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5/15/2017

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Robert A. Cramer

Associate Professor Microbiology and Immunology

MSG ERC Asilomar September 22nd, 2016

Treatise on Air and Fire – 1775 ( pub. 1777) Carl

Wilhelm Scheel

Swedish Pharmacist Carl Wilhelm

Scheel – O2 experiments in 1772

1775 – An Account of

Further Discoveries in

Air – Joseph Priestly

"The feeling of it to my lungs was

not sensibly different from that of

common air, but I fancied that my

breast felt peculiarly light and easy

for some time afterwards.”

-Mice incubated in this

“dephlogisticated air’were more

active and lived longer

Rev. Joseph Priestly, Portrait by

Ellen Sharples 1794

Fungi, Oxygen, and Evolution

Figure Modified From: Thannickal, 2009, Am J. Respir Cell Mol Biol 40, 507-10

Fungi Diverge

from animalsFungal

Explosion

Ascomycota

and Basidiomycote

Diverge

Watercolor Painting

By: July Sigma

Eh (Redox Potential) and pH Largely Determine the

Members of Microbial Communities

Billen 1973, Stumm 1966, Heintze 1934, Kimbrough et al. 2006, Seo and DeLaune 2010

Eh usually measured in millivolts. Eh measurements were common and important in the

early investigations (pre-molecular genetics) era of Microbiology/Microbial pathogenesis.

-Strong Reducing Potentials were often associated with microbial virulence

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Image Modified From:Abad et al. 2010 Rev Iberoam Micol. 2010 27(4)

Temporal Aspects of Microbial Pathogenesis are critical --- when/how you intervene

Most Healthy Tissue Eh Values are POSITIVE = Slightly Oxidizing, Robust Immune Function = Resistance or tolerance

Most Damaged Tissue Eh Values are NEGATIVE = REDUCING

Chemotherapy, immune

modulation, genetic

mutations, alters Redox

Homeostasis

Redox Homeostasis

Redox Imbalance

Host Environments

Immune Response

Ho

st D

amag

e

Why are certain microbes “Opportunistic” Pathogens?

Hypothesis: Microbial Bioenergetics = Virulence Rheostat

Host Genetics and Immune Responses Drive/Maintain Redox Tissue Homeostasis to Prevent Host Damage and

microbe proliferation

”Complementary Bioenergetics”

Met

abo

lic P

ow

er

Microbial and Host Bioenergetics: A Key to Improved

Infectious Disease Outcomes?

Aspergillus fumigatus produces Ethanol During Mammalian

Infection !

Uninfected Mouse

Collaborators: Dr. Jeffrey Macdonald and Dr. Michael Gamcsik UNC

Aspergillus Inoculated Animals

Mock (PBS) Inoculated Animals

Grahl et al. 2011, PLoS Pathogens 7

Tissue Damage/Inflammation Leads to Hypoxia and

Low Eh values

Healthy Lung – pO2 = 100 – 110 mm Hg

Resident Macrophages

Diseased Lung – pO2 = severe drop, hypoxia,

2.5 mm Hg CF !

Recruited Macrophages, Neutrophils

Mucus, Pus, Edema, Vascular leakage

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Hypoxia Occurs In Vivo During

Invasive Pulmonary Aspergillosis

Grahl et a. 2011 PLoS Pathogens

Steroid Murine Model of IPA

Hypoxyprobe = can detect ≤ 1.5% Oyxgen levels in tissue.

