enterocyte damage. Importantly, these synergistic and antagonistic effects were additionally influenced by oxygen concentrations, a factor that is highly variable in the host. We will further focus our attention on differences between selected strains from the two species to identify mechanisms that mediate antagonism in vitro and enhanced enterocyte damage.

165

P22B

Genome-wide profiling of histone modifications in Candida albicans using Spike-in ChIP-Seq.

R. Jordan Price, Alessia Buscaino University of Kent, Canterbury, Kent, UK

Repetitive DNA regions, such as those found at telomeres, centromeres and the rDNA locus, are assembled into transcriptionally repressed chromatin, known as heterochromatin. The presence of heterochromatin at these regions suppresses recombination and promotes stability of the genome. We have shown previously that the histone deacetylase Sir2 is required for hypoacetylated heterochromatin formation at telomeres, subtelomeres and the rDNA locus in the human fungal pathogen Candida albicans. Furthermore, we have shown that Sir2 regulates C. albicans genomic stability at subtelomeric regions. To gain a deeper understanding of the role of Sir2 as an epigenomic regulator in this organism, we have performed for the first time genome-wide profiling of histone modifications H3K9ac and H4K16ac in BWP17 (wt) and sir2-/- mutant strains. Conventional ChIP-Seq methodologies only provide information on genomic distributions of histone modifications, which makes it difficult to compare occupancies between samples derived from different cell types or between cells that have experienced genomic alterations. To reveal quantitative changes in the occupancy of these modifications between strains, we used a spike-in ChIP-Seq normalisation method. Using this methodology allowed us to detect subtle epigenomic changes and we have identified specific loci where Sir2 is required to prevent hyperacetylated chromatin.

166

P23B

Fungal Transformers: Tracking a Moving Target

Delma Childers1, Gabriela Avelar1, Judith Bain1, Arnab Pradhan1, Daniel Larcombe1, Mihai Netea2, Lars Erwig1 ,3, Neil Gow1, Alistair Brown1 1University of Aberdeen Centre for Medical Mycology, Aberdeen, UK, 2Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands, 3GlaxoSmithKline, London, UK

In human hosts, the opportunistic fungal pathogen Candida albicans often proliferates in sugar-poor niches (e.g. the gut), but is exposed to sugars in other niches (e.g. the bloodstream). Carbon sensing regulates several aspects of fungal physiology including metabolism, cell wall architecture and elasticity, and virulence. In addition, yeast cell division exposes pathogen-associated molecular patterns (PAMPs) at the cell surface that are known to be immune-stimulatory (e.g. β-glucan). Host niches, morphogenesis and antifungal drugs have also been implicated in PAMP exposure. However, little is known about which host signals influence PAMP exposure and the molecular mechanisms that modulate this exposure. We have recently shown that lactate, an alternative carbon source present in mucosal niches and produced by activated innate immune cells, acts as a signalling molecule to modulate β-glucan exposure (Ballou et al., Nature Micro, 2016). Lactate-induced β-glucan masking is driven via a non-canonical signal transduction pathway resulting in a calcineurin-independent activation of the transcription factor Crz1. However, it remains to be elucidated whether the reduction in β-glucan exposure is the result of PAMP camouflaging by other cell wall components, by PAMP modification, or a combination of both. Therefore, we are characterizing the signalling pathways and downstream effectors that affect PAMP exposure in response to different carbon sources and other key environmental inputs that C. albicans encounters in host niches. We are also examining the impact of PAMP exposure on human disease.

167

P24B

The dynamic influence of the novel antifungal F901318 on growth and morphogenesis of Aspergillus fumigatus

Saskia du Pré1 ,2, Nicola Beckmann1, Graham Sibley1, Nick Read2, Jason Oliver1, Mike Birch1 1F2G Ltd, Manchester, UK, 2University of Manchester, Manchester, UK

Each year around 1.5 million people die as a result of an invasive fungal infection. Limited treatment options and the emergence of fungal strains that have become resistant to the available antifungals are an increasing problem, which is why there is a high need for new antifungals with a novel mode of action.

F901318 is a new antifungal compound from a new class of antifungal drugs, the Orotomides, acting on a novel target. It inhibits dihydroorotate dehydrogenase (DHODH), the fourth enzyme in the pyrimidine biosynthesis pathway and is highly active against all pathogenic Aspergillus species. F901318 is currently in clinical development.

In this study the influence of F901318 on the growth and morphogenesis of Aspergillus fumigatus was studied with live-cell confocal microscopy. Strains were pre-incubated for 8-16h in Vogel’s Minimal Media before being exposed to 0.1 µg/ml F901318 (approximately 2x the Minimal Inhibitory Concentration (MIC)). DHODH was tagged with GFP to study its localisation before and after treatment with F901318. Co-localisation with MitoTracker Red FM demonstrated that A. fumigatus DHODH is situated at the mitochondria, as is thought to be the case for most, but not all, eukaryotic species DHODH. In addition, to compare cell wall contents in treated and untreated hyphae, the cell wall was stained with aniline blue (β-glucan) and calcofluor white (chitin). Chitin content in the cell wall was increased upon exposure to F901318. Finally, using the fluorescent dye CMAC, F901318 treatment was shown to significantly increase the size of the vacuoles.

168

P25B

Calcineurin orchestrates lateral transfer of Aspergillus fumigatus during macrophage cell death

Anand Shah, Shichina Kannambath, Susanne Herbst, Matt Fisher, Serge Mostowy, Darius Armstrong-James Imperial College London, United Kingdom, UK

Aspergillus fumigatus (Af) is a lethal fungal pathogen in transplant recipients. We report a major role for the calcineurin pathway, which is the primary target of transplant immunosuppression, in initial fungal control of Af in human macrophages. Calcineurin was required for phagocytosis, reactive oxygen species production, and killing of Af. Ultimately, progressive fungal germination within macrophages triggered calcineurin-dependent necroptotic cell death, and was coupled to lateral transfer (metaforosis) of Af to recipient macrophages to enable fungal control. Metaforosis occurred through a vasodilator-stimulated phosphoprotein (VASP)-actin encapsulated late endosomal compartment. We confirmed the relvance of this phenomenom in vivo in a zebrafish model of invasive aspergillosis. These observations with a model mould pathogen identify an entirely novel calcineurin-dependent form of programmed cell death as a primary innate response to fungi in the lung, which facilitates cooperative control of infection by macrophages through a new cell-cell transfer mechanism know as metaforosis. To our knowledge this is the first description of a host-mediated pathogen cell to cell transfer mechanism. These findings yield novel insights into host innate immunity to Aspergillus fumigatus in the lung, extend current understanding of the pathogenesis of pulmonary aspergillosis in organ transplantation, and have broader relevance to transplant immunity and chronic inflammatory lung disease.

169

P26B

A new role for C. glabrata drug:H+ antiporters: in the crossroad between drug resistance and virulence

Mafalda Cavalheiro1 ,2, Rui Santos1 ,2, Daniela Romão1 ,2, Dalila Mil-Homens1 ,2, Pedro Pais1 ,2, Catarina Costa1 ,2, Mónica Galocha1 ,2, Arsénio Fialho1 ,2, Miguel Cacho Teixeira1 ,2 1Institute for Bioengineering and Biosciences, Lisbon, Portugal, 2Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

The ability to fight Candida glabrata infections is limited by the lack of knowledge on the mechanisms of persistence and virulence displayed by this fungal pathogen, that exhibits an unusual ability to tolerate antifungal drugs and stress imposed by the host’s immune system. In this study, a novel role for the drug:H+ antiporters (DHA) of the Major Facilitator Superfamily in C. glabrata virulence is described. This is particularly the case of the DHA transporters CgTpo1_1 and CgTpo1_2. These plasma membrane transporters had been previously found to contribute to azole drug resistance, through a direct role in drug extrusion [1], a function shared by other transporters of the same family [2]. However, the expression of either CgTPO1_1 or CgTPO1_2 was found to lead to increased virulence against the infection model Galleria mellonella [3]. Their role in C. glabrata virulence was found to be consistent with their ability to confer resistance to antimicrobial peptides, in the case of CgTpo1_1, and to survive upon phagocytosis by G. mellonella hemocytes, in the case of CgTpo1_2. Interestingly, our preliminary data suggests that additional members of the C. glabrata DHA family, so far uncharacterized, are also involved in these phenomena. Altogether, the C. glabrata DHA multidrug transporters appear to be at the crossroad between multidrug resistance and pathogenesis, thus constituting promising new targets for antifungal therapy.

[1] Pais et al, Mol Cell Proteomics, 15:57, 2016; [2] Costa et al, Front Physiol, 5:197, 2014; [3] Santos et al, Cell Microbiol, doi: 10.1111/cmi.12686.

170

P27B

Investigating Candida albicans Colonisation of Voice Prosthesis Following Laryngectomy

Daniel Pentland1, Campbell Gourlay1, Fritz Muhlschlegel1 ,2 1Kent Fungal Group, University of Kent, Canterbury, Kent, UK, 2East Kent Hospitals University NHS Foundation Trust, Kent, UK

A total laryngectomy is a surgical procedure for people with advanced laryngeal cancer which involves the removal of the entire larynx (including the vocal cords). Speech can be obtained using a small silicone valve, called a voice prosthesis, which is inserted between the trachea and oesophagus. Voice prostheses are susceptible to biofilms which, if left to grow, eventually block the valve, causing the voice prosthesis to fail. One of the main microorganism species colonising these voice prostheses, and certainly the primary fungal pathogen, is Candida albicans. Here we show that CO2 plays a significant role in the promotion of Candida albicans biofilm growth on voice prostheses.

The level of CO2 in exhaled breath is approximately 150x that in normal air (~5% compared to 0.03%) which acts as a powerful inducer of the Candida albicans yeast-to-hyphae switch. The yeast-to-hyphae switch is crucial for biofilm growth because hyphal-specific adhesins are important for cell attachment. Furthermore, Candida albicans cells locked in either yeast or filamentous growth states produce altered biofilms with reduced biomass and cell density. We have conducted a screen of 165 Candida albicans mutants each missing a non-essential transcription factor in an attempt to identify the regulatory elements required for biofilm formation on voice prosthesis. Our results indicate there is an important role for iron regulation in this Candida albicans biofilm formation, with several of the transcription factors identified having a function in iron homeostasis.

171

P28B

The Snf1-activating kinase Sak1 is a key regulator of metabolic adaptation and in vivo fitness of Candida albicans.

Bernardo Ramírez-Zavala1, Austin Mottola1, Julia Haubenreißer1, Sabrina Schneider1, Thomas Krüger2, Stefanie Allert3, Sascha Brunke3, Knut Ohlsen1, Bernhard Hube3, Olaf Kniemeyer2, Joachim Morschhäuser1 1Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany, 2Department of Molecular and Applied Microbiology, Hans Knöll Institute, Jena, Germany, 3Department of Microbial Pathogenicity Mechanisms, Hans Knöll Institute, Jena, Germany

The metabolic flexibility of Candida albicans is important for colonization and infection of host niches. Complex regulatory networks, in which protein kinases play central roles, link metabolism and virulence-associated traits, and thereby control commensalism and virulence. By screening a protein kinase deletion mutant library generated in the present work, we found that mutants lacking orf19.3840 exhibited filamentous growth defects in response to glucose limitation. The ortholog of orf19.3840 in Saccharomyces cerevisiae is SAK1, which encodes one of the upstream activators of the protein kinase Snf1, a highly conserved regulator of nutrient stress responses. In C. albicans, Snf1 is essential for viability, and we found that activation of Snf1 by phosphorylation of T208 was virtually abolished in the absence of Sak1. The sak1Δ mutants failed to grow on many alternative carbon sources and were hypersensitive to cell wall/membrane stress. These phenotypes were mirrored in mutants lacking other subunits of the SNF1 complex and partially compensated by a hyperactive form of Snf1. Transcriptional profiling of sak1Δ mutants showed that Sak1 ensures basal expression of glyoxylate cycle and gluconeogenesis genes. A phosphoproteomic analysis identified the protein kinase Rim11 as a novel downstream target of Sak1/Snf1. Mutants lacking Rim11 or Sak1 had overlappping phenotypes, and RIM11 overexpression rescued the filamentation defect of sak1Δ mutants. In a mouse model of gastrointestinal colonization, sak1Δ mutants were rapidly outcompeted by wild-type cells. Our results show that Sak1 is a crucial activator of Snf1 and a key regulator of metabolic adaptation and in vivo fitness of C. albicans.

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P29B

CRISPR-Cas9-engineered C. glabrata strains for the study of virulence factors in animal models.

Yacine Dahman1, Ludovic Enkler1, Julie Denis2, Delphine Richer1, Ermanno Candolfi2, Fabrice Jossinet1 1Institut de Biologie Moleculaire et Cellulaire (I.B.M.C), UPR 9002, Strasbourg, France, 2Institut de Parasitologie et de Pathologie Tropicale de Strasbourg, EA 7292, Strasbourg, France

Recent estimates suggest that about 1 billion people worldwide suffer from disease related to fungi. Among them, the opportunistic fungal pathogen Candida glabrata is a major public health concern. This increases the need to develop innovative therapeutic strategies to circumvent its high rates of resistance to antifungals. To better understand the pathogenicity of C. glabrata, we studied its transcriptional landscape during systematic infection in immunocompromised Drosophila melanogaster. Using RNAseq, we identified several gene candidates differentially expressed and potentially involved in the host adaptation and/or the infection process. To validate these genes as potential virulence factors, we developed a recombinant strain of C. glabrata expressing the CRISPR-Cas9 system. Several candidates have been knocked-out and were challenged for infection fitness in flies. In parallel, we’re using this genetic tool to investigate one of the most striking molecular adaptation of C. glabrata. It has been observed that the RNA component of its nuclear RNase P harbors three large additional domains. Such insertions have already been observed as a driving force in the evolution of non-coding RNAs. Frequently, they allow the interaction with proteins leading to novel biological functions. In order to test this hypothesis, we have produced a CRISPR-Cas9 variant of this RNase P. It allows us to purify the ribonucleoprotein complex and to identify its protein components by mass spectrometry. We have tested and compared the composition of this complex between several experimental conditions: liquid cultures and during infection of models such as human macrophages and Drosophila melanogaster.

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P30B

The unique iron homeostasis system of Candida glabrata

Franziska Gerwien1, Fabrice Hille1, Abu Safyan1, Bernhard Hube1 ,2, Lydia Kasper1, Sascha Brunke1 1Microbial Pathogenicity Mechanisms, Hans Knöll Institute, Jena, Germany, 2Friedrich Schiller University, Jena, Germany

Iron acquisition and homeostasis are important contributors to fungal virulence in the human host. Siderophores, high-affinity iron uptake systems and surface ferric reductases are found throughout the pathogenic fungi. The regulatory networks for iron homeostasis are mostly conserved from Aspergillus to Candida species and many other pathogenic and apathogenic fungi, although interestingly Saccharomyces cerevisiae uses a highly derived iron regulation system.

We have investigated the iron uptake system and its regulation in C. glabrata and found some striking difference to other pathogenic fungi like C. albicans. First, on the regulatory side, C. glabrata employs a hybrid system for transcriptional control. This comprises the Aft1 activator of iron uptake on the one side, in a pathway highly similar to S. cerevisiae. On the other side, an ortholog of the C. albicans Sef1 activator is also required for a full transcriptional response to iron starvation and thus, for full virulence in a human blood model.

On the effector side, C. glabrata differs from both C. albicans and S. cerevisiae in its absence of surface ferric reductase activity. This activity is mediated by a large, expanded gene family in C. albicans, which contributes to its virulence. In C. glabrata this family is lacking, and we instead found evidence of a secreted, low molecular weight factor that mediates iron reduction in the medium.

Overall, C. glabrata differs from many other fungi both in its strategies for iron uptake and homeostasis, and these specific adaptations likely have important effects on its interactions with the host.

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P31B

NET induction by candidalysin

Sujan Yellagunda1, Selene Mogavero2, Julian Naglik3, Bernhard Hube2, Constantin Urban1 1Umea University, Umea, Sweden, 2Leibniz Institute for Natural Product Research and infection biology, Jena, Germany, 3King’s college London, London, UK

Bacteria and viruses have long been known to possess protein and peptide toxins that are capable of damaging host cells and modulate the host immune system. These toxins constitute a major class of virulence factors associated with severe pathogenesis. In contrast, existence of peptide toxins in fungal pathogens has been shrouded. As a consequence, fungal infections were considered less pathogenic despite the emergence of virulent, drug resistance strains. Association of virulence with Candida albicans filamentation is a well-known virulence trait. However, a virulent toxin secreted by C. albicans had not been discovered until recently.

Myoes etal have shown that candidalysin acts similar to an invasin and at lytic concentration candidalysin is capable of damaging host epithelium concomitant with release of lactate dehydrogenase and calcium influx. In addition, candidalysin induces the release of cell damage associated cytokines like IL-1α. However, a complete network of mechanisms underlying this host-pathogen interplay at molecular level is yet to be unraveled.

In this regard, our experimental data demonstrate that candidalysin induces Neutrophil Extracellular Traps (NETs). The induction was dose dependent and in comparison to control peptide, ECE1-III KR peptide induced NETs more rapidly than ECE1-III peptide. In addition, NET induction by C. albicans strain ece1Δ/Δ was four and six fold less compared to the revertant strain ece1Δ/Δ+ECE1 and the parental strain BWP17+Clp30, respectively.

In summary, we provide first evidence that a peptide secreted from C. albicans can induce NETs.

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P32BFrom + to - : How O2 deprivation can contribute to C. albicans survival by hampering neutrophil function

Pedro Lopes1 ,2, Sandra Holmberg1 ,2, Emelie Backman1 ,2, Cecilia Heimer1 ,2, Rolf Claesson4, Constantin Urban1 ,2 1Department of Clinical Microbiology- Umeå University, Umeå, Sweden, 2The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå, Sweden, 3Umeå Centre for Microbial Research (UCMR), Umeå, Sweden, 4Section Oral Microbiology, Department of Odontology, Umeå University, Umeå, Sweden

Human fungal pathogens require molecular oxygen for several essential biochemical reactions, yet they are known to thrive under low oxygen or completely anoxic environments. In Candida albicans for example important virulence attributes, such as biofilm formation are regulated by genes important for low oxygen adaption. To halt C. albicans infection, the host needs to adapt to these hostile conditions. Neutrophils are innate immune cells working as a first line of defence once pathogens enter the host. Neutrophils kill and prevent growth of pathogens via three processes: phagocytosis, degranulation of cytoplasmic granules, and neutrophil extracellular traps (NETs). We have characterized the effect of anoxia on neutrophil responses encountering C. albicans under the form of different plantonic morphotypes and under biofilm form. Neutrophils have a reduced cytokine response that is mainly mediated by calprotectin and IL-1β inflammasome activation. In normoxia, reactive oxygen species (ROS) activate the NADPH-oxidase signalling leading to NET-formation. Oxygen presence seemed therefore a fundamental requisite for NET-formation. However anoxic NET-formation follows a ROS-independent mechanism mediated by the recognition of C. albicans by Syk tyrosine kinase.

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P33B

Physicochemical characterization and antifungal activity against Cryptococcus spp. of human and cryptococcal melanins

Nestor Correa1 ,2, Cristian Covarrubias1, Paula I Rodas3, German Hermosilla1, Fabien Magne1 ,4, Veronica Olate5, Cristian Valdes5, Wieland Meyer6, Cecilia V Tapia1 ,7 1Universidad de Chile, Santiago/Region Metropolitana, Chile, 2Universidad del Desarrollo, Santiago/Region Metropolitana, Chile, 3Universidad Andres Bello, Santiago/Region Metropolitana, Chile, 4Laboratoire de Biologie, Conservatoire National des Arts et Métiers, Paris/Île-de-France, France, 5Universidad de Talca, Talca/Region del Maule, Chile, 6University of Sydney, Sydney, Australia, 7Clinica Davila, Santiago/Region Metropolitana, Chile

Melanin is a negative charged and hydrophobic pigment, found in all the biological kingdoms. It plays a key role in protection against UV radiation, oxidant agents and ionizing radiation damage. A new biological function reported is the antimicrobial activity. Cryptococcus spp. is a capsulated yeast causing cryptococcosis and melanin is an important virulence factor. Here, we characterized physicochemically human and cryptococcal melanins and evaluated their antifungal activity against Cryptococcus spp. Methods: Melanin extracts were obtained from human hair and 8 genetically characterized strains of C. neoformans (n=4) and C. gattii (n=4). Synthetic melanin was used as a control. All the extracts were studied by: Infrared spectroscopy (FT-IR); Scanning electron microscopy (SEM); Energy Dispersive X ray spectroscopy (EDX); Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometer (MALDI-TOF-MS) and High Performance Liquid Chromatography (HPLC). The antifungal activity was determined by broth microdilution test. Results: By FT-IR, differences were observed in shape and intensity of signals related to C-C bonds of the aromatic ring of melanin monomer associated with a differential antifungal activity. On the other hand, by SEM a different conformation was observed between human (compact) and fungal (amorphous) melanin, but not related to the antifungal

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activity. The average/range of MICs was 16.6/7.8- 31.2 µg/mL for human melanin; 320 /125- >500 µg/mL for C. neoformans melanin and 140.6/62.5-250 µg/mL for C. gattii melanin. Conclusions: This results show that human melanin is more active than fungal melanins against Cryptococcus. The differences in activity could be explained by chemical differences between melanins from different sources.

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P34B

Linking hyphal maintenance and quorum sensing in Candida albicans

Melanie Polke1, Marcel Sprenger2, Kirstin Scherlach3, Franziska Wittig1, Christine Dunker1, María Cristina Albán-Proaño1 ,5, Ronny Martin2 ,6, Christian Hertweck3 ,4, Bernhard Hube2 ,4, Ilse D. Jacobsen1

,4 1Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Jena, Germany, 2Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Jena, Germany, 3Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Jena, Germany, 4Friedrich Schiller University, Jena, Germany, 5Unidad de Genética y Molecular, Hospital Carlos Andrade Marin IESS, Quito, Ecuador, 6Septomics Research Center, Friedrich Schiller University and Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany

In Candida albicans the morphologic plasticity, an important virulence attribute of the fungus, is governed by complex regulatory circuits including the fungal quorum sensing molecule (QSM) farnesol and the hyphal maintenance related gene EED1. Here we show that deletion of EED1 leads to a significantly increased sensitivity to farnesol, identifying the first C. albicans mutant hypersensitive to the fungal QSM. As farnesol decreases the transient filamentation of an eed1Δ strain without inducing cell death, we provide evidence that these are two separate mechanisms mediated by farnesol. We further demonstrate that the observed hypersensitivity of eed1Δ to farnesol is likely not caused by significant changes in important farnesol regulated signaling pathways, namely the Ras1-Cyr1-cAMP signaling pathway, or Nrg1 and Czf1 signaling, respectively. Interestingly, the eed1Δ strain displayed increased farnesol production independent of

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the pyrophosphatase Dpp3. When eed1Δ was grown under continuous medium flow conditions, to remove accumulating QSMs from the supernatant, maintenance of eed1Δ filamentation, although not restored, was significantly prolonged, indicating a link between farnesol sensitivity, production, and the hyphal maintenance-defect in the eed1Δ mutant strain. We furthermore show that the secondary yeast subset which forms under increased farnesol signaling during the reverse morphogenesis of eed1Δ, is different to the planctonic eed1Δ and wild type yeast, with respect to oxidative stress sensitivity and cell wall structure. Thus, we provide evidence that the altered farnesol signaling in eed1Δ may additionally affect further virulence attributes than merely morphogenesis.

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P35B

Dermal Fibroblasts Play a Central Role in Skin Model Protection against C. albicans Invasion

Andreas Kühbacher1, Philip Stevens3, Helena Henkel1, Christian Grumaz1, Doris Finkelmeier1, Anke Burger-Kentischer1 ,2, Kai Sohn1, Steffen Rupp1 ,2 1Fraunhofer IGB, Stuttgart, Germany, 2University of Stuttgart, Stuttgart, Germany, 3Medical University of Vienna, Vienna, Austria

Research efforts in the last decades have shed light on mechanistic interactions of fungal pathogens with host organisms down to the molecular level. Although considerable progress has been made in the last decades, the function of the individual components of epithelial barriers with regard to host-microbial interaction is not fully understood.

The fungal pathogen Candida albicans colonizes basically all human epithelial surfaces including the skin. Under certain conditions, such as immunosuppression, invasion of the normally protected epithelia occurs. In natural skin these infections are only superficial but not much is known about defense mechanisms against C. albicans in subepithelial layers such as the dermis.

Using immune cell-supplemented 3D skin models we could define a new role for fibroblasts in the dermis and identify a minimal set of cell types for skin protection against C. albicans invasion. Dual RNA-Seq of individual host cell populations and C. albicans revealed that dermal invasion is directly impeded by dermal fibroblasts. They are able to integrate signals from the pathogen and CD4+ T cells and shift towards an antimicrobial phenotype with broad specificity that is dependent on TLR2 and IL-1β. Skin model protection could be induced by addition of IL-1β even in the absence of T cells. TLR2 activation in dermal fibroblasts which occurs through C. albicans, results in induction of IL-1β expression, but it is secreted only in the presence of CD4+ T cells. These results highlight a central function of dermal fibroblasts for skin protection opening new possibilities for treatment of infectious diseases.

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P36B

Identification of tomatidine, a plant natural product targeting fungal human pathogens

Stephane Dorsaz1, Tiia Snäkä1, Quentin Favre-Godal2, Nathalie Boulens2, Eric Allemann2, Howard Riezman3, Emerson F. Queiroz2, Jean-Luc Wolfender2, Dominique Sanglard1 1University of Lausanne and University Hospital, Lausanne, Switzerland, 2University of Geneva and University of Lausanne, Geneve, Switzerland, 3University of Geneva, Geneve, Switzerland

Candida albicans is the major cause of fungal diseases in humans and its increasing resistance against available drugs is of major concern. In an attempt to discover novel antifungal agents, we initiated a small scale screening of a library of 240 natural plant compounds (NPs). In vitro profiling experiments identified 40 NPs exhibiting activities against C. albicans (MIC50 ≤ 32 µg/ml) among which one was promising: a sterol alkaloid named tomatidine from Solanum lycopersicum (Tomato) with high fungistatic activity against Candida species (MIC50 ≤ 1 µg/ml) and no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine treated-cells revealed a major alteration (up-regulation) of ergosterol genes suggesting that the ergosterol pathway could be targeted. Sterol content analysis in tomatidine-treated C. albicans cells indeed showed a dual inhibition of both Erg6 and Erg4. A forward genetic approach in Saccharomyces cerevisiae coupled with whole genome sequencing identified two misssense mutations in ERG6 gene (amino acids: D249G and G132D) as responsible for tomatidine resistance. Our results therefore identified unambiguously Erg6, a sterol C-24 methyltransferase which is absent in mammals, as the main direct target of tomatidine. To confirm the high potential of tomatidine as a new antifungal therapeutic agent, we tested the in vivo efficacy of the compound in a mouse model of C. albicans systemic infection. Treatment with an intraperitoneal nano-crystal pharmacological formulation of tomatidine successfully decreased the fungal burden in the infected kidney as compared to placebo and thus confirmed the potential of tomatidine as a therapeutic agent.

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P37B

The interplay between Neutrophil Extracellular Traps and Candida albicans virulence factors.

Marcin Zawrotniak, Maria Rapala-Kozik Department of Comparative Biochemistry and Bioanalytics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

Neutrophils, are the first line of defence against pathogens. They use a recently described mechanism of combating microorganisms, based on the release of neutrophil extracellular traps (NETs). These traps, made of the chromatin decorated with antimicrobial factors successfully catch and kill pathogens. Process of NET release, called netosis can be induce by a number of biological agents, e.g. fungi Candida albicans. This microorganism is an opportunistic pathogen that, using a number of virulence factors causes dangerous, hard to treatment infections. In our study, we determine the relationship between progress of C. albicans infection and response of neutrophils. The results showed, that release of NETs depends on the morphological form of fungi, which correlate with the stage of fungal infection. The study was focused also on the role of selected virulence factors of C. albicans in activation of netosis. The non-protein compounds of pathogen cell wall, like mannans and glucans, are able to trigger two netosis pathway, dependent or independent on reactive oxygen species (ROS). Most of aspartic proteases (Sap), secreted by C. albicans during infection, activated the classical ROS-depedent netosis pathway, but two of them used additionally ROS-independent netosis process. Moreover, the same virulence factors were able to inhibit the first mechanism of netosis by inactivation of NADPH oxidase, a key enzyme, responsible for ROS production. The receptors, involved in activation of netosis by these fungal compounds were also identified and the possible signal mediators involved in triggering of netosis by C. albicans virulence factors were proposed.

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P38B

Fast in vivo super-resolution microscopy and background reduction in Candida albicans.

Wouter Van Genechten, Liesbeth Demuyser, Peter Dedecker, Patrick Van Dijck KU Leuven, Leuven, Belgium

Optical and electron microscopy are extremely valuable tools in life sciences, but both techniques also have their disadvantages. One of the drawbacks of optical microscopy is the restriction on resolution enhancement due to the diffraction limit of light. Electron microscopy on the other hand, can only be performed on fixed, and thus dead, cells impairing real-time visualization of the subject molecule.

State-of-the-art microscopy techniques such as super-resolution optical fluctuation imaging (SOFI) combine solutions for both problems. They overcome the diffraction limit of light using temporal fluctuations of photoswitchable fluorescent proteins visualized with normal in vivo imaging techniques. So far these techniques have been performed and developed for use in mammalian cells yet not, as far as we know, in any of the Candida species. In this work we present the development and application of photoswitchable fluorescent proteins (PSFP) for use in the major human fungal pathogen Candida albicans.

Two Candida-optimized variants of a green PSFP have shown to be very promising for use in super-resolution microscopy. Attaching these fluorescent proteins to several structural and signaling components showed precise localization. A second major advantage of this system is the ability to reduce the background fluorescence of C. albicans. The latter is a well-known problem when visualizing proteins with low abundancy. Combining the increase in resolution and consequent possibility to do accurate co-localization assays with the reduction of the autofluorescence, renders this an extremely valuable tool for any researcher performing fluorescence microscopy on C. albicans.

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P39B

A coarse mapping of phenotypes of Candida glabrata reveals highly individualistic adaptations to a fluctuating environment

Reinhard Beyer1, Birgit Willinger2, Joseph Strauss1, Christoph Schüller1 1University of Natural Resources and Life Sciences, Vienna, Austria, 2Medical University of Vienna, Vienna, Austria

Candida glabrata is a successful commensal and able to adapt to a variety of environmental situations. It is further a cause of recurring vulvo-vaginal candidiasis and shows high resistance to antifungals. To explore how this flexibility is possible without a sexual cycle, we investigated the phenotypic variation of several hundred C. glabrata clinical isolates collected by the Vienna General Hospital. Our quantitative high-throughput determination of growth parameters revealed a broad variation of phenotypes. For example temperature influenced growth rate in an unexpected range. We found that many strains tolerated either higher or lower temperatures while all strains had a temperature optimum at around 39°C. The collection was also scored for resistance to low pH, weak acid, osmotic stress, carbon source utilization and resistance to antifungals. Furthermore, about 15% of our strains are efficiently forming biofilms. We also investigated the stability of the observed phenotypes. We tried to manipulate fluconazole resistance by adapting sensitive strains to intermediate fluconazole concentrations. This succeeded but lead to a simultaneous post-adaption loss of growth rate on rich medium, indicating that adaptation to fluconazole comes at a cost. Genome sequencing of selected phenotypically eye-catching strains revealed that their genomic profiles differed in up to 105 bases making specific assignments difficult. We conclude that, C. glabrata exhibits a broad phenotypic spectrum. It is further able to quickly and transiently adapt in vitro to fluconazole. Further investigation of the genetic or epigenetic basis of these adaptations is required to understand the success of C. glabrata as an opportunistic commensal.

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P40B

Phenotypic changes following echinocandin microevolution

Csaba Gergo Papp, Csaba Vágvölgyi, Attila Gácser Department of Microbiology, University of Szeged, Szeged, Hungary

In vitro microevolution in laboratories has been used previously to investigate azole resistance mechanisms with genome and transcriptome sequencing. In our microevolutionary study, we have grown the Candida parapsilosis CLIB 214 parental echinocandin sensitive strain in the presence of increasing concentration of different echinocandins. We aimed to investigate the phenotypic consequences of the created echinocandin resistant strains. The three echinocandin evolved strains showed increased MIC for the echinocandin which were used to evolve the particular strain. Additionally, the anidulafungin evolved strain were resistant to all three kind of echinocandins and the caspofungin evolved strain were resistant to caspofungin and micafungin, however the micafungin evolved strain were only resistant to micafungin. We also examined the growth capability of the parental and evolved strains on YPD plates complemented with different osmotic-, oxidative stressors and cell wall-, membrane perturbing agents. The evolved strains were susceptible to cell wall perturbing agents, this phenomena were more prominent on 37 °C and the micafungin evolved strain were unable to growth in the presence of calcofluor white and congo red on both 30 °C and 37 °C. Interestingly the micafungin evolved strain were the only one (including parental strain) which were able to grow without any defect in the presence of SDS. To investigate virulence properties of the echinocandin evolved strain we have used the unconventional Galleria mellonella model. We find that the micafungin evolved strains showed attenuated virulence as the infected wax moth larvae survived better than those infected with other strains.

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P41B

Oxidative stress resistance and modulation of macrophage pro-inflammatory response by Candida albicans MAM33

Manju Chauhan, Selvakumar Subbian, Neeraj Chauhan Rutgers, the State University of New Jersey, Newark, NJ, USA

Mitochondria are key contributors to fungal pathogenesis. However, the biological role of most Candida albicans mitochondrial proteins is poorly understood. In the current study, we present data on the characterization of, Mam33, a putative mitochondrial protein of C. albicans. The subcellular localization of Mam33 was confirmed by fluorescence microscopy. Disruption of MAM33 causes reduced resistance to oxidative stress and hypersensitivity to SDS, suggesting cell surface defects in these mutants. To determine the role of Mam33 in interaction with host immune cells, we performed invasion (10 min) and entry/survival (90 min) assays by using the murine J774 macrophage like cells. Our results show that compared to the wildtype, mam33∆/∆ null mutant strains were defective for invasion and/or entry/survival. We observed a reduction in the level of expression of proinflammatory molecules, including Ccl2, Cxcl10, IL6, IL12a, IL12b and Tnfa, in J774 cells infected with mam33∆/∆ compared to the wildtype. Consistent with the down regulation of proinflammatory markers, expression of IL10, an immune-suppressing cytokine, was upregulated in J774 cells upon infection by mam33∆/∆. In addition, transcriptional profiling revealed significant down-regulation of genes involved in the glycolytic and N-acetylglucosamine metabolism pathways. However, genes involved in the TCA cycle and ATP synthesis were enriched more than 11-folds in mam33∆/∆, relative to the wildtype. To provide, further insight into the role of MAM33 in fungal virulence, current experiment using a murine model of invasive candidiasis and metabolomics approaches are on-going. Together, these results reveal a novel role of fungal mitochondrial proteins in host-pathogen interactions.