Steroid treatment and fungal inoculation have large

effects on host metabolism

Significantly changed

metabolites

Steroid only

Healthy control

Steroid +

FungusSteroid only

Total metabolites

(p≤0.05)

242 168

Metabolites () 175|67 34|134

630 detected metabolites

LIPIDS dramatically increase with steroid treatmentWorking Hypothesis: Hypoxic Fungal Bioenergetics

are detrimental to immune compromised hosts

Dying Achilles at Achilleion, Corfu Greece, Ernst

Herter 1884

Aspergillus fumigatus

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Hypoxia induces significant changes in the transcriptome of


Collaborators: Dr. William Nierman, Dr. Liliana Losada et al. JCVI

8025

genes

1416

genes

522

genes

Genes with < 4 fold change

Genes with Increased mRNA Abundance ≥ 4

fold

Genes with decreased mRNA Abundance ≥ 4

fold

7114

genes

2166

genes

683

genes

Hypoxia alters transcript levels for ~30% of the

genome in 30 minutes!

Hy

po

xia

CEA10 Af293 W72310 47-4 47-57 47-10

No

rmo

xia

Defining the Aspergillus fumigatus

hypoxia transcriptome

1. Shake Flask Cultures 2. Chemostat Cultures

A. Microarray and B. RNA-SEQ C. Proteomics

Hypoxia Increases Transcripts/Proteins For:

1. Ergosterol Biosynthesis

2. Amino Acid Metabolism

3. Respiration – early time points !?

4. Metal Uptake

5. Various Transporters

6. Cell Wall biosynthesis

7. Fermentation Pathways

8. Transcriptional Regulators

Hypoxia Reduces Transcripts/Proteins For:

1. Ribosome Proteins

2. Nucleic Acid biosynthesis

3. Cell Wall biosynthesis

4. Transcriptional Regulators

5. Various Transporters

Collaborators: Natalie Federova, Bill Nierman, Remert Pieper, Olaf Kniemeyer, Martin Voedisch

AFUB_024220 'C6 transcription factor (G5) 6.636885221 27.36168536 2.043578867

AFUB_034630'fungal specific transcription factor (2A5) 1.656582934 6.882257869 2.054671518

AFUB_031000 'AflR-like C6 transcription factor (H8) 0.424739497 1.76815379 2.057593584

AFUB_018340 HLH transcription factor, SrbA (F1) 12.19969841 54.11056239 2.149064752

AFUB_043270 C6 transcription factor Fcr1, (2D1) 97.573105 492.4017983 2.335280587

AFUB_025200fungal specific transcription factor(lethal?) 22.75310161 114.8376241 2.335460262

AFUB_086990 C6 transcription factor, (3G12) 4.038159802 20.67627293 2.356206243

AFUB_091540 C2H2 finger domain protein (5H6) 9.745031892 50.88073792 2.384380782

AFUB_097300fungal specific transcription factor (4B3) 0.272391627 1.516017782 2.476532412

AFUB_004210 C6 transcription factor, (A7) 6.873424829 40.34945825 2.553448267

AFUB_096150transcriptional activator (PtaC), (lethal?) 38.78129556 260.4551517 2.747602068

AFUB_019830 C2H2 finger domain protein (5E12) 20.44382959 145.6763684 2.833029486

AFUB_046410 C6 transcription factor Ctf1B-like (2E7) 13.02379062 97.66512354 2.906694053

AFUB_099050 C6 transcription factor Ctf1A, (4B7) 22.81782155 173.0661882 2.923090927

AFUB_088380 C6 transcription factor, (3H4) 1.846041506 16.41415958 3.152433989

AFUB_079810fungal specific transcription factor (3E10) 3.977779026 46.06768714 3.533720129

AFUB_037000 C6 transcription factor (2B1) 15.49840347 212.1873158 3.775146904

AFUB_088390 C6 transcription factor (3H5) 0.88465823 17.06634813 4.269890365

AFUB_013240 C2H2 transcription factor,Rpn4, (C9) 11.07227024 221.6558196 4.323298276

AFUB_099590HLH DNA binding domain protein, SrbB (6A5) 147.1849553 3234.70886 4.457933751