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P42B

Mitochondrial proline metabolism is key to arginine-induced morphogenesis in Candida albicans

Fitz Gerald Silao, Kicki Ryman, Per Ljungdahl Stockholm University, Stockholm, Sweden

Amino acids (AA) are among the best-established inducers of morphogenesis in the human fungal pathogen Candida albicans, yet a clear mechanistic understanding of how AA initiate the yeast-to-filamentous switch is lacking. Using a combination of genetic and biochemical approaches we have shown that the inducing signal from several AA is derived from their catabolism within cells rather than being sensed in the extracellular environment. Specifically, proline, one of the most potent inducers of morphogenesis, is exclusively metabolized in the mitochondria, generating the reduced electron carriers NADH and FADH2 as catabolic by-products. These reduced carriers are oxidized by the mitochondrial electron transport chain, a process coupled to the generation of ATP. Interestingly, strongly inducing AA such as arginine and ornithine are also catabolized through proline utilization pathway. These results align well with a recent model that elevated intracellular levels of ATP is key to the induction of the Ras1/cAMP/PKA signaling cascade; this pathway positively controls the activity of the morphogenic effector transcription factor Efg1. Our findings are inconsistent with the accepted model that arginine induces morphogenesis primarily through the generation of CO2. Taken together, our results provide the basis of an alternative unifying model in which the catabolism of arginine (and ornithine) to glutamate via proline in the mitochondria leads to elevated ATP levels, which in turn stimulate the Ras1/cAMP/PKA pathway, positively activing Efg1 and thereby inducing the genetic program that engages the yeast-to-filamentous switch.

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P43B

Analyzing the role of the Candida albicans Hog1 MAPK pathway during tunicamycin-induced Endoplamic Reticulum stress

Farha Husain1, Elvira Roman2, Jesus Pla2, Sneh Lata Panwar1 1School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India, 2Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid-28040, Spain

Endoplasmic reticulum (ER) homeostasis is critical for normal cellular functions. Eukaryotic cells respond to the accumulation of misfolded proteins in the ER by activating the unfolded protein response (UPR), which is a conserved signaling pathway that activates the expression of genes coding for chaperones and folding enzymes. Studies pertaining to UPR pathway have shown that UPR is important for antifungal resistance and virulence; therefore, the pathway could be a noble therapeutic drug target. Candida albicans, a human pathogenic fungus, faces ER stress when it invades human tissues. The host microenvironment not only accounts for UPR-triggering oxidative damage and cell wall/membrane stress, but also increases demand on the secretory pathway in these fungal pathogens. In S. cerevisiae, the high osmolarity glycerol (HOG) pathway plays a role in ER stress resistance, while the role of this pathway in ER stress remains uncharacterized in C. albicans. We are particularly interested in exploring the role of the HOG pathway during ER stress in C. albicans. We show that strains lacking the MAPK Hog1p and other components of the HOG pathway displayed sensitivity to the ER stessors; tunicamycin and DTT. This effect seems to be mediated by the basal activity of the Hog1p MAPK as tunicamycin exposure does not trigger the phosphorylation of Hog1p and its nuclear import. Besides, all these effects seem to be independent of the classical Hac1p-mediated UPR pathway. Moreover, tunicamycin exposure leads to overproduction of glycerol, the osmoprotectant, and that this is dependent on a functional HOG pathway.

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P44B

The role of the cell wall integrity and the contribution of the dectin-1 receptor in the recognition of Candida parapsilosis

Katalin Csonka1, Bianca Schulze2, Christine Dunker2, Ilse D. Jacobsen2, Attila Gacser1 1Department of Microbiology, University of Szeged, Szeged, Hungary, 2Hans Knöll Institute, Jena, Germany

The invading microorganisms are recognized by a number of pattern recognition receptors (PRRs) of the innate immune system, antifungal immunity appears to be mediated primarily by members of the C-type lectin receptor (CLR) family, including Dectin-1, which receptor recognizes β-1,3-glucans in the cell walls. Here, we examined the role of cell wall integrity in the immune sensing of C. parapsilosis in in vivo experiments.The effects of N-linked mannosylation were explored in the C. parapsilosis och1∆/∆ (Cpoch1∆/∆), which mutant strain displays reduction of about 67% in the mannan and increment in the β-glucan and chitin level.

The Cpoch1∆/∆ showed significantly reduced fungal loads in kidney, liver, and spleen of the wild-type mice compared to CpWT. Furthermore, the Cpoch1∆/∆ infection of wild-type mice stimulated higher recruitment of neutrophils, macrophages and dendritic cells to the kidney compared to the CpWT strain. In addition, we also observed the lower induction of TNFa, IL-1b, IL-6, INF-g cytokines and GM-CSF in the kidney of mice after the Cpoch1∆/∆-challenge in comparison to CpWT. Infection with the CpWT strain eventuated in similar fungal burden in the wild-type and Dectin-1-/- mice. Nevertheless, no differences in the composition of immune cells were found between the wild-type and Dectin-1 receptor mutant mice after the CpWT stimulation.

Taken together, our results demonstrate that N-linked mannosylation significantly affects the virulence and the specific steps of innate immune response against this pathogen. Interestingly, in contrast to C. albicans, our observations disclose the redundant role of Dectin-1 in the recognition of wild-type C. parapsilosis.

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P45B

Pathophysiology of systemic candidiasis depends on both the phenotypic properties of Candida albicans and the immune status of the host.

Louise Basmaciyan1 ,2, Fabienne Bon1, Ralitsa Atanasova1, Marie-Elisabeth Bougnoux3 ,4, Christophe D’enfert3, Amandine Ducreux1, Coralie L’Ollivier5, Frédéric Vincent1, Frédéric Dalle1 ,2 1UMR PAM, Univ Bourgogne-Franche Comté, Bâtiment EPICURE 1 Esplanade Erasme - 21000 Dijon, France, 2Centre Hospitalier Universitaire, Service de Parasitologie Mycologie, 2 Rue Angélique Ducoudray, F-21079 Dijon Cedex, France, 3Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, INRA, USC2019, F-75015 Paris, France, 4Laboratoire de Parasitologie-Mycologie, Service de Microbiologie, Hôpital Necker-Enfants Malades, Université Paris Descartes, Faculté de Médicine, F-75015 Paris, France, 5Laboratoire de Parasitologie-Mycologie, Aix-Marseille Univ. Marseille; AP-HM, CHU Timone, F-13385 Marseille cedex 05, France

Candida albicans is a member of the commensal flora residing on mucosal surfaces in healthy humans. C. albicans commensalism and pathogenicity result from a balance between host defences and fungal virulence factors. In a previous study, we randomly selected 84 C. albicans isolates according to their clinical (i.e. invasive bloodstream isolates, non-bloodstream isolates) or commensal origin. Whereas these isolates displayed a wide phenotypic diversity in vitro and ex vivo, we were not able to establish a clear correlation between their phenotypic properties and their MLMT genotypes. However we observed that mortality in mice intravenously infected with C. albicans isolates was the result of different infection processes depending on the intrinsic phenotype of the isolate tested. For instance, isolate Ca926 defective for hyphae production in vitro (i.e. in filament-inducing conditions) and ex vivo (i.e. unable to invade and damage intestinal epithelial cells) induced lower mortality in immunocompetent systemically-infected mice as compared to the reference isolate CaSC5314,

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corroborating the contribution of the yeast-to-hyphae transition in C. albicans virulence. Strikingly however, in systemically-infected immunosuppressed mice, no significant difference in mortality induced by the two isolates was observed but the mechanism of infection differed. Altogether, these data suggest that interactions between C. albicans and the host are influenced by the natural phenotypic variations of C. albicans infecting strains and the immune status of the host. Comparative studies of organ fungal burden, histology and cytokine expression are under investigation aimed at specifying the mechanisms underlying these differences.

192

P1C

Understanding how the Hsp90 kinase interactome affects Candida albicans virulence

Leenah Alaalm1 ,2, Carolyn Williamson1 ,2, Kangzhen Dong1 ,2, Stephanie Diezmann1 ,2, Ranjith Rajendran3, Gordon Ramage3 1Department of Biology & Biochemistry, University of Bath,, Bath, UK, 2The Milner Centre for Evolution, University of Bath,, Bath, UK, 3Glasgow Dental Hospital & School, University of Glasgow Dental School, Glasgow, UK

Systemic infections caused by Candida albicans are often life-threatening and difficult to treat due to therapeutic limitations. Here, we are focusing on the essential and central regulator, the heat shock protein 90 (Hsp90) hence it plays a key role in regulating several virulence traits of C. albicans. Yet, treatment combining standard antifungal therapy and Hsp90 inhibition resulted in severe host toxicity in the murine model of systemic candidiasis. Thus, novel drug targets are urgently needed. A chemical genomic screen revealed that Hsp90 regulates 226 C. albicans genes, with 35 encoding kinases, which are considered attractive drug targets. We propose that combinatorial therapy comprising Hsp90 inhibitors, which are already in clinical trials as anti-cancer drugs, and C. albicans-specific kinase inhibitors could provide an effective therapeutic strategy for the treatment of systemic candidiasis. To identify suitable kinases, we study two sets, those that regulate Hsp90 function (Ck2 kinase complex) and those that require Hsp90 for proper function (Hsp90-target kinases). So far, we identified novel physical interactions between Hsp90 and its kinase-targeting co-chaperone Cdc37 and the Ck2 kinase complex. We selected nine Hsp90-target kinases, four highly conserved and five uncharacterized, fungal-specific for further study of their virulence potential. Gene deletion mutants are characterized in a series of in vitro and in vivo assays. So far, we identified three kinases affecting morphogenesis, and two kinases regulating Fluconazole resistance in an Hsp90-dependent fashion. Understanding how the Hsp90-kinase circuitry regulates virulence will be a crucial milestone in our efforts to identify novel drug targets.

193

P2C

Methionine synthases as drug targets for antifungal drug development

Jennifer Scott, Elaine Bignell, Jorge Amich Manchester Fungal Infection Group, University of Manchester, UK

There are only three classes of antifungals, echinocandins, polyenes and azoles, and only the last can be orally administered. The continued and widespread emergence of drug resistance highlights a desperate need to develop new antifungal treatments. We recently demonstrated that methionine synthase (MetH) is essential for Aspergillus fumigatus viability and virulence, which lead us to propose it as a promising drug target. MetH is also essential in Candida albicans and Cryptococcus neoformans, suggesting a broad spectrum of action for possible novel drugs. To validate the target further, we have constructed a metH_tetOFF regulatable strain and investigated the effects of down-regulating gene expression for A. fumigatus growth. This system has the invaluable advantage that expression can be shut down in mycelia, which mimics the action of a drug on an established infection and therefore overcomes the flaw of using null mutants for virulence studies. We have observed that as little as 0.1 µg/ml doxycycline (dox) prevents colony formation, suggesting effective transcriptional repression. Indeed, 30 minutes after addition to mycelia, dox produces a sustained reduction in metH expression, which causes a strong reduction in fungal growth on both solid and liquid media. We are currently testing the use of the tetOFF system in murine models of aspergillosis, which will hopefully allow us to demonstrate, with unprecedented certainty, that methionine synthase is a valid drug target for the development of novel antifungals. The tetOFF system may be an invaluable new tool to validate targets in vivo without the requirement of an actual drug.

194

P3C

The intraspecies diversity of C. albicans triggers qualitatively and temporally distinct hostresponses that determine the balance between commensalism and pathogenicity.

Salome Leibundgut Universtiy of Zurich, Zurich, Switzerland

The host immune status is critical for preventing opportunistic infections with C. albicans. Whether the natural fungal diversity that exists between C. albicans isolates also influences disease development remains unclear. Here, we used an experimental model of oral infection to probe the host response to diverse C. albicans isolates in vivo and found dramatic differences in their ability to persist in the oral mucosa, which inversely correlated with the degree and kinetics of immune activation in the host. Strikingly, the requirement of IL-17 signaling for fungal control was conserved between isolates, including isolates with delayed induction of IL-17. This underscores the relevance of IL-17 immunity in mucosal defense against C. albicans. In contrast, the accumulation of neutrophils and induction of inflammation in the infected tissue was strictly strain-dependent. The dichotomy of the inflammatory neutrophil response was linked to the capacity of fungal strains to cause cellular damage and release of alarmins from the epithelium. The epithelium thus translates differences in the fungus into qualitatively distinct host responses. Together, this study provides a comprehensive understanding of the antifungal response in the oral mucosa and demonstrates the relevance of evaluating intraspecies differences for the outcome of fungal-host interactions in vivo.

195

P4C

Candidalysin interferes with members of the commensal microbiota

Toni Förster, Selene Mogavero, Bernhard Hube Hans-Knöll-Institut, Jena, Germany

The opportunistic pathogen Candida albicans is a common and mostly harmless inhabitant of human mucosal surfaces. Only under predisposing conditions like immunosuppression, damaged barriers or antibiotic treatment, C. albicans can switch to a pathogenic state. Given the importance of a protective bacterial microbiota, it is likely that interactions with other members of the microbiota contribute to its commensal state. The recently discovered peptide-toxin Candidalysin is an important virulence factor of C. albicans. Here we investigate whether Candidalysin is also active against bacteria of the human microbiota. We screened a total of 35 human associated bacteria for growth defects in the presence of synthetic Candidalysin and found that the toxin inhibits the growth of distinct bacteria. This effect was confirmed by measuring minimal bactericidal concentrations (MBC) and metabolic activity. We also see that Candidalysin leads to perturbations in membranes of selected bacteria. Moreover, we have hints that Candidalysin, which is not totally soluble and forms aggregates in aqueous solutions, co-aggregates also with bacteria leading to the formation of large peptide-bacteria clumps. We additionally tested derivatives of Candidalysin for antimicrobial activity and found that an N-terminal truncation of Candidalysin considerably improves its antimicrobial activity. These truncated versions are more toxic and are active against more bacterial species than the original Candidalysin. In conclusion, we showed that Candidalysin exerts multiple effects against selected bacteria, such as growth inhibition, clumping or membrane perturbation and modified versions of the peptide may even have increased activity.

196

P5C

Acetylation of Eaf1 controls merge of NuA4-SWR1 complexes and cell fate plasticity

Jiangye Chen, Xiongjun Wang, Wencheng Zhu, Chang Peng Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China

Phenotypic plasticity is common in development. The dynamic process of cell fate specification is determined and maintained by a dual-layer network which consists of transcriptional factors and chromatin structure. Candida albicans, a major opportunistic fungal pathogen of humans, possess the unique ability to achieve rapid and reversible cell fate between unicellular form (yeast) and multicellular form (hypha) in response to environmental cues. This ability makes it an outstanding model to investigate the mechanisms of cell plasticity and cell fate determination. In this study, we show that a dynamic merge and separation of NuA4 and SWR1 controls the reversible cell fates between yeast and hypha in Candida albicans. In yeast growth state, the two complexes merge together via anchoring of Yaf9 to acetylated Eaf1 at K173. During yeast to hypha development, the two complexes separate each other via departure of Yaf9 from de-acetylated Eaf1. When hyphae cells are dedifferentiated, the two separated complexes re-merge again to promote cell proliferation. During the reversible morphological change, the acetylation level of Eaf1 is dynamically regulated by cooperation of Esa1 and Hda1, which in turn to regulate its association with the Yaf9 and further mediate the interplay between NuA4 and SWR1. To our knowledge, we first observed a dynamic fusion and separation of NuA4 and SWR1 and provide an evidence to support the evolution of two chromatin modifying complexes NuA4 and SWR1 into a large merger TIP60 and reveal a molecular mechanism for cell fate control.

197

P6C

Functional annotation of transcription factors of the major mould pathogen, Aspergillus fumigatus

Norman van Rhijn, Takanori Furukawa, Panos Papastamoulis, Sayema Khan, Lea Gregson, Michael Bromley, Magnus Rattray, Elaine Bignell University of Manchester, Manchester, UK

Aspergillus fumigatus is the main causative agent of aspergillosis, causing over 90% of infections. A. fumigatus remains poorly characterised at the molecular level but previous studies have identified transcriptional regulation as being critical for mammalian infection. In particular, the ability of the pathogen to respond to host-imposed stress has repeatedly proven to be essential for pathogenicity. In order to define the genomic cohort of A. fumigatus gene products required for pathogenicity, we have used a high throughput gene deletion approach. An in vitro screening methodology was devised to analyse relative fitness of 401 A. fumigatus transcription factor null mutants in the presence and absence of host-imposed stress. Firstly, growth curves were analysed for each of the 401 null mutants to identify mutants suffering general fitness defects. Fungal growth was then measured for all mutants in parallel under each of the following host imposed stresses: iron starvation, temperature stress, pH tolerance, and zinc acquisition. Data analysis was automated to convert optical density readings collected directly from the spectrophotometer into fitness indices. Our study identifies, for the first time, the full cohort of A. fumigatus transcription factors which govern responses to significant host-imposed stresses and provides a first genome-scale functional annotation of transcription factors in A. fumigatus.

198

P7C

The proteomic response of Candida species to antifungal drugs

Giuseppe Buda De Cesare, Carol Munro, David Stead University of Aberdeen, Aberdeen, Scotland, UK

C. albicans, an opportunistic fungal pathogen, causes systemic and superficial infections, especially in immunocompromised patients. C. albicans is a normal constituent of the human microflora and when it escapes from its normal host niches is responsible for the majority of Candida bloodstream infections (candidemia), with an increasing percentage of infections caused by Candida glabrata. One of the emerging problems related to these pathologies is the acquisition of resistance of clinical isolates to azoles and echinocandins, the most commonly used antifungals to treat patients. Several molecular mechanisms that lead to antifungal resistance have been elucidated. The main aim of this study is to identify antifungal resistance biomarkers in the major pathogenic Candida species. Mass spectrometry analyses have been performed to detect changes in protein expression between drug resistant and sensitive C. albicans and C. glabrata isolates. The cultures have been grown in different conditions (in the presence or absence of caspofungin or fluconazole) and samples taken at different time points (to define the differences in protein expression over time). Different cellular fractions (cytoplasmic, cell wall and secreted) have been isolated and the expression profiles examined in order to define the trends characterizing the resistant strains and the response to drugs. This information will be applied for future development of the Bruker MALDI Biotyper technology. A parallel future aim is to isolate llama antibodies against selected biomarkers for development of drug resistance diagnostics.

199

P8C

Uncovering new links between mitochondria and hyphal growth of Candida albicans

Barbara Koch, Adele Barugahare, Traude Beilharz, Ana Traven Monash University, Melbourne, Australia

The morphological switch between yeast and hyphae is a key virulence factor for Candida albicans. Here we investigated the roles of mitochondrial dynamics in hyphal growth of C. albicans using a small molecule inhibitor. Strikingly, the inhibitor represses both the yeast-to hyphae transition and ongoing filamentation, and its effects on morphogenesis can be uncoupled from general growth inhibition. RNAseq experiments of inhibitor-treated cells coupled with Candida mutant analyses suggest the existence of a novel mechanism of action to represses hyphal growth. The inhibitor was protective to host cells, increasing the survival of bone-marrow derived macrophages in ex vivo macrophage-Candida infection assays, indicating it has potential as a therapeutic. Our work has implications for uncovering novel biology that governs C. albicans filamentation. Our study is also relevant for identifying new therapeutic avenues with distinct mechanism of action compared to the antifungal drugs used in the clinic at present. Since Candida sp cause an estimated 400, 000 life-threatening infections per year worldwide, and the mortality remains in the range of 40%, identifying new avenues for treatment is an urgent need

200

P9C

Mutation of a 6 member MNN1 gene family in Candida albicans using CRISPR Cas9 to elucidate the role of fungal cell wall α-1,3-mannosides on the activation of the human immune response

Katja Schaefer, Bhawna Yadav, Neil A.R. Gow The Aberdeen Fungal Group, MRC Centre for Medical Mycology, School of Medicine, Medical Sciences & Nutrition, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK

The cell wall of Candida albicans harbors many molecules that can activate host immune responses. Mannan the major component of the outer surface of the cell wall is critical for the adherence of the fungus to host cells and the activation of host immune responses. Mannans are synthesized by a series of families of glycosyl transferases that catalyze the addition of mannose sugars via different linkages to the developing oligosaccharides that glycosylate cell wall proteins. Because specific mannosylation events are catalyzed by multigene families, it is usually necessary to achieve the deletion of each of the single genes, as well as the entire complement of genes in each family, to be able to dissect the single and combined functions of different classes of mannosyltransferase. Here we analyse the six-membered MNN1 gene family that is predicted to act in the terminal stages of glycosylation by adding α-1,3-mannan residues to the side chains of N-linked mannans. Because these residues represent the terminal sugars of the outer cell wall we predicted they may have major roles in host-fungus interactions. We created sextuple MMN16 mutants by disrupting all twelve MNN1 alleles using CRISPR-Cas9 genome editing technology. Removal of the outermost N-linked α-1,3-mannosides resulted in alterations in yeast cell shape and in the profile of the immune response in macrophage cell cultures as well as human peripheral blood monocytes. These results suggest that the α-1,3-mannosides of C. albicans N-mannan is important in cell wall morphogenesis and immune recognition by cells of the innate immune system.

201

P10C

The Kennedy Pathway: A rheostat for virulence in Candida albicans

Robert Tams1, Chelsi Cassilly1, Sanket Anaokar2, William Brewer1, Justin Dinsmore1, Joseph Chen3, Jana Patton-Vogt2, Todd Reynolds1

1University of Tennessee, Knoxville, Tennessee, USA, 2Duquesne University, Pittsburgh, Pennsylvania, USA, 3National Taiwan University, Taipei, Taiwan

Phospholipids are important structural lipids of cellular membranes that are also involved in cell signaling. We previously reported that phosphatidylethanolamine (PE) synthesis by the de novo biosynthetic pathway is required for virulence in Candida albicans. Further downstream, PE can be methylated to form phosphatidylcholine (PC), and the role of PC synthesis in virulence is unknown. We hypothesized that PE conversion to PC would be required for virulence. However, we found that a mutation that blocks PE conversion to PC increased virulence. This led to the hypothesis that increased PE synthesis may cause an increase in virulence. To test this, we developed a strategy to increase or decrease PE/PC synthesis independently of the de novo pathway using the Kennedy pathway. The Kennedy pathway involves three successive enzymatic steps that synthesize PE or PC from diacylglycerol and exogenous ethanolamine or choline, respectively. By modulating the gene for the final step in the pathway, EPT1, we have decreased or increased PE/PC synthesis. We have found that an ept1Δ/Δ knockout strain has decreased cytotoxicity to FaDu tissue culture cells, and decreased fungal burden in the kidneys of infected mice. In contrast, overexpression of EPT1 causes increased cytotoxicity to FaDu cells, and mice infected with the EPT1 overexpression mutant succumb to infection more rapidly than those infected with the wild type. Thus, tuning the levels of PE/PC correlates with levels of virulence, suggesting that this pathway is able to regulate virulence in mice. We are currently exploring the mechanism by which PE/PC regulation impacts virulence.

202

P11C

Heat Shock factor 1 (HSF1) in Candida albicans is essential under iron deprivation and drug defense

Remya Nair1 ,2, Mohd. Shariq1 ,3, Sanjiveeni Dhamgaye2, Chinmay Mukhopadhyay3, Shamim Shaikh1, Rajendra Prasad4

1Rajiv Gandhi Institute of IT & Biotechnology, Bharati Vidyapeeth University,, Pune, India, 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, India, 3Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India, 4Amity Institute of Integrative Sciences and Health and Amity Institute of Biotechnology, Gurgaon, India

Recently, we have reported that the conditional mutant of the heat shock factor-1 (HSF1) in Candida albicans displays enhanced susceptibility not only towards a plant alkaloid, berberine, but also to diverse antifungal drugs. The present study attempts to identify additional phenotypes highlighting the non-heat shock responsive roles of HSF1 that could be correlated with the enhanced drug susceptibility. We uncover an intricate relationship between cellular iron and HSF1 mediated drug susceptibility of C. albicans. Interestingly, at 30℃, the conditional deletion of HSF1 while presented no growth defect, exhibited low intracellular iron. Notably, exogenous supplementation of iron reversed growth defects of HSF1 mutant when grown at 37℃. We provide evidence that the HSF1 mutant presents interesting phenotypes at basal conditions and are implicated in enhanced drug susceptibilities, dysfunctional mitochondria, decreased resistance towards oxidative stress and compromised cell wall integrity, all of which could be fully reversed upon iron supplementation. The HSF1 mutant also displayed defective filamentation at basal conditions under various solid hypha inducing media. Further, chelation of iron of HSF1 mutant cells led to severe growth defects and apparently triggers an iron starvation signal in the cell thus, demonstrating that HSF1 is essential for C. albicans cells to tolerate the iron deprivation stress. Together, apart from the well-established roles of HSF1 in reciprocation of thermal stress, this study extends its role under basal conditions and provides molecular insights into the role of HSF1 in iron deprivation and drug defense of C. albicans.

203

P12C

The Rab GTPase Ypt6 is critical for Candida albicans invasive growth and virulence

Rohan S. Wakade1, Daniele Stalder1, Scott G. Filler2, Norma V. Solis2, Robert A. Arkowitz1, Martine Bassilana1 1Universite Cote d’Azur, CNRS, Inserm, ibV, Nice, France, 2Los Angeles Biomedical Research Institute at Harbor-UCLA Medical center, Torrance, CA, USA

Candida albicans dimorphic switch requires cytoskeleton reorganization and sustained membrane trafficking. Rab (Ras related in Brain) GTPases play a central role in membrane trafficking, yet little is known regarding their function and regulation during hyphal growth and virulence. In C. albicans, Sec4 and its activator Sec2 localize at the Spitzenkörper (1, 2) and Cdc28/Hgc1-dependent Sec2 phosphorylation is critical for hyphal growth (2). Golgi polarization during hyphal growth is also regulated via the Rab GAP (GTPase Activating Protein) Gyp1 (3). Here, we focused on Ypt6 and Ypt31, the human Rab6 and Rab11 homologs, respectively. Our results indicate that these two proteins are critical for cell wall integrity, yet only Ypt31 is critical for antifungal sensitivity. Furthermore, both Ypt31 and Ypt6 are required for invasive hyphal growth. During hyphal growth, we show that Ypt6 localizes to the Golgi while Ypt31 localizes predominantly to the Spitzenkörper. With respect to the ypt6 deletion mutant, we also show that it produces shorter hyphae and is reduced for virulence in two murine candidiasis models. Further characterization of this ypt6 mutant shows that this defect in hyphal growth maintenance is associated with an alteration of the endocytic site distribution, upon filament extension. We are currently investigating the molecular mechanism underlying this defect and Ypt6 regulators and/or effectors during hyphal growth.

(1) Jones & Sudbery, 2010, Euk. Cell, 9, 1455. (2) Bishop et al., 2010, EMBO J., 29, 2930. (3) Huang et al., 2013, Euk. Cell, 12, 998.

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P13C

Ahr1 contributes to control of ALS3 and ECE1 expression in Candida albicans

Sophia Ruben1, Joachim Morschhäuser2, Ronny Martin1 ,3, Oliver Kurzai1 ,3 1Septomics Research Center, Leibniz Institute for Natural Product Research and Infection Biology –Hans Knoell Institute, Jena, Thuringia, Germany, 2Institute for Molecular Infection Biology and Research Center for Infectious Diseases (ZINF), Julius Maximilians Universität, Würzburg, Bavaria, Germany, 3Center for Sepsis Control and Care, Jena University Hospital, Jena, Thuringia, Germany

Formation of filamentous forms from yeast cells is a crucial step during infection with Candida albicans. This morphological switch is accompanied by induced transcription of core filamentation response genes, including ALS3 and ECE1. These two genes are not required for hyphal growth, but contribute to filament-associated traits like adhesion to host cells, invasion and cell damage. Several transcription factors contribute to the regulation of these two genes. A screening of regulatory mutants for below wild type level transcription of ALS3 and ECE1 identified Ahr1 as a putative regulator. So far, Ahr1 was known to be involved in processes like white opaque switching and biofilm formation. Our findings show that deletion of AHR1 in C. albicans leads to reduced ECE1 and ALS3 expression, even though the mutant forms normal hyphae. Interestingly, a hyperactive AHR1 allele induced pseudohyphal growth under conditions which normally trigger yeast cell formation. This correlated with a significant up-regulation of ECE1 and ALS3 up to levels identical to those found in wild type hyphae. This up-regulation was independent from Cph1 and Efg1. Indeed, hyperactive Ahr1 could even induce filamentation in the cph1∆/efg1∆ double mutant, which is usually only able to form yeast cells. These results indicate that Ahr1 is involved in the complex regulation of ALS3 and ECE1 and also contributes to the formation of hyphae. Further experiments will focus on the molecular mechanism of this regulation.

205

P14C

Role of plasma membrane PI(4)P in Candida albicans filamentous growth

Rocio Garcia-Rodas, Martine Bassilana, Robert A. Arkowitz Université Côte d’Azur, CNRS, Inserm, iBV, Nice, France

Candida albicans capacity to switch from a yeast form to a filamentous invasive form is critical for virulence. Membrane lipids and, in particular, the phosphatidylinositol phosphates PI(4)P and PI(4,5)P2, are important for C. albicans filamentous growth (1, 2). We have been investigating PI(4)P at the plasma membrane, which is generated by the PI-4-kinase Stt4. In budding yeast, two plasma membrane proteins that colocalize with Stt4 at cortical punctate structures are important for the stability of this lipid kinase (3) and have been shown to play a role in C. albicans filamentous growth (4). We have analyzed these two proteins, Efr3 and Ypp1, in C. albicans by investigating their localization as well as their effect on different phospholipids distribution. We show that these proteins localize to discrete regions of the plasma membrane and deletion mutants have reduced levels of plasma membrane PI(4)P and an altered phosphatidylserine distribution. In contrast, Golgi PI(4)P and plasma membrane PI(4,5)P2 levels are not altered in the efr3 and ypp1 mutants. Together our results indicate that plasma membrane PI(4)P has specific functions, independent of its role as a PI(4,5)P2 precursor, that are critical for filamentous growth.

(1) Vernay et al. (2012). J Cell Biol. 198: 711.

(2) Ghugtyal et al. (2015). PNAS. 112: 8644.

(3) Baird et al. (2008). J Cell Biol. 183: 1061.

(4) O’Meara et al. (2016). PLoS Genet. 12:e1006142.

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P15CCandida albicans responds to glycostructure damage by Ace2-mediated feedback regulation of Cek1 signaling

Lasse van Wijlick1 ,2, Marc Swidergall1, Joachim F. Ernst1 ,2 1Molecular Mycology, Heinrich-Heine-University, Dusseldorf, Germany, 2Manchot Graduate School Molecules of Infection, Dusseldorf, Germany

The transcription factor Ace2 is found in many fungal genomes and positively regulates expression of genes involved in cell separation and cell wall biogenesis. Distinct from its conserved function Ace2 was shown to regulate transcription of PMT genes encoding protein-O-mannosyltransferases in the human fungal pathogen Candida albicans. Protein O-mannosylation affects various cellular processes, but its most important function is the maintenance of an intact cell wall, which is an essential structure in fungi. C. albicans uses the Cek1 MAPK pathway to restore cells from damage of its cell wall glycostructures. Defective protein N- or O-glycosylation activates Cek1 and the transcription factor Ace2 as its downstream target, to upregulate genes encoding protein O-mannosyltransferases. Genome-wide chromatin immunoprecipitation (ChIP) on chip analyses were used to explore binding of Ace2 to target sequences. Surprisingly, genes encoding components of the Cek1 pathway including MSB2, CST20, HST7, CEK1 and ACE2 were also identified as Ace2 targets indicating Ace2-mediated transcriptional amplification of pathway genes under N-glycosylation stress. In this condition, physical interaction of the Ace2 protein with the upstream MAPKKK Cst20 was detected. Cst20-GFP showed stress-induced import from the cytoplasm into the nucleus and phosphorylation of Ace2. Interestingly, forced nuclear localization of Cst20 inhibited Cek1-Ace2 signaling, while forced cytoplasmic localization of Cst20 retained full signaling activity, suggesting that nuclear Cst20 downregulates the Cek1 pathway. Collectively, the results indicate that Ace2 is a versatile multifunctional transcriptional regulator, which activates glycostress responses of C. albicans by both positive forward and negative feedback regulation of Cek1 signaling.

207

P16C

The absence of TLR3 controls paracoccidioidomycosis with increase of cytotoxic CD8+ T cells

GRASIELLE JANNUZZI1, JOSÉ ROBERTO DE ALEMIDA1, GILBERTO KAIHAMI2, LAVINIA ROMERA1, SANDRO ROGÉRIO DE ALMEIDA1, KAREN FERREIRA3 1Faculty of Pharmaceutical Sciences, University of São Paulo, SÃO PAULO, Brazil, 2IQ-USP - Institute of Chemistry, University of São Paulo, SÃO PAULO, Brazil, 3Institute of Environmental Sciences, Chemical and Pharmaceutical, of Federal University of São Paulo, DIADEMA,SÃO PAULO, Brazil

Toll-like receptors comprise the best-characterized pattern-recognition receptor family that can activate different immune responses, depending on which receptor and adaptor set are utilized. TLRs, such as TLR2, TLR4 and TLR9, and their signaling have been implicated in the recognition of P. brasiliensis and regulation of the immune response, however, the role of TLR3 remains unclear. Thus, understanding the endosomal function of TLR3 in experimental PCM is crucial. We used in vitro and in vivo models of infection by P. brasiliensis, WT and TLR3-/- mice, to assess the contribution of TLR3 on development of infection. We show absence of TLR3 leads to increase nitric oxide and phagocytic ability by macrophages, the first 4 hours of interaction with yeast P. brasiliensis. We further show that TLR3-/- mice plays a protective role after 30 days of intratracheal infection with P. brasiliensis, showing decrease of CFU grown, increase of pro-inflammatory cytokines production, as well as increase of cytotoxic T CD8+ cells producers of IFN-γ and IL-17. In early stages, we observed increase tlr9 expression in lungs of TLR3-/- mice in first 4 hours post infection and later (72 hours) enhanced recruitment of neutrophil in the lung followed by increased inflammatory cytokines. Recent studies indicate that TLR3 is important receptor for the immune response in mycosis and its absence favors the fungal infection. In contrast, our findings show that in the case of PCM, the TLR3 is deleterious to the host, suggesting that activation of TLR3 can be a possible escape mechanism of P. brasiliensis.