AFUB_078160 C6 transcription factor (3E4) 3.44754916 83.34817509 4.595507583

RPKM N RPKM H Log2 H/N

21 transcription factors induced by initial hypoxia exposure

SrbA, a SREBP ortholog

in A. fumigatus

Hypothesis: SrbA is a

major regulator of


hypoxia bioenergetics

and fitness

A. fumigatus SrbA is required for hypoxia fitness

and pathogenicity

Willger et al. 2008, PLoS Pathogens

WT

ΔsrbA

ΔsrbA + srbA

1% O2

Mock

WT

ΔsrbA

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SrbA is Required for Fluconazole and Triazole Drug Tolerance

Willger et al. 2008, PLoS Pathogens, Blosser and Cramer 2012 AAC

CEA10 srbA srbA + srbA

Fluconazole

Voriconazole

How does SrbA Mediate Hypoxia Fitness,

Virulence and Drug Tolerance?

DEFINE THE SrbA GENETIC

NETWORK

• Sterol Biosynthesis

• Iron Acquisition

• Oxygen Consumption

• Heme Biosynthesis

• Carbon/Nitrogen Metabolism

ChIP SrbA Ab WT

Input Control WT

SrbA/ SrbB Target Gene Alcohol Dehydrogenase (AlcC) is

important for virulence

*

*

CEA10 ∆alcC

Day 3

Day 4

Grahl et al. 2011, PLoS Pathogens

Hypoxic driven fermentation is

immunosuppressive and contributes to in vivo

fungal growth

Fungal SREBPs coordinate Hypoxia Bioenergetics to

Promote Fungal Virulence

VIRULENCE

CreA

Chung, Barker et al. 2014 PLoS Pathogens Nov 6;10(11) Beattie et al. 2016, In Revision

Rationale: Fungal Specific SREBP Targets and/or Regulatory factors ==

promising antifungal drug targets

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A population of five distinguishable

environmental isolates at 0.2% O2

Significant Genotypic and Phenotypic

Heterogeneity Exists in the A. fumigatus

population

Kowalski, Beattie, et al. 2016 mBio In Press

Virulence Heterogeneity of Aspergillus fumigatus

Strains

Kenalog Murine Model

Is this at all linked to the “bioenergetics” of the

Strains?

Hypoxia Fitness is Heterogeneous and Correlates with

Virulence

2 4 6 8 10 12 140.0

0.1

0.2

0.3 r = -0.7867

p=0.0003

Median Survival

H/N

Ra

tio

Environmental Isolates

2 4 6 8 10 12 140.00

0.05

0.10

0.15

0.20

0.25

0.30 r = -0.7031p=0.0268

H/N

Ra

tio

Clinical Isolates

2 4 6 8 10 120.00

0.05

0.10

0.15

0.20

0.25

0.30 r = -0.9429p= 0.0167

Median Survival

H/N

Ra

tio


Experimental evolution is a tool to identify

mechanism of hypoxia adaptation and virulence

Attenuated

WT strain

“Host-like”

Conditions(0.2% O2, glucose minimal medium)

Survival

Experiment

Caitlin Kowalski, In Preparation

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After 20 passages (3 months) …..

EVOL20 Hypoxic Fitness Increases

AF293

EVOL20 AF293

EVOL20

0.0

0.1

0.2

0.3

0.4

0.5

0.00

0.01

0.02

0.03

0.04

Normoxia Hypoxia

Strain:

Condition:

***

*

Dry

We

igh

t (g

) Dry

We

igh

t (g)


…. and increased virulence!105 Dose in CD-1 Corticosteroid Model with Conidia from Reducing Conditions

0 5 10 150

20

40

60

80

100Mock

AF293

EVOL20

*Mantel-Cox Test p = 0.0296*Gehan-Breslow-Wilcoxon Test p = 0.0332

Days Post Infection

Pe

rce

nt s

urv

iva

l

AF29

3

EVO

L20

0.00

0.05

0.10

0.15

Ra

tio

of H

/N B

iom

ass

105 conidia I.N.