208

P17C

Counteracting nutritional immunity: zinc assimilation and homeostasis in Candida albicans

Aaron Crawford, Dhara Malavia, Omran Alamir, Laura Lehtovirta-Morley, Duncan Wilson University of Aberdeen, Aberdeen, UK

The mammalian immune system has evolved sophisticated mechanisms to withhold micronutrients such as zinc from potential invaders in a process known as nutritional immunity. In spite of this defence mechanism, pathogens still thrive and cause disease. Therefore, pathogenic microbes have, in turn, evolved mechanisms to circumvent nutritional immunity. Despite the fundamental importance of this host-pathogen “tug-of-war”, its underlying mechanisms, and how they can be exploited to prevent disease remain poorly understood. We are exploring how Candida albicans adapts to fluctuations in zinc availability and how this essential, yet potentially toxic cation is mobilised within the fungal cell. We have found that environmental zinc has profound impacts on C. albicans morphology. Cellular assimilation of zinc occurs via transporter- or extracellular scavenger (zincophore) mediated mechanisms and phylogenetics has revealed examples of extensive expansion and loss of zinc assimilation genes in contemporary fungal species. Following uptake, zinc is stored in distinct subcellular compartments, including the fungal vacuole. Interestingly these organellar stores can be mobilised when the fungus faces subsequent zinc starvation, serving as a nutrient source. We are now using a combination of intracellular zinc probes, together with targeted deletion of zinc transporters to gain a complete picture of how C. albicans assimilates, compartmentalises and mobilises this essential micronutrient and the impact these processes have on pathogenicity.

209

P18C

Impaired phagocytosis directs human monocyte activation by particulate fungal β-glucan

Giorgio Camilli1, David L. Williams2, Vishu Kumar Aimanianda Bopaiah1, Jessica Quintin1 1Institut Pasteur, Paris, France, 2East Tennessee State University, Tennessee, USA

Monocytes are key regulators of early host responses to invading infectious agents. They also act in concert with macrophages and dendritic cells to detect, capture and process various pathogens at the site of infection. Microbes are sensed by monocytes via several innate immune receptors that are able to recognize conserved chemical motifs known as Pathogen-Associated Molecular Pattern (PAMPs). Among these, the recognition of the β-glucan polymer, a major component of the cell wall of fungi, is primarily mediated by Dectin-1 and triggers multiple antifungal responses. β-glucan recognition by Dectin-1 on myeloid phagocytes triggers phagocytosis and production of ROS that are important in protection against fungal infection. However, the ability of particulate β-glucan to mediate Dectin-1-dependent cytokine production remains unclear if not controversial. Here we show that the particulate β-glucan isolated from the cell wall of yeast does not induce the production of pro-inflammatory cytokines by primary human monocytes. However, a Dectin-1-dependent cytokine production occurs when actin polymerization is inhibited. Of note, the actin-conditional induction of pro-inflammatory cytokines triggers NF-κB and is dependent on the structure, solubility and source of the glucan ligand. A better understanding of the signaling pathway needs further investigation and is now under investigation. Of note, the “frustrated phagocytosis” induced by actin inhibition decreases the inflammatory properties of heat-inactivated-Candida albicans. This discrepancy in our observation is reminiscent of the differential cellular activation and subsequent inflammation induced upon yeasts vs. the non-phagocytosable hyphae encounter.

210

P19C

Druggable spots on the Candida albicans ribosome: combination of in silico and in vitro results

Marcela SABOU1, Hélène GASS2, Julie THOMPSON2, Marat YUSUPOV3, Ermanno CANDOLFI1 1Institut de Parasitologie et de Pathologie Tropicale, Strasbourg, France, 2ICube, UMR CNRS 7357, Strasbourg, France, 3Biologie Structurale Intégrative, Institut de Génétique, Biologie Moléculaire et Cellulaire, Illkirch, France

Candida albicans is an opportunistic and nosocomial agent that has become a cause of major health concerns. It has emerged as an important cause of morbidity and mortality in immunocompromised or immunocompetent patients, being responsible of unacceptably high mortality rates. Prevention and treatment have led to a widespread use of antifungal drugs, leading to the emergence of resistant strains. There is a need for new selective and less toxic antifungal molecules. Moreover, the drug companies’ antifungal pipeline is mostly dry, urging different approaches of exploring different targets. The ribosome is an interesting target: it represents the cellular machinery responsible for protein biosynthesis and it can be blocked by small molecules as antibiotics for example. Our study set out to compare the latest 80S ribosome structures from Saccharomyces cerevisiae and C. albicans in order to show interesting druggable spots on the C. albicans ribosome. These spots have been tested by specific inhibitors to identify the differences between the lab model, S. cerevisiae, and C. albicans. Interesting spots will be used for further comparison with the human ribosome in order to develop and design new ribosome inhibitors directed specifically against C. albicans.

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P20C

Variability of adhesin presentation in the cell walls of Candida glabrata and Candida parapsilosis

Emilia Gomez-Molero1 ,2, Ana Moreno2, Henk Dekker3, Ichsan .1, Toni Gabaldon4, Attila Gacser5, Uwe Groß1, Michael Weig1, Piet de Groot2, Oliver Bader1 1Institute for Medical Microbiology, University Medical Center Göttingen, Goettingen, Germany, 2Regional Center for Biomedical Research, Albacete Science and Technology Park, University of Castilla-La Mancha, Albacete, Spain, 3Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands, 4Comparative Genomics Group, CRG-Centre for Genomic Regulation, Barcelona, Spain, 5Department of Microbiology, University of Szeged, Szeged, Hungary

Non-Candida albicans Candida species have gained significant importance over the past decades becoming one of the principal causes of nosocomial and bloodstream-invasive Candidiasis. The increased prevalence of drug resistance and high capacity to form biofilms on abiotic surfaces makes Candida parapsilosis and Candida glabrata important targets for the development of specific diagnostic or treatment strategies. Both genomes encode a number of potential adhesins whose roles remain uncharacterized. We hypothesized that these might be modulated in clinical isolates conferring different adhesion properties and biofilm formation intensities. We therefore analyzed biofilm formation capacity, presence of putative-novel adhesins and azole susceptibility in clinical isolates of both species. Cell-wall proteomic analyses of two hyperadhesive isolates C. glabrata demonstrated the incorporation of previously undescribed adhesion molecules in addition to a core cell wall proteome. Routine screening of adhesion showed that approx. 20% of clinical C. glabrata and 24% of C. parapsilosis isolates presented a hyperbiofilm-forming phenotype, but no correlation with body site of isolation became evident. An increased number of different adhesins were detected in the cell walls

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of hyperbiofilm-forming isolates of both species. Downstream analyses showed distinct correlations of phenotypes such as altered virulence or morphology with adhesin presentation, as well as genomic alterations in hyper biofilm-forming isolates. In conclusion, differences in C. glabrata and C. parapsilosis cell wall properties among clinical isolates reflect variations in infection-relevant parameters.

213

P21C

Coinfection of Candida albicans and Proteus mirabilis boosts the swarmer’s virulence

M. Joanna Niemiec1 ,2, Mario Kapitan1 ,2, Stephanie D. Himpsl3, Harry L. Mobley3, Ilse D. Jacobsen1 ,2 1Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Insitute, Jena, Germany, 2Center for Sepsis Control and Care, Jena, Germany, 3University of Michigan Medical School, Ann Arbor, USA

The human gut, as the organ harboring the highest density of microbes, is a relevant source of life-threatening infections. Numerous opportunistic bacteria and fungi colonize the gut and can disseminate into the bloodstream (BS) upon immunosuppression or impairment of barrier function. While the mechanisms promoting translocation of Candida albicans into the BS are not fully understood, recent studies estimate up to 29 % of Candida BS infections to be in fact polymicrobial - implying a role for Candida-bacteria interactions. In order to understand the causality between co-colonization and coinfection, we investigated the interplay of C. albicans and the gram-negative opportunistic pathogen Proteus mirabilis, an emerging cause of bacterial BS infections. In the absence of host cells, fungal numbers were reduced during long-term coincubation with P. mirabilis while bacterial survival was enhanced, suggesting antagonistic interactions benefitting P. mirabilis. More strikingly, we observed significantly increased damage of enterocytes during in vitro coinfections with C. albicans and four different P. mirabilis strains compared to summed-up single-species damage. This synergistic effect was strongly diminished by deletion of P. mirabilis hemolysin. To investigate whether Candida filamentation and damage were essential for boosting host cell damage, we tested C. albicans mutants with impaired hypha formation (ΔEFG1, ΔHGC1, ΔCAP1, ΔEDE1) or lacking candidalysin (ΔECE1). Surprisingly, neither candidalysin nor filamentation were required for the Candida-Proteus synergism. Experiments aimed to determine (i) whether C. albicans affects hemolysin production of P. mirabilis, and (ii) if synergistic virulence occurs in in vivo models are currently performed.

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P22C

Characterization of the role of Candida albicans GPI-anchored proteins in biofilm formation.

Mennat El Ghalid, Sophie Bachellier-Bassi, Christophe d’Enfert Institut Pasteur, Paris, France

Infections due to Candida albicans have emerged over the past three decades as a considerable threat to human health. Nowadays, treatment of C. albicans infections relies on a limited therapeutic arsenal including drugs whose efficacy is hampered by a lack of selectivity, some toxicity, and the development of resistance. Since fungi are eukaryotes, one great challenge is to characterize new anti-fungal targets, which are fungal-specific. GPI-anchored proteins (GAPs) may represent suitable targets in the development of novel antifungals, immunotherapeutics and vaccines. GAPs are cell wall components, which play a crucial role in the biology of C. albicans. However there is a lack of information on their specific role. Thus, our objective is to understand the contributions of such proteins in the biology and pathogenicity of C. albicans. Previous data obtained in our laboratory showed that overexpressing some GAP-encoding genes impacts adherence and biofilm formation (eg members of the Ihd1 family1,2, PGA22 and PGA592). We are currently testing the biofilm formation ability and other phenotypes of KO mutants deleted for several members of the Ihd1-family. We are also attempting to characterize the specific role of selected GAPs by producing their recombinant mature domains in E. coli and P. pastoris, in order to determine their 3D structure and mine these structures to understand how these proteins contribute to adherence. Finally, we aim to identify the domains that should be targeted in order to inhibit their function in adherence.

1 Sosinska, G. (2012)

2 Cabral, V. et al. (2014)

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P23C

Mating-type switching in Candida glabrata and related species.

Cecile Fairhead, Stéphanie Boisnard, Youfang Li Zhou Université Paris Sud, Orsay, France

Although it seems to have lost the capacity for sexual reproduction, the genome of Candida glabrata contains three MAT-like cassettes, and the HO gene, encoding the endonuclease that initiates the mating-type interconversion event. We have published that S. cerevisiae Ho induces mating-type switching in C. glabrata; that this results in massive cell death, and that Ho cuts the additional loci, HMR and HML. Examination of strains in our collection shows mating-type interconversion events, and rearrangements of MAT-like loci. We now show that it is not the additional cleavage of MAT-like loci that induces cell death, but only cleavage of MAT. We also show that this situation is peculiar to C. glabrata, since the closely-related Nakaseomyces delphensis species can undergo mating-type interconversion by expression of the S. cerevisiae HO gene, without the massive cell death observed in C. glabrata. We will discuss these observations and propose hypotheses.

216

P24C

Fungal isocyanide synthases: Discovery of the xanthocillin biosynthetic pathway in the opportunistic human pathogen, Aspergillus fumigatus

Fang Yun Lim1, Tae Hyung Won2, Joshua Baccile2, Jennifer Wisecaver3, Abigail Lind3, Antonis Rokas3, Frank Schroeder2, Nancy Keller1 1University of Wisconsin-Madison, Madison, WI, USA, 2Cornell University, Ithaca, NY, USA, 3Vanderbilt University, Nashville, TN, USA

Isocyanide-containing natural products are of immense interest in the biological and chemical milieu owing to their repertoire of bioactivities and capability for metal coordination; these functions, attributed to the highly reactive isocyano- functional groups tethered to structurally diverse carbon scaffolds. Isocyanide production has been implicated in the pathogenesis of various bacterial and fungal pathogens. In the entomopathogenic bacterium, Xenorhabdus nematophila, the isocyanide, rhabduscin, is capable of disabling the innate immune defense of insects via inhibition of the cuproenzyme, phenoloxidase. In contrast to the plethora of isocyanides isolated to-date, knowledge on their biosynthetic machineries is still in its infancy with only four characterized biosynthetic gene clusters (BGCs) in prokaryotes, and unprecedented in eukaryotes. The identification of isocyanide synthases (ICS) in the opportunistic human fungal pathogen Aspergillus fumigatus marks the first description of eukaryotic ICSs and lead to the discovery of the xanthocillin (xan) gene cluster, the first reported biosynthetic pathway dedicated to this family of isocyanides since its isolation over half a century ago. Further characterization of xanthocillin production in A. fumigatus suggests evidence of pathway redundancy between two distinct ICS gene clusters: the xan and a copper-responsive metabolite (crm) gene cluster containing a novel fungal-specific ICS-NRPS enzyme. We will further assess the impact of isocyanides towards the pathogenesis of invasive aspergillosis. As this family of ICS-NRPS enzyme is highly enriched in fungal pathogens of humans, insects, and plants, characterization of isocyanide BGCs in these fungal pathogens may provide new insights into their role(s) during plant and human infections.

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P25C

Modifications of transfer RNAs represent an epigenetic link to virulence traits in Candida species.

Bettina Böttcher1 ,2, Bianka Morgner1, L. Peter Sarin3 ,6, Stefanie Allert1, Ilse D. Jacobsen7, Hannes Drexler3, Sebastian Leidel3 ,4, Sascha Brunke1 ,5 1Hans Knoell Institute, Department of Microbial Pathogenicity Mechanisms, Jena, Germany, 2Center for Innovation Competence (ZIK) SEPTOMICS, Junior Research Group Host Fungal Interfaces, Jena, Germany, 3Max Planck Institute for Molecular Biomedicine, Research Group for RNA Biology, Münster, Germany, 4University of Münster, Cells-in-Motion Cluster of Excellence, Münster, Germany, 5University Hospital Jena, Jena, Germany, 6University of Helsinki, Department of Biosciences, Faculty of Biological and Environmental Sciences, Helsinki, Finland, 7Hans Knoell Institute, Research Group Microbial Immunology, Jena, Germany

tRNA molecules are bridges between nucleic acids and proteins. Post-transcriptional modifications of tRNAs are conserved in all living cells, as they provide translational effectiveness and fidelity. Defects in tRNA modification can thus slow down the protein biosynthesis apparatus with detrimental consequences for the cell. Although pioneering studies have shown a causative relationship between modification levels and the viability and virulence potential of pathogenic bacteria, their importance in human fungal pathogens is still completely unknown.

A common agent in disseminated fungal disease is Candida albicans. Its closest sister species, Candida dubliniensis, is only infrequently associated with life-threatening candidiasis. With this in mind, we performed a genomic cross-species comparison to identify potential C. albicans virulence factors. We found that integration of C. albicans HMA1 changed the morphological transitions of C. dubliniensis under nitrogen starvation.

The involvement of Hma1 in nutritional signaling was further demonstrated

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by an altered resistance against TOR pathway antagonistic substances. Phenotypic characterization of a C. albicans hma1∆ mutant showed a species-specific impact on filamentation in vitro and during infection of human oral epithelial cells. In an embryonated egg infection model, the C. albicans hma1∆ mutant induced significantly reduced mortality compared with its wild type strain. The Hma1 ortholog in Saccharomyces cerevisiae is Tcd2: a tRNA-modifying enzyme that targets an adenosine adjacent to the anticodon. Our preliminary data indicates that the same activity is catalyzed by the C. albicans enzyme. Hence, we have shown for the first time a connection between tRNA modifications and the species-specific virulence of C. albicans.

219

P26C

Fingerprinting of Candida spp. blood infections

Philipp Kämmer1, Sylvie Schulze2, Thomas Wolf2, Kerstin Hünniger3, Jörg Linde2, Oliver Kurzai3, Reinhard Guthke2, Bernhard Hube1 ,4, Sascha Brunke1 1Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany, 2Research Group Systems Biology and Bioinformatics, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany, 3Septomics Research Center, Friedrich Schiller University and Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany, 4Friedrich Schiller University, Jena, Germany

Candida species are important agents of invasive fungal infections. Among them, Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis account for the majority of all systemic Candida infections. To mimic the initial phase of systemic Candida infections, we here established an ex vivo whole blood infection model for all these species. This model allowed the investigation of interdependent transcriptional responses of both human host and fungal pathogen, based on a dynamic dual-species RNAseq approach.

We found a predominantly uniform human response to infection with the four species, with very few species-specific regulations. These strongly time-dependent transcriptional changes are governed by processes of the immune system response, in particular by genes involved in TNF and NF-κB signaling.

On the fungal side, the three more closely related species C. albicans, C. tropicalis, and C. parapsilosis each regulate a noticeable fraction of their genomes in response to blood exposure, while C. glabrata surprisingly only reacts with minor transcriptional adjustments. Common fungal responses comprise the shutdown of protein biosynthesis and the activation of heat shock responses. In contrast, genes involved in oxidative stress response

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are broadly up-regulated by C. albicans, C. tropicalis, and C. parapsilosis, but not by C. glabrata. The regulation of several other processes, like iron homeostasis or cell cycle progression, is even more species-specific. This led us to the conclusion that different Candida species pursue discrete strategies to survive within the harsh bloodstream environment, while the transcriptional host response is much more uniform.

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P27C

Evolution of fluconazole-resistant Candida albicans strains exhibiting multiple resistance mechanisms by drug-induced sexual recombination

Christina Popp, Joachim Morschhäuser University of Würzburg, Würzburg, Germany

Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is commonly caused by mutations in the target enzyme Erg11 or by gain-of-function mutations in the transcription factors Mrr1, Tac1, and Upc2, which result in overexpression of multidrug efflux pumps and ergosterol biosynthesis genes. Many fluconazole-resistant isolates are homozygous for the mutated genes and exhibit multiple resistance mechanisms. The loss of heterozygosity is frequently accompanied by MTL homozygosity, which enables the cells to switch to the mating-competent opaque cell form. To investigate if sexual recombination may be involved in the evolution of highly drug-resistant strains, we propagated a set of isogenic strains that were heterozygous for ERG11, MRR1, TAC1 and UPC2 alleles with resistance mutations in the presence of fluconazole. This resulted in the emergence of derivatives with increased resistance that had become homozygous for the mutated allele and the mating type locus. When mixing MTLa/a and MTLα/α cells of these strains in all possible combinations, we could isolate mating products containing the genetic material from both parents. The initial mating products did not exhibit higher drug resistance than their parental strains, but further propagation under selective pressure resulted in the loss of the wild-type alleles and increased fluconazole resistance. Our results demonstrate that fluconazole treatment not only selects for resistance mutations but also promotes genomic alterations that confer mating competence. This allows cells in an originally clonal population to exchange individually acquired resistance mechanisms by sexual recombination and generate highly drug-resistant progeny.

222

P28C

Adaptation to acidic environments induces unmasking of beta-glucan in Candida albicans

Sarah Sherrington, Rebecca Hall University of Birmingham, Birmingham, UK

Candida albicans is a common commensal pathogen with the ability to thrive in a variety of host niches. These different body sites vary dramatically in environmental factors including pH, CO2 levels and the presence of quorum sensing molecules. Although these environmental parameters have been extensively investigated for the ability to regulate morphogenesis and white-opaque switching of C. albicans, their role in regulating cell wall biosynthesis is largely unknown. Here we investigate how adaptation to acidic environments affects the architecture and biosynthesis of the fungal cell wall. Growth in acidic environments (pH4) resulted in significant exposure of the underlying beta-glucan, which was associated with enhanced innate immune recognition mediated via Dectin-1. Investigation into the conventional signalling pathways that regulate adaptation to environmental pH and cell wall stress, suggested that this unmasking of beta-glucan is regulated via a non-canonical signalling mechanism. To elucidate key genes involved in this phenotype we have screened a library of C. albicans mutants for their ability to undergo pH dependent beta-glucan unmasking. This screen identified several genes that may function in the unmasking of beta-glucan in response to environmental stimuli, and also identified genes that function in general cell wall biogenesis. We are now beginning to uncover the molecular mechanisms driving β-glucan unmasking, in the hope to better understand the dynamic nature of the Candida cell wall within different host environments.

223

P29C

Distinct mechanisms of poacic acid and caspofungin inhibition of fungal β-1,3-glucan synthesis

KEUNSOOK KATHY LEE1, KAREN KUBO2, JEHAN ABDELMONEIM ABDELAZIZ1, XIAOLIN CHEN2, HIROKI OKADA2, YOSHIKAZU OHYA2, NEIL A.R. GOW1 1The Aberdeen Fungal Group, MRC Centre for Medical Mycology, School of Medicine, Medical Sciences & Nutrition, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK, 2Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan

Seeking new antifungal agents for the treatment of human fungal infections is urgently required. Recently a novel plant extract called poacic acid (PA) was identified and found to have antifungal activity against a range of plant pathogens and Saccharomyces cerevisiae. Like the echinocandins, PA targets the β-1,3-glucan synthesis enzyme Fks1 and has potential utility in the treatment of clinically important fungal pathogens. However, the antifungal activity of PA against Candida species has not been explored and the defined mode of action of this compound is not fully understood. Here we demonstrate that PA sensitivity of Candida species varied, and that the susceptibility correlated with the predicted topology of the target β-1,3-glucan synthase protein (Fks1) in the membrane. C. albicans, C. dubliniensis, C. glabrata, and C. tropicalis were less susceptible to PA than C. guiliermondii, C. orthopsilosis, and C. parapsilosis. A hotspot mutation in Fks1 had different effects on caspofungin and PA sensitivity. In addition, dysfunction in a single allele of C. albicans Fks1 led to a decreased susceptibility to PA. PA-sensitive strains shared a common topology with a key domain on the inner-face membrane. In PA-insensitive strains this domain was predicted to be on the outer-face membrane in. These data demonstrate significant differences in the mode of action of PA and caspofungin in inhibiting β-1,3-glucan synthesis and suggest that PA may be potentially useful as a topical antifungal for the treatments of some, but not all, superficial Candida infections.

224

P30C

Iron restriction inside the phagosome of macrophages is an essential host defense mechanism against Mucorales

Angeliki M. Andrianaki1 ,6, Irene Kyrmizi1 ,5, George Samonis6, Dimitrios P. Kontoyiannis2, Ashraf S. Ibrahim3 ,4, George Chamilos1 1Department of Clinical Microbiology and Microbial Pathogenesis, School of Medicine, University of Crete, Heraklion, Crete, Greece, 2Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA, 3Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USA, 4David Geffen School of Medicine at UCLA, Los Angeles, California, USA, 5Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Crete, Greece, 6Department of Internal Medicine, Infectious Diseases Unit, University of Crete Medical School, Heraklion, Crete, Greece

Mucorales are emerging airborne fungal pathogens with unique virulence attributes and incompletely understood pathogenesis. We found that as opposite to Aspergillus fumigatus, Mucorales have a selective “tropism” for alveolar macrophages (AMs), which accounts for the establishment of prolonged intracellular “persistence” in the lungs of immunocompetent mice. Although Mucorales conidia are fully susceptible to the effector mechanisms of macrophages, they avoid killing and establish intracellular “persistence” via the induction of phagosome maturation arrest. Mechanistically, lack of intracellular cell wall swelling of Mucorales conidia leads to surface retention of melanin and subsequent blockade in early critical steps of phagosome biogenesis. On the other site, inhibition of germination of Mucorales conidia inside macrophages is an essential host defense mechanism, as AM depletion significantly increases susceptibility of mice to mucormycosis. Nutritional immunity via iron restriction is the principal mechanism of inhibition of Mucorales growth inside arrested

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phagosomes of macrophages. Importantly, increased iron availability via iron or deferoxamine supplementation leads to germination of Mucorales conidia inside macrophages, which is reversed upon genetic ablation of essential components of iron assimilation pathways of the fungus. Collectively, our findings reveal novel aspects of cellular immunity against Mucorales that are critical in pathogenesis of mucormycosis and represent future potential targets for therapeutic interventions.

226

P31C

Characterization of novel small C. albicans proteins induced by macrophage phagocytosis

Elisa Vesely, Michael Lorenz University of Texas Health Science Center at Houston, Houston, Texas, USA

The interaction between Candida albicans and mammalian phagocytes is a key determinant of disease initiation and progression and has proven to be a valuable experimental tool to uncover key virulence traits. To gain further insight into the fungal response to phagocytosis, we used RNA-sequencing to characterize the transcriptome of C. albicans after phagocytosis by murine bone marrow-derived macrophages. We detected expression of 99.4% of protein-coding genes, through which we confirmed earlier observations of a significant reorganization of carbon metabolism, downregulation of translation and cell cycle progression, and induction of responses to oxidative and nitrosative stresses and DNA damage repair. Surprisingly, the set of upregulated genes includes a dramatic over-representation of transcripts encoding proteins of less than 100 amino acids, genes omitted from earlier microarray-based studies. Because such small proteins are often important as secreted mediators of host-pathogen interactions, we have begun a genetic analysis 13 small ORFs upregulated at least 4-fold in phagocytosed cells. Preliminary investigations of the first two genes to be characterized, SMA1 and SMA2, have identified host-relevant phenotypes. A sma1∆ strain has increased susceptibility to oxidative stressors and is deficient in survival upon contact with macrophages. Sma2 shows features of a putative adhesin, and mutants are defective in adhesion to surfaces. Future studies are characterizing their mechanism of action and contribution to C. albicans virulence in the context of the mammalian host.

227

P32C

The ‘Beast From The East’: 20th Century Out-of-Asia Origin of a Panzootic Threat to Global Amphibian Biodiversity

Simon O’Hanlon1 ,4, Adrien Rieux3, Matteo Fumagalli1, Francois Balloux2, Trent Garner4, Lola Brooks4, Gonçalo Rosa4, Matthew Fisher1 1Imperial College London, London, UK, 2University College London, London, UK, 3Cirad, Réunion, France, 4The Zoological Society of London, London, UK

Discovery of the emerging infectious chytrid fungus Batrachochytrium dendrobatidis (Bd) was a turning point in understanding why amphibian species worldwide are in rapid decline. Phylogenomic studies to date have identified four genetic lineages of Bd of which one, the hypervirulent Global Panzootic Lineage (BdGPL), has driven the signature declines across Australia, the Americas and Europe. However, the spatiotemporal origin of BdGPL remains one of the most contentious and elusive secrets in disease ecology. To answer this question, we developed approaches to recover fungal DNA from preserved samples alongside genome-sequencing and molecular dating from a pan-global panel of Bd isolates. Here, we report a 4-fold higher nucleotide diversity in strains isolated from frogs native to South Korea than in the rest of our representative collection of global Bd strains diversity. Korean Bd isolates belong to a highly diverse ancestral population at mutation-drift equilibrium, which seeded all Bd lineages currently in circulation through transcontinental jumps and subsequent hybridisation events. Root-to-tip calibrated mitochondrial clocks date the expansion of BdGPL to the mid 20th Century, coinciding with the expansion of commercial amphibian trade worldwide, and consistent with the earliest histological confirmation of amphibian infections. Our data show that Asia is a hotspot for Batrachochytrium biodiversity and likely includes the modern ancestors of all lineages infecting amphibians worldwide.

228

P33C

Investigating the mechanisms by which the transcription factor Hsf1 regulates Candida albicans morphogenesis

Amanda Veri1, Teresa O’Meara1, Rebecca Shapiro2, Faiza Tebbji3, Sang Hu Kim1, Juan Colazo1, Nicole Robbins1, Malcolm Whiteway4, Koon Ho Wong5, Leah Cowen1 1University of Toronto, Toronto, Ontario, Canada, 2Broad Institute, Boston, Massachusetts, USA, 3Université Laval, Québec City, Québec, Canada, 4Concordia University, Montréal, Québec, Canada, 5University of Macau, Macau SAR, China

Invasive fungal infections caused by Candida albicans are among the leading causes of death in immunocompromised individuals. One key virulence trait of C. albicans is its ability to transition from yeast to filamentous growth, which is typically regulated by environmental cues at the physiological temperature of 37°C. The heat shock transcription factor Hsf1 orchestrates the C. albicans temperature response in part through regulation of Hsp90, a molecular chaperone involved in governing morphogenesis. Here, we focus on elucidating the role of Hsf1 in the yeast-to-filament transition. We discovered that overexpression and depletion of HSF1 induces filamentation independent of the temperature requirement of 37°C. However, the mechanisms by which differing Hsf1 levels regulate the morphogenetic program appear distinct. HSF1 depletion compromises Hsp90 function even when HSP90 expression is driven by a constitutive Hsf1-independent promoter. This suggests that HSF1 depletion alters the expression of a critical regulator of Hsp90, leading to compromised chaperone function and filamentous growth. Overexpression of HSF1 tightly links morphogenesis with temperature as moderate overexpression allows filamentation at 34°C while strong overexpression drives filamentation at 30°C. HSF1 overexpression enables morphogenesis through overexpression of positive regulators of filamentation such as Brg1 and Ume6. To determine if these are direct Hsf1 targets and to provide a global analysis of Hsf1-targets critical for morphogenesis, we are performing chromatin immunoprecipitation coupled to sequencing and RNA sequencing analyses. This work illuminates a novel role for Hsf1 in regulating morphogenesis and provides a fascinating example of a gene whose overexpression and depletion induces C. albicans filamentation.

229

P34C

Is the Candida albicans Hsp90 co-chaperone complex essential for virulence?

Naomi Lyons, Stephanie Diezmann University of Bath, Somerset, UK

Genes regulating microbial virulence are often located close to the telomere, such as the TLO genes in Candida albicans (Anderson et al., 2012) and the VAR genes in Plasmodium falciparum (Su et al., 1995). C. albicans Hsp90 co-chaperone genes are near the telomeres. When considering that homologs in the benign baker’s yeast are spread evenly across the chromosome, could Hsp90 co-chaperones play a key role in virulence of C. albicans?

C. albicans is a leading fungal pathogen of humans, killing ~700 people in the UK annually (Brown et al., 2012). Life-threatening systemic infections are difficult to treat due to the limited number of antifungal drug targets and the increasing prevalence of antifungal resistance. Hsp90 co-chaperones may recommend themselves as a solution to this problem if essential for virulence, but not for commensal growth.

In order to ascertain whether co-chaperones could be exploited as novel drug targets their role in C. albicans virulence must first be assessed. Homozygous deletion mutants were generated and examined for growth and virulence using assays such as iron sequestration, survival of oxidative stress, biofilm formation, and killing of an invertebrate host model. Data thus far shows Ssa1 and Sba1 play a role in survival of oxidative stress, Ssa2 and Sba1 are necessary for biofilm formation, and Sti1 plays a role in the heat shock response.

230

P35C

Candida albicans evades innate immunity by hijacking two distinct host responses to trigger macrophage cell death

Timothy Tucey, Jiyoti Verma, Tricia Lo, Thomas Naderer, Ana Traven Infection and Immunity Program and the Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Australia

In Candida albicans, morphogenetic switching between yeast and hyphal forms is crucial for evasion of innate immunity. We have established single-cell live cell imaging of infected macrophage populations to monitor and quantify the activation of inflammasome responses and host cell death during the course of infection. With this, we show that C. albicans-induced macrophage killing can be mechanistically separated into two phases. Initially, the switch from yeast to hyphae inside the phagosome triggers NLRP3/caspase-1 inflammasome mediated pyroptosis, which enables fungal escape. Inflammasome activation can be hugely delayed in response to mutants that form hyphae but display reduced virulence, indicative of signal threshold-dependent activation linked to fungal pathogenicity. After pyroptosis, escaped C. albicans triggers another, inflammasome-independent form of cell death that eliminates the remaining macrophages in a fast wave of killing. We now show that this second mechanism of macrophage killing is regulated by metabolic interactions between host and pathogen and is accompanied by the reverse hyphae-to-yeast switch that could aid in dissemination. Using various clinical isolates of C. albicans, pharmacological regulators and fungal mutants, coupled with live cell imaging, RNAseq and biochemical experiments, here we report the mechanistic details of host and fungal pathways involved in the two phases of host cell death. Our study shows that C. albicans takes advantage of two distinct host responses to successfully eliminate macrophages, shedding light on the morphogenetic and metabolic switches that regulate the host-pathogen interaction. This provides a new understanding by which microbial pathogens can “hijack” host responses to successfully evade immunity.

231

P36C

Identification of mediators of antifungal tolerance in Candida albicans

Eric Delarze1, Fabio Maranzano1, Marion Patxot-Bertran1, Sadri Znaidi2, Mélanie Legrand2, Maiken Cavling Arendrup3, Carol Munro4, Christophe d’Enfert2, Dominique Sanglard1 1University of Lausanne and University Hospital Center, Lausanne, Vaud, Switzerland, 2Pasteur Institute, Paris, France, 3Statens Serum Institut, Copenhagen, Denmark, 4University of Aberdeen, Aberdeen, UK

Antifungal tolerance can be defined as the ability of C. albicans cells to survive at high drug concentrations but without acquiring mutations associated with resistance. The mechanisms mediating drug tolerance are still not well understood. In this study we aimed to identify mediators of tolerance to fluconazole (FLC) using two different approaches. First, the tolerance profile of 27 clinical C. albicans isolates against FLC using the EUCAST drug susceptibility assay was assessed. Tolerant strains were identified by their ability to sustain residual growth after 24h at a drug concentration above the FLC resistance breakpoint (> 4µg/ml). This screening revealed different profiles grouped from low to high tolerance. These FLC-tolerant strains are candidates for further characterization by genetic and transcriptomic approaches and will be used to test the importance of drug tolerance in animal models. In a second approach, in order to identify genetic mediators of tolerance, we used a collection of 582 tetracycline-inducible overexpression barcoded strains, which were pooled and maintained under FLC pressure for five days of repeated subcultures. This strategy was used to enrich the pool in FLC-resistant and/or FLC-tolerant strains. After amplification and sequencing of all barcodes from the cultures, the fractional index of each strain in the population was calculated. The FLC susceptibility profile of enriched strains yielded the identification of two potential tolerance mediators. When overexpressed, these genes contributed to increase tolerance to FLC as compared to controls. The potential role of these two genes upon tolerance is now under investigation.