*


EVOL20 consumes less oxygen per minute

than the parental strain

0 20 40 600

100

200

300

400

500

Oxygen consumption in strains

AF293

EVOL20

10 mM

Oligomycin

Time (minutes)

OC

R (p

mo

les

/min

)

Optimization of Bioenergetics in Low

Redox/Oxygen Conditions ---

promotes fungal proliferation

Can the infection site microenvironments be manipulated

to alter fungal and host metabolism to reduce damage?

Collaboration Dr. Jay Buckey, Dartmouth

Hitchcock Hyperbaric Medicine

Hypoxia

Host

DAMAGE

Fungal PathogenesisImmuno-

Pathogenesis

+Hyperbaric

Oxygen

Improved Invasive

Fungal Infection

Outcomes?*Expand the therapeutic window?

*Fungicidal activity?*Immunomodulatory?

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Hyperbaric Oxygen Shuts down in vitro fungal

metabolism and slows growth

Sourabh Dhingra, PhD., unpublished data

XTT Metabolic Activity

Assay

Effect of HBO on survival in leukopenic model of

IPA

Survival analysis

0 5 10 150

50

100

150Norm Mock

HBO Mock

Norm Infected

HBO Infected

Days PI

Pe

rce

nt su

rviv

al

*P = 0.03

Sourabh Dhingra, PhD Post-Doctoral Fellow unpublished data

Hyperbaric oxygen Combination Therapy?

Survival of Data 1:Survival proportions

0 5 10 150

50

100

150Norm WT

Norm SOD

Days PI

Pe

rce

nt su

rviv

al


Superoxide Dismutase is Critical for

Growth in Normoxia

WT

△SOD

Norm Hypoxia N H


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SOD Mutant Is More

Fit in Hypoxia

Combination of Oxidative Stress Mutant and

HBO significantly reduces murine mortality

Norm SOD vs HBOT SOD:Survival proportions

0 5 10 150

50

100

150Norm SOD

HBOT SOD

Days PI

Pe

rce

nt su

rviv

al

*P = 0.04

HBO therapy

stopped


*P = 0.004

Redox Homeostasis

Redox Imbalance

Host Environments

Immune Response

Ho

st D

amag

e

Why are certain microbes “Opportunistic” Pathogens?

Hypothesis: Modulation of Fungal AND/HOST bioenergetics will improve IFI treatment outcomes

FUNGUS HYPOXIA

Red

uci

ng

Pow

er

Microbial and Host Bioenergetics: Therapeutic Opportunity

Grahl et al. PLoS Pathogens

Acknowledgements

Collaborators on these data:

• Dartmouth

– Dr. Chao Cheng

– Dr. Jay Buckey and laboratory

– Dr. Joshua Obar

– CCMR Staff

– Immune Monitoring CORE

• Memorial Sloan Kettering

– Dr. Tobias Hohl and Dr. Yin-Wei Tang

• Univ. California Riverside

– Dr. Jason Stajich

• Strains – Shawn Lockhart CDC, Mihalis LionakisNIH, Paul Dyer Nottingham, Jean Paul Latge, Pasteur

• Duke University Medical Center

– Dr. John Perfect

• The Cramer Lab:

•Arsa Thammahong, Ph.D. Candidate., Sarah Beattie, Ph.D. Candidate, SourabhDhingra, Ph.D. , Caitlin Kowalski, Ph.D. Candidate, Nancy Pohl, Ph.D. Student, Katie Bultman Lab Technician

•Former lab members that contributed to these data: Sven Willger, Bridget Barker, Ph.D, Dawoon Chung, Ph.D., Ph.D., Nora Grahl, Ph.D., Jean Cornish, Ph.D. Sara Blosser, Ph.D. and Kelly Shepardson, Ph.D.

Funding:

Geisel School of Medicine, Dept. Microbiology and Immunology

Dartmouth Lung Biology Center

Funded by a CFF RDP and the NIH

IDeA Program

Investigator in the Pathogenesis of Infectious

Diseases

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