232

P37C

Forces in Candida albicans filamentous growth

Darren Thompson3, Xavier Noblin2, Charles Puerner1, Agnese Seminara2, Martine Bassilana1, Robert A. Arkowitz1 1Université Côte d’Azur, CNRS, Inserm, iBV, Nice, France, 2Université Côte d’Azur, CNRS, LPMC, Nice, France, 3Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, UK

Little is known regarding the physical forces that are involved in the filamentous growth of human fungal pathogens, as essentially non-pathogenic yeasts have been examined thus far (1). Candida albicans is a major human fungal pathogen, causing both superficial and systemic infections. Filamentous growth and its ability to invade solid surfaces, such as medical implants and human tissues are critical for its pathogenicity. Our goal is to determine the biophysical properties of C. albicans hyphae, which are crucial for substrate penetration and invasive growth. Towards this goal, we have generated transparent PDMS microchambers (2), in which we have entrapped C. albicans and subsequently monitored hyphal growth. Furthermore, we have altered the stiffness of these PDMS microchambers to mimic that of human cells and tissues. The analysis of the behavior of these inert microchambers during hyphal growth provides a read-out of fungal mechanical properties. Specifically, we are determining the deformation of either the hyphae or the PDMS micro-chambers during filamentous and invasive growth. We have also followed hyphal penetration into the substrate and measured differences in growth inside and on the surface of PDMS. These studies provide a framework for analyzing the interplay between mechanical constraints and polarized growth during the transition to fungal filamentous growth.

(1) Minc et al. (2009) Curr Biol. 19: 1096-101.

(2) Minc (2014). Meth Cell Biol. 120: 215-26.

233

P38C

The role of mistranslation in the yeast-to-hyphal transition of C. albicans cells

Ana Rita Bezerra1, Carla Oliveira1, Edgar Lopes1, Sílvia Rocha2, Gabriela Moura1, Manuel Santos1 1Department of Medical Sciences, iBiMED – Institute of Biomedicine, University of Aveiro, Aveiro, Portugal, 2Departament of Chemistry - QOPNA, University of Aveiro, Aveiro, Portugal

Candida albicans is a major opportunistic fungal pathogen of humans. Its capacity to switch between yeast and hyphal morphologies is critical for its pathogenesis and survival in its hosts. Hyphae have invasive properties that promote tissue penetration and evasion of immune cells, whereas yeast cells are suited for dissemination in the bloodstream. This pathogen exhibits a unique translational system, misincorporating Leu (3%) at the atypical Ser-CUG codon. Exposure of cells to macrophages increases Leu misincorporation levels from 3% to approximately 50% and these hypermistranslators display remarkable morphological plasticity. Here, we used recombinant strains that constitutively mistranslate within 3%-60% range to elucidate the molecular mechanism by which mistranslation triggers filamentation. Germ-tube (GT) assays were performed using rich medium (in non-inducing conditions) and approximately 50% of cells of hypermistranslator strains showed germinating characteristics. In the control strain, only 1.4% of cells showed the same features. We then studied the metabolomic changes associated with the differential morphogenesis using GC-MS based methods. By comparing the concentration of metabolites in hypermistranslators against control cells, 20 metabolites were detected at lower concentrations. Among such metabolites was farnesol, a quorum-sensing molecule that controls hyphal initiation through the N-end rule pathway-mediated protein degradation. These preliminary results suggest that mistranslation may trigger filamentation by altering the quorum-sensing mechanism of this human pathogen.

This work is supported by iBiMED (UID/BIM/04501/2013), FCT and Portugal2020 (FEDER) through grant SFRH/ BPD/96741/2013 and project PTDC/IMI-MIC/5350/2014.

234

P39C

Candida albicans: a model organism to study mechanisms involved in eukaryotic genome integrity

Mélanie Legrand1, Adeline Feri1, Timea Marton1, Raphaël Loll-Krippleber1, Corinne Maufrais2, Christophe d’Enfert1 1Institut Pasteur, INRA, Fungal Biology and Pathogencity Unit, Paris, France, 2Institut Pasteur, center for Informatics in Biology, Paris, France

Genomics studies in Candida albicans have highlighted various degrees of tolerance to genome plasticity. In different instances, genome rearrangements have been correlated with adaptation of the cells to various stress both in vivo and in vitro. In particular, loss-of-heterozygosity (LOH) events, aneuploidies and isochromosome formation have been shown to contribute to increasing the resistance to azole antifungals. Genomics studies have also revealed that some chromosome haplotypes cannot be found in the homozygous state, suggesting the presence of recessive lethal alleles on certain chromosome homologs of C. albicans.

Because DNA double-strand breaks (DSBs) have been shown to be very potent initiators of recombination in yeast and other organisms, we investigated the molecular mechanisms involved in the repair of a targeted, I-SceI enzyme-induced DNA DSB in C. albicans. Our data indicated that most of the I-SceI-induced DNA DSBs are repaired by gene conversion in C. albicans. Interestingly, the precise characterization of the progenies that arose from a targeted DSB on Chromosome 4 (Chr4) or Chr7 led to the identification of recessive lethal and deleterious alleles on Chr4 and Chr7 of the C. albicans SC5314 laboratory strain. In addition, high-resolution analysis of an induced DNA DSB revealed template switching events and, unexpectedly, bidirectional homozygosis in C. albicans. Our data suggest that C. albicans is a valid model to study mechanisms involved in eukaryotic genome integrity.

235

P40C

The long pentraxin PTX3 regulates the pathogenic potential of Trichosporon asahii via differential induction of IL-10

Cláudio Duarte-Oliveira1 ,2, Samuel Gonçalves1 ,2, Fernando Rodrigues1 ,2, Cecilia Garlanda3, Gustavo Goldman4, Alberto Mantovani3 ,5, Agostinho Carvalho1 ,2, Cristina Cunha1 ,2 1Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal, 2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal, 3Humanitas Clinical and Research Center, Rozzano (Milan), Italy, 4Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil, 5Humanitas University, Rozzano (Milan), Italy

Most species of the Trichosporon genus are commensals of the human skin and gastrointestinal tract. Trichosporon asahii is however an increasingly recognized causative agent of superficial and invasive fungal disease in humans. How T. asahii interacts with and subverts the host immune system remains largely unknown. The long pentraxin 3 (PTX3) is a soluble pattern-recognition receptor and an essential component of the humoral arm of innate immunity. Because PTX3 plays a non-redundant role in resistance against selected microbial pathogens, we evaluated its contribution to the immune response to T. asahii by resorting to cellular and in vivo models of infection. We found that PTX3 expression was induced during infection and that PTX3 could bind the cell surface of blastoconidia from T. asahii. Accordingly, bone marrow-derived macrophages (BMDM) from Ptx3-/- mice and human macrophages from PTX3-deficient individuals displayed a marked defect in fungal internalization and killing. Cytokine production upon infection in these cells remained largely intact, apart from IL-10, which was almost undetectable. In a model of invasive trichosporonosis, Ptx3-/- animals were highly susceptible to disseminated infection, with increased fungal burdens in the liver, kidney, and spleen, and extensive inflammatory pathology in these target organs. Decreased levels of IL-10 were observed in organ

236

homogenates, whereas proinflammatory cytokines presented comparable levels to the wild-type controls. Finally, the treatment of Ptx3-/- animals with recombinant PTX3 reverted the IL-10 deficiency and susceptibility to trichosporonosis. Our results suggest that PTX3 regulates IL-10 production during T. asahii infection, eliciting mechanisms of antifungal resistance with controlled immunopathology.

237

P41C

An activating NLRP3 inflammasome mutation predisposes to fungal disease by impairing antifungal effector functions of macrophages

Samuel Gonçalves1 ,2, Cláudio Duarte-Oliveira1 ,2, Joana Gaifem1 ,2, Frank van de Veerdonk3, Fernando Rodrigues1 ,2, Egídio Torrado1

,2, Ricardo Silvestre1 ,2, Mihai Netea3, António Campos Jr.4, João Lacerda5 ,6, Cristina Cunha1 ,2, Agostinho Carvalho1 ,2 1Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal, 2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal, 3Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands, 4Serviço de Transplantação de Medula Óssea, Instituto Português de Oncologia do Porto, Porto, Portugal, 5Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisboa, Portugal, 6Serviço de Hematologia e Transplantação de Medula, Hospital de Santa Maria, Lisboa, Portugal

Fungal infections occur in over a billion people each year globally and recent evidence suggests that the rate is increasing. Despite significant progress in their diagnosis and treatment, invasive fungal infections are currently a leading cause of morbidity and mortality among immunocompromised hosts. Risk of infection and its clinical outcome vary considerably even among patients with similar predisposing clinical conditions, and recent evidence has implicated several genetic factors in the development of invasive aspergillosis (IA). Protection against fungi is conferred mainly through phagocytes that recognize pathogen motifs through pattern recognition receptors, including NOD-like receptors (NLRs). These molecules can form inflammasomes, a group of multimeric protein complexes consisting of an inflammasome sensor molecule, the adaptor ASC and caspase-1. Here, we have screened multiple NLR genes for nonsynonymous variants and analyzed associations with susceptibility

238

to IA in a multicenter cohort of stem-cell transplant recipients and their donors. We report an activating mutation in the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome leading to increased risk of infection. Mechanistically, the mutation was correlated with a striking fungicidal defect of macrophages, notwithstanding the enhanced levels of IL-1β produced upon fungal infection. The presence of the mutation paradoxically restrained the production of reactive oxygen species (ROS) elicited by infection, a cell-intrinsic mechanism likely in place to prevent further pathogenic NLRP3 activation, but that may compromise ROS-dependent clearing mechanisms, including LC3-associated phagocytosis. Our results suggest that hyperactivation of the NLRP3 inflammasome is detrimental to antifungal immunity and contributes to susceptibility to IA in immunocompromised patients.

239

P42C

Role of transcription factor Sef1 in regulating iron homeostasis in a mitochondrial biogenesis mutant in Candida albicans

Shivani Ror, Edwina Thomas, Sneh Lata Panwar School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India

In a previous study from our laboratory, we characterized FZO1, a key component of the mitochondrial biogenesis apparatus, in order to analyze the pleiotropic effects of dysfunctional mitochondria in C. albicans (Thomas E et al., 2013). Data gleaned from the transcriptome profiling of fzo1∆/∆ showed up regulation of genes involved in iron-uptake in iron replete conditions. In wild type cells, up regulation of iron-uptake genes generally occurs in situations of iron deprivation whereas these genes remain repressed under iron replete conditions. Several iron regulon genes (involved in iron assimilation/transport) induced in fzo1∆/∆ such as SIT1, RBT5, CFL2 and CFL5 are shown to be direct gene regulatory targets of the transcription factors, Sef1 and Sfu1 in C. albicans. Therefore, the objective of the current study is to delineate the regulatory mechanism of iron homeostasis in the fzo1∆/∆ mitochondrial mutant and explore its implications on associated cellular functions. Our study demonstrates a new mechanism of iron regulation, wherein, firstly we show that the iron levels are high in the fzo1∆/∆ cells, due to perturbed iron-sulfur cluster biogenesis. Secondly, we show that the defective iron-sulfur cluster biogenesis in fzo1∆/∆ cells serves as a signal for transcription factor, Sef1, enabling it to localize in the nucleus thereby causing constitutive expression of the iron regulon in this mitochondrial mutant. Furthermore, fzo1∆/∆ maintains significantly high levels of Sef1 protein under iron replete conditions, consistent with its nuclear localization indicative of an improper feed forward loop between Sef1, Sfu1 and the iron regulon genes.

240

P43C

Sialic acids on the surface of Aspergillus fumigatus conidia impacts pathogen recognition and anti-fungal response by PBMCs

Intan M Dewi1 ,2, Martin Jaeger1, Berenice Roesler1, Mark S Gresnigt1, Frank L van de Veerdonk1 1Radboud University Medical Center, Nijmegen, The Netherlands, 2Universitas Padjadjaran, Bandung, Indonesia

Aspergillus fumigatus is a ubiquitous fungus that causes invasive pulmonary aspergillosis, ABPA, and CPA. In the past decade there have been reports on IPA associated with influenza. Influenza virus harbors neuraminidase on its surface, and most of these patients had received neuraminidase-inhibitors during the course of the influenza infection.

Previous studies have identified the presence of sialic acids in A. fumigatus conidial surface, which mediate adhesion to fibronectin and basal lamina in pulmonary epithelial cells. We hypothesized that sialic acids could also be important for immune recognition of this pathogen. Our study aims to determine whether the removal of sialic acids from A. fumigatus conidia and the presence of neuraminidase-inhibitors affects the host’s immune response.

Peripheral blood mononuclear cells were stimulated with A. fumigatus conidia pre-treated with neuraminidase. In a different set of experiments, Oseltamivir was pre-incubated with PBMCs for 2 hours prior to stimulation with conidia. TNFα, IL-1β, and IL-6 were measured from culture supernatants after 24 hours of incubation at 37˚C. Removal of sialic acids of A. fumigatus conidia significantly increased the capacity to induce TNFα, IL-6 and IL-1β in human PBMCs. Reversibly, pre-exposure of PBMCs to neuraminidase-inhibitor prior to stimulation with live conidia decreased the cytokine response by these cells.

These findings indicated that sialic acid removal from A. fumigatus conidia prior to stimulation may increase pro-inflammatory cytokines by PBMCs and possibly affect the host’s anti-fungal defense. This study might help us to understand further about the role of fungal sialic acids in the host-pathogen interface.

241

P44C

Host factor adenosine modulates inflammatory responses in systemic candidiasis

Ava Hosseinzadeh1, Munender Vodnala1, Madhu Shankar1, Mattias Carlström2, Anders Hofer1, Constantin Urban1 1Umeå University, Umeå, Sweden, 2Karolinska Institute, Stockholm, Sweden

Invasive fungal infections may be accompanied by overwhelming inflammatory responses in the host, often at the time of immune recovery, resulting in immune reconstitution inflammatory syndrome (IRIS). In search for host factors which reduce inflammatory responses in systemic mycoses we investigated adenosine. The nucleoside is produced in many tissues, such as for instance by endothelium. Adenosine is considered as anti-inflammatory molecule conveying function via specific receptors, classified in four different subtypes A1, A2A, A2B and A3. The role of adenosine in regulating inflammatory processes during microbial infection is poorly understood. We showed that adenosine levels increase more than ten-fold in plasma of mice systemically infected with Candida albicans. Neutrophils are recruited to sites of C. albicans infection in large numbers and contribute to an inflammatory milieu by production of ROS, secretion of pro-inflammatory cytokines and release of neutrophil extracellular traps (NETs). Interestingly, we found that adenosine-treated neutrophils released less amounts of cytokines and NETs upon Candida stimulation. Adenosine-triggered regulation was mediated via adenosine receptor A3 expressed on neutrophils. The signal was further conveyed by PI3 kinase and protein kinase B (AKT). Pharmacological activation of A3 in a murine model of systemic candidiasis reduced inflammation and markers for organ damage in plasma. We are currently investigating A3 knockout mice for confirmation of pharmacological data. In summary, our findings identify adenosine as important modulator of inflammation during systemic candidiasis and suggest that pharmacological targeting of A3 receptor could be a promising adjunct therapy for IRIS during episodes of invasive mycoses.

242

P45C

Syk and CARD9: minor players in resistance to Candida parapsilosis?

Erik Zajta1, Adél Tóth1, Katalin Csonka1, Janka Zsófia Csepregi2, Tamás Németh2, Attila Mócsai2, Attila Gácser1 1Department of Microbiology, University of Szeged, Szeged, Hungary, 2Department of Physiology, Semmelweis University School of Medicine and MTA-SE ‘Lendület’ Inflammation Physiology Research Group of the Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary

An important antimicrobial intracellular pathway initiated by C-type lectin receptors leads through Syk and CARD9. Several major fungal pathogens activate this route. Candida parapsilosis is a regular cause of candidaemia and threatens especially neonates. Despite its clinical relevance, little is known about the immunological processes during C. parapsilosis infections. Our goal was to examine the role of Syk and CARD9 in C. parapsilosis infections. We generated bone marrow chimeras with wild type, Syk- or CARD9-deficient hematopoietic systems. Mice were infected intravenously with C. parapsilosis or C. albicans and fungal burden was determined from organs and blood. Peritoneal macrophages and bone marrow derived macrophages were cultured and infected with C. parapsilosis or C. albicans and supernatants were analysed for cytokine content and LDH activity. Phagocytosis of C. parapsilosis by macrophages and fungal survival in co-cultures were assessed. The absence of Syk or CARD9 in macrophages led to reduced IL-1β, TNFα and KC secretion upon C. albicans and C. parapsilosis infections. There was no difference in phagocytic activity between WT and CARD9-deficient BMDMs nor in their LDH release. Both Syk- and CARD9-deficient mice were highly susceptible to C. albicans. Notably however, Syk- and CARD9-deficient mice were only slightly more sensitive to C. parapsilosis. Our data suggest that both Syk and CARD9 influence C. parapsilosis induced host responses in vitro. However, unlike in the case of C. albicans, these proteins are not key players in the immune-control of systemic C. parapsilosis infection.

Zajta E was supported by the National Talent Programme (NTP-NFTÖ-16-0569).

243

Index

Presenter, Institution and email

Alaalm, Leenah (University of Bath) l.alaalm@bath.ac.uk P1CAllert, Stefanie (Hans-Knöll-Institute) stefanie.allert@leibniz-hki.de P12BAmich, Jorge (University of Manchester) jorge.amichelias@manchester.ac.uk

P2C

Andrianaki, Angeliki M. (University of Crete) aggandr@yahoo.gr P30CAndrianopoulos, Alex (University of Melbourne) alex.a@unimelb.edu.auArmstrong-James, Darius (Imperial College London) d.armstrong@imperial.ac.uk

P25B

Bader, Oliver (University Medical Center Goettingen) obader@gwdg.de

P20B, P20C

Ballou, Elizabeth (University of Birmingham) e.r.ballou@bham.ac.uk S2/5Bantel, Yannick (University of Stuttgart) yannick.bantel@igb.fraunhofer.de

P37A

Basmaciyan, Louise (UMR PAM, Univ Bourgogne-Franche Comté)louise.basmaciyan@chu-dijon.fr

P45B

Batarce, Christian R (Universidad de Chile) christianbafa@hotmail.com

P33A

Bennett, Richard (Brown University) richard_bennett@brown.eduBerman, Judith (Tel Aviv University) judithberman11@gmail.com S2/1Beyer, Reinhard (University of Natural Resources and Life Sciences, Austria) reinhard.beyer@boku.ac.at

P39B

Bezerra, Ana Rita (University of Aveiro) armbezerra@ua.pt P38CBignell, Elaine (University of Manchester) elaine.bignell@manchester.ac.uk

P19A

Böttcher, Bettina (Hans Knöll Institute) bettina.boettcher@leibniz-hki.de

P25C

Brand, Alexandra (University of Aberdeen) a.brand@abdn.ac.uk S6/1Brown, Alistair J.P. (University of Aberdeen) al.brown@abdn.ac.uk K1Brunke, Sascha (Hans Knöll Institute) sascha.brunke@hki-jena.de P30B

Buda De Cesare, Giuseppe (University of Aberdeen) giuseppe.budadecesare@abdn.ac.uk

P7C

244

Buscaino, Alessia (University of Kent) A.Buscaino@kent.ac.uk P25AButler, Geraldine (University College Dublin) gbutler@ucd.ie S1/1Camilli, Giorgio (Institut Pasteur) giorgio.camilli@pasteur.fr P18CCaplan, Tavia (University of Toronto) tavia.caplan@mail.utoronto.ca P10ACarvalho, Agostinho (University of Minho) agostinhocarvalho@med.uminho.pt

S5/4

Cassilly, Chelsi (University of Tennessee, Knoxville) ccassill@utk.edu P10BCavalheiro, Mafalda (University of Lisbon) mafalda_cavalheiro@outlook.com

P26B

Chamilos, Georgios (University of Crete, and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology) hamilos@imbb.forth.gr

S5/3

Chang, Zanetta (Duke University, USA) zc37@duke.edu P8BChauhan, Neeraj (Rutgers, the State University of New Jersey) chau-han1@njms.rutgers.edu

P41B

Chen, Jiangye (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) jychen@sibcb.ac.cn

P5C

Childers, Delma (University of Aberdeen) delma.childers@abdn.ac.uk

P23B

Cornet, Muriel (CHU Grenoble Alpes) mcornet@chu-grenoble.fr P34ACosta, Catarina (University of Lisbon) catarinaspcosta@gmail.com P41ACrawford, Aaron (University of Aberdeen) a.crawford@abdn.ac.uk P18ACsonka, Katalin (University of Szeged) katalin.csonka88@gmail.com P44BCunha, Cristina (University of Minho) cristinacunha@med.uminho.pt

P38A

d’Enfert Christophe (Institut Pasteur) denfert@pasteur.frDahman, Yacine (Institut de Biologie Moleculaire et Cellulaire, UPR 9002) yacine.dahman@gmail.com

P29B

Dai, Baodi (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) bddai@sibcb.ac.cn

P7A

Dawson, Thomas (Agency for Science, Technology, and Research, Institute of Medical Biology) thomas.dawson@imb.a-star.edu.sg

S4/2

Delarze, Eric (University of Lausanne and University Hospital Center) eric.delarze@chuv.ch

P36C

Denis, Julie (Université de Strasbourg) julie.denis@chru-strasbourg.fr

P29A

245

Dewi, Intan M (Radboud University Medical Center) intan.dewi@radboudumc.nl

P43C

Donovan, Paul (University College Dublin) 09470158@ucdconnect.ie

P11A

dos Santos, Suelen (Universidade de São Paulo) suelen.silvana.santos@gmail.com

P17A

Dromer, Francoise (Institut Pasteur) francoise.dromer@pasteur.fr S3/2du Pré, Saskia (F2G Ltd) sdupre@f2g.com P24BDuarte-Oliveira, Cláudio (University of Minho) claudiooliveira@med.uminho.pt

P40C

El Ghalid, Mennat (Institut Pasteur) el-ghalid@pasteur.fr P22CFairhead, Cecile (Université Paris Sud) cecile.fairhead@u-psud.fr P23CFogaça de Almeida, José Roberto (University of São Paulo) joalmeida@usp.br

P45A

Förster, Toni (Hans-Knöll-Institut) toni.foerster@leibniz-hki.de P4CGaffen, Sarah (University of Pittsburgh) sarah.gaffen@pitt.edu S5/1Garbe, Enrico (Septomics Research Center, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute) enrico.garbe@hki-jena.de

P15B

Garcia-Rodas, Rocio (Université Côte d›Azur, CNRS, Inserm, iBV) rocio.rodas@gmail.com

P14C

Gonçalves, Samuel (University of Minho) samuel@med.uminho.pt P41CHaas, Hubertus (Medical University Innsbruck) hubertus.haas@i-med.ac.at

S2/3

Hall, Rebecca (University of Birmingham) r.a.hall@bham.ac.ukHuang, Guanghua (Institute of Microbiology, Chinese Academy of Sciences) huanggh@im.ac.cn

P9A

Husain, Farha (Jawaharlal Nehru University) farhahusain786@gmail.com

P43B

Icheoku, Uju Joy (University of Manchester) uju.icheoku@postgrad.manchester.ac.uk

P12A

Iracane, Elise (University College Dublin) elise.iracane@ucd.ie P31AJabra-Rizk, Mary Ann (University of Maryland) mrizk@umaryland.edu

P16A, P19B

Jacobsen, Ilse (Leibniz Institute for Natural Product Research and Infection Biology) ilse.jacobsen@hki-jena.de

P3A

246

Janbon, Guilhem (Institut Pasteur) janbon@pasteur.fr S1/4Jannuzzi, Grasielle (University of São Paulo) grasi_jannuzzi@hotmail.com

P16C

Jenull, Sabrina (Medical University of Vienna) sabrina.jenull@meduniwien.ac.at

P39A

Kämmer, Philipp (Leibniz Institute for Natural Product Research and Infection Biology) philipp.kaemmer@leibniz-hki.de

P26C

Kapitan, Mario (Hans-Knöll-Institute) mario.kapitan@hki-jena.de P21BKlein, Bruce (University of Wisconsin-Madison) bsklein@wisc.edu S6/2Koch, Barbara (Monash University) barbara.koch@monash.edu P8CKoh, Andrew (University of Texas Southwestern Medical Center) andrew.koh@utsouthwestern.edu

S4/1

Kousser, Courtney (University of Birmingham) cak500@bham.ac.uk P42AKrappmann, Sven (University Hospital Erlangen and Friedrich-Alexan-der University) sven.krappmann@uk-erlangen.de

S6/3

Krysan, Damian (University of Rochester) damian_krysan@urmc.rochester.edu

S3/1

Kurzai, Oliver (Hans-Knoll Institute) oliver.kurzai@leibniz-hki.deLabbaoui, Hayet (Université Côte d’Azur, CNRS, Inserm, iBV) hlabbaoui@unice.fr

P11B

Lee, Keunsook Kathy (University of Aberdeen) k.k.lee@abdn.ac.uk P29CLegrand, Mélanie (Institut Pasteur) mlegrand@pasteur.fr P39CLeibundgut, Salome (Universtiy of Zurich) salome.leibundgut-landmann@uzh.ch

P3C

Leong, Cheryl Kit Mun (University and University Hospital Zurich) lkmcheryl@gmail.com

P6B

Lim, Fang Yun (University of Wisconsin-Madison) fylim@wisc.edu P24CLombardi, Lisa (University College Dublin) lisa.lombardi@ucd.ie P27ALongo, Larissa (Universidade Federal de São Paulo) larissa_guilhen@yahoo.com.br

P17B

Lopes, Pedro (Umeå University) pedro.lopes@umu.se P32BLorenz, Michael (University of Texas McGovern Medical School) mi-chael.lorenz@uth.tmc.edu

S2/2

Lyons, Naomi (University of Bath) naomislyons@gmail.com P34CMa, Li-Jun (University of Massachusetts Amherst) lijun@biochem.umass.edu

S1/3

247

MacCallum, Donna (University of Aberdeen) d.m.maccallum@abdn.ac.ukMalavia, Dhara (University of Aberdeen) d.malavia@abdn.ac.uk P4AMartin, Ronny (Septomics Research Center, Leibniz Institute for Natural Product Research and Infection Biology –Hans Knöll Institute) ronny.martin@leibniz-hki.de

P14A

Martin-Yken, Helene (LISBP INRA Universite de Toulouse) helene.martin@insa-toulouse.fr

P3B

McCann, Bethany (University of Manchester) bethany.mccann@postgrad.manchester.ac.uk

P35A

Mody, Christopher (University of Calgary) cmody@ucalgary.ca S5/5Mogavero, Selene (Hans Knöll Institute) selene.mogavero@leibniz-hki.de

P5A

Montelongo-Jauregui, Daniel (The University of Texas at San Antonio) daniel.montelongo@utsa.edu

P8A

Morschhäuser, Joachim (University of Würzburg)joachim.morschhaeuser@uni-wuerzburg.de

P27C

Munro, Carol (University of Aberdeen) c.a.munro@abdn.ac.ukNaglik, Julian (King’s College London) julian.naglik@kcl.ac.uk S6/5Nemeth, Tibor Mihaly (University of Szeged) narvaltm@gmail.com P43ANielsen, Kirsten (University of Minnesota) knielsen@umn.edu S6/4Niemiec, M. Joanna (Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Insitute) joanna.niemiec@leibniz-hki.de

P21C

Novohradská, Silvia (Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute) silvia.novohradska@hki-jena.de

P5B

O’Hanlon, Simon (Imperial College London) simon.ohanlon@imperial.ac.uk

P32C

Otieno-Odhiambo, Patricia (University of Cape Town) otnpat001@myuct.ac.za

P23A

Pais, Pedro (University of Lisbon) pedrohpais@tecnico.ulisboa.pt P18BPapp, Csaba Gergo (University of Szeged) papp.cs66@gmail.com P40BPavelka, Norman (Agency for Science, Technology, and Research, Singapore) norman_pavelka@immunol.a-star.edu.sg

S1/5

Pentland, Daniel (University of Kent) drp25@kent.ac.uk P27B

˝

248

Petosa, Carlo (Université de Grenoble Alpes, CEA, CNRS) carlo.peto-sa@ibs.fr

S3/4

Polke, Melanie (Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute) melanie.polke@hki-jena.de

P34B

Prasad, Priya (Indian Institute of Technology, Bombay) priya8810@gmail.com

P4B

Price, R. Jordan (University of Kent) rp426@kent.ac.uk P22BPuccia, Rosana (Universidade Federal de São Paulo) ropuccia@gmail.com

P30A

Puerner, Charles (Université Côte d›Azur, CNRS, Inserm, iBV) charles.puerner@unice.fr

P37C

Quintin, Jessica (Institut Pasteur) jessica.quintin@gmail.com S5/2Ramírez-Zavala, Bernardo (University of Würzburg) b.ramirez@uni-wuerzburg.de

P28B

Richard, Mathias (INRA, Micalis Institute) mathias.lavie-richard@inra.fr

S4/3

Richardson, Jonathan (King’s College London) jonathan.richardson@kcl.ac.uk

P32A

Rogiers, Ona (VIB-KU Leuven Center for Microbiology, KU Leuven Laboratory of Molecular Cell Biology) ona.rogiers@kuleuven.vib.be

P13B

Rokas, Antonis (Vanderbilt University) antonis.rokas@vanderbilt.edu

S1/2

Romani, Luigina (University of Perugia) luigina.romani@unipg.it

S4/4

Ror, Shivani (Jawaharlal Nehru University) shivaniror20@gmail.com

P42C

Ruben, Sophia (Septomics Research Center, Leibniz Institute for Natural Product Research and Infection Biology –Hans Knöll Institute) sophia.ruben@leibniz-hki.de

P13C

Rupp, Steffen (Fraunhofer IGB) steffen.rupp@igb.fraunhofer.de

P35B

SABOU, Marcela (University of Strasbourg) amsabou@unistra.fr

P19C

Sanglard, Dominique (University of Lausanne and University Hospital) dominique.sanglard@chuv.ch

P36B

249

Schaefer, Katja (University of Aberdeen) katja.schaefer@abdn.ac.uk

P9C

Sen, Rishika (Indian Statistical Institute) rishika21sen@gmail.com P1BSherrington, Sarah (University of Birmingham) s.sherrington@bham.co.uk

P28C

Shevchenko, Marina (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS) mshevch@gmail.com

P28A

Sil, Anita (University of California San Francisco) anita.sil@ucsf.edu S2/4Silao, Fitz Gerald (Stockholm University) fitzgerald.silao@su.se P42BSilva, Patrícia M. (Université Côte d›Azur, CNRS, Inserm, iBV) pmdeoliveira@unice.fr

P16B

Sohn, Kai (Fraunhofer IGB) kai.sohn@igb.fraunhofer.de P44ASpyrou, Maria (University of Aberdeen) m.spyrou@abdn.ac.uk P24ATams, Robert (University of Tennessee) rtams@vols.utk.edu P10CTan, Tze Guan (Singapore Immunology Network, Agency for Science, Technology, and Research, Singapore) tgtan01@gmail.com

P40A

Tapia, Cecilia V (Universidad de Chile) cvtapiap@gmail.com P33BTeixeira, Miguel C. (University of Lisbon) mnpct@tecnico.ulisboa.pt P9BThevissen, Karin (CMPG, KU Leuven) karin.thevissen@kuleuven.be S3/3Traven, Ana (Monash University) ana.traven@monash.eduTroyanova, Natalia (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS) troyanatali@gmail.com

P26A

Trzaska, Wioleta (University of Birmingham) w.j.trzaska@bham.ac.uk

P14B

Tucey, Timothy (Monash University) timothy.tucey@monash.edu P35CUrban, Constantin (Umeå University) constantin.urban@umu.se P44CUsher, Jane (University of Exeter) J.Usher@exeter.ac.uk P1AVan Dijck, Patrick (VIB-KU Leuven Center for Microbiology) patrick.vandijck@kuleuven.vib.be

P20A

Van Genechten, Wouter (VIB-KU Leuven Center for Microbiology) wouter.vangenechten@kuleuven.vib.be

P38B

van Rhijn, Norman (University of Manchester) norman.vanrhijn@manchester.ac.uk

P6C

van Wijlick, Lasse (Institut Pasteur) lasse.vanwijlick@pasteur.fr P15CVeri, Amanda (University of Toronto) amanda.veri@mail.utoronto.ca

P33C

250

Verma, Jiyoti (Monash University) jiyoti.verma.gaur@monash.edu P35AVesely, Elisa (University of Texas Health Science Center at Houston) elisa.vesely@uth.tmc.edu

P31C

Vidan, Nikolina (Ruder Boskovic Institute) nikolina.vidan@gmail.com

P15A

Vylkova, Slavena (The University of Texas Health Science Center at Houston) slavena.vylkova@hki-jena.de

P2A

Wakade, Rohan S. (Universite Cote d’Azur, CNRS, Inserm, ibV) rohan.wakade@unice.fr

P12C

Wallace, Edward (The University of Edinburgh) ewjwallace@gmail.com

P13A

Wang, Wenjuan (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) wangwenjuan@sibcb.ac.cn

P7B

Wheeler, Robert (University of Maine) robert.wheeler1@maine.edu P2BWilson, Duncan (University of Aberdeen) duncan.wilson@abdn.ac.uk

P17C

Yang, He (University of Massachusetts Amherst) heyang@mcb.umass.edu

P21A

Yellagunda, Sujan (Umea University) sujan.yellagunda@umu.se P31BZajta, Erik (University of Szeged) writetotono@gmail.com P45CZawrotniak, Marcin (Jagiellonian University) marcin.zawrotniak@uj.edu.pl

P37B

Zhang, Xing (Centre d’Immunologie de Marseille) xzhang@ciml.univ-mrs.fr

P22A

Zhu, Wencheng (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) zhuwencheng2014@sibcb.ac.cn

P6A