Virus and host interaction: lipidomics and applications

Ampa Suksatu, Ph.D.Department of Microbiology

Faculty of Science, Mahidol University

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Family Flaviviridae• 2 important genera: Flavivirus, and

Hepacivirus (HCV)• Enveloped with a single positive-sense RNA

• Flavivirus genus consists of >50 species, many of them are arthropod-borne human pathogens

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Amplifying host Vector

Birds Mosquito (Culex)

Mosquito (Aedes)

Primates

TickRodents

Flavivirus

Cellular lipids & Flavivirus life cycle

3Martín-Acebes MA. et al. Prog Lipid Res. 2016;64:123-137.

Vesicle packet

Vesicle packets

Vesicle packet

Convoluted membrane

virion

Flavivirus infection and lipid metabolism(cell culture-based)

• WNV infected cells increased proportion of glycerophospholipids and sphingolipids (Martin-Acebes et al. 2014)

• WNV virions enriched in sphingolipids and reduced PC (Martin-Acebes et al., 2014)

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• DENV infections altered size and abundance of lipid droplets (Heaton and Randell 2010, Zhang et al. 2018)

• Evidence of alterations in lipid profiles of mosquito cell lines and midgut cells in response to DENV infection (Perera et al. 2012, Chotiwan et al. 2018)

• DENV NS3 recruited FASN to the replication site, inhibition of FASN reduced viral infection and virion production (Heaton at al. 2010, Tongluan et al., 2017)

Multiple connections of flaviviruseswith lipid metabolism

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Flavivirusinfection

Intracellular membrane remodeling

(ER)

Interaction with

peroxisomes

Interaction with lipid droplets

Regulation of cellular

lipid metabolism

Modulation of

autophagy(lipophagy)

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Cholesterol metabolism

Sphingolipid metabolism

GPL metabolism

Fatty acids

Innate immune evasionMembrane rearrangement

Virion morphogenesis

Energy sourceLipid biosynthesisViral replication

Virion morphogenesisMembrane rearrangement

Virion morphogenesisInnate immunity

Martín-Acebes MA. et al. Prog Lipid Res. 2016;64:123-137.

Flavivirus interaction with lipid metabolism

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Metabolomics & Lipidomics• Aim to study the global changes of small molecule metabolites

and lipids in biological systems in response to biological stimuli or perturbations

• Provide global views of metabolome and lipidome landscapes and implicate metabolic to disease phenotype

1. Potential serum biomarkers of infection/severity

2. Platform of antiviral strategies

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Applications:

Mass Spectrometer(DIMS/LC-MS/GC-MS)

2. Ion separation

1. IonizationElectrospray ionization (ESI) – great for polar

Atmospheric pressure chemical ionization (APCI) – great for nonpolar

There is a trade-off between speedand mass accuracy/resolution

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Ion separation, the big 3 techniques

- Very fast (up to 500 spectra/sec)

- Low mass resolution- Low mass accuracy

Quadrupole

- Fast (10-100 spectra/sec)

- Medium/high mass resolution

- Excellent mass accuracy

Time-of-flight(TOF)

- Slow (1-20 spectra/s)- Very high resolution- Excellent mass

accuracy

Orbitrap

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Untargeted vs. Targeted MS

- Analysis of all the measurable metabolites in a sample, including chemical unknowns

- Usually applied to hypothesis generation

- Rely on high resolution, accurate mass measurement and fragmentation data

- Suitable for Q-TOF/Orbitrap type instrument

Untargeted- Focus on specific compounds of

interest, which means you already know them

- Usually applied to validate novel discovery of a hypothesis

- Rely on characteristic fragmentation of molecules

- Low resolution instruments such as triple quadrupole (QQQ)

Targeted

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Selected metabolites from publications on human infections with DENV

11Byers NM. et al. Viruses. 2019;11(3). pii: E225.

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Healthy Febrile Defervescence convalescenceTemporal serum metabolome profiles with DENV infection

Metabolome was significantly perturbed

during early acute phaseand normalized to control

levels at convalescent stage

Prognostic markers of the disease

60 metabolites

Cui L. et al. PLoS Negl Trop Dis. 2013;7(8):e2373.

Q-TOF

Correlation of serum lipids with specific serological parameters

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SM => Early host response lysoPE => Late recovery response

Cui L. et al. PLoS Negl Trop Dis. 2013;7(8):e2373.

QQQ

Total and LDL-cholesterol levels are lower in serum of patients with severe dengue

14Lima W.G. et al. Rev Med Virol. 2019;29(5):e2056.

Selected metabolites from publications on CHIKV, WNV, and ZIKV

15Byers NM. et al. Viruses. 2019;11(3). pii: E225.

Lipid biomarkers from serum analysis of ZIKV-infected patients

16Melo CFOR. et al. Front Microbiol. 2017;8:1954.

DIMS Orbitrap

Serum concentration of molecular species of PE and pPE in ZIKV infected and controls patients

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6 PE molecular species

12 pPE molecular species

Queiroz A. et al. Front Microbiol. 2019;10:753.

Triple TOF

• Broad variation in response to the infection among individuals makes it difficult to identify markers of severity

• Different control groups, sample preparation methods, and sample collection time

• Types/abilities of mass spectrometer• Various studies showing the association between circulating

metabolite/lipid levels and the severity of infection so far lack consistency

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Problems in metabolome/lipidome studies from human infections with flaviviruses

Examples of lipid-related anti-flaviviral strategies

19Martín-Acebes MA. et al. Prog Lipid Res. 2016;64:123-137.

Sterols

Others

Sphingolipids

Fatty acids

Glycerophospholipids

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Effect of D609 on endogenous SM contents

Treatment with 50 μM of D609 for 16 hrsreduced total SM levels 19.50-43.73% in MEFs

SM with C16 acyl chain length was drastically suppressed by D609

↓23.39%

↓57.20%*

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*

QQQ

Suksatu A. et al. in publication process

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Effect of D609 treatment on infectivity of different flaviviruses

Suksatu A. et al. (in publication process)

ZIKV

WNV Kunjin

• Repositioning/repurposing drugs that already licensed, i.e., statins (cardiovascular disease), metformin (diabetes), imipramine (antidepressant),could lead to more affordable broad spectrum, lipid-targeting compounds

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The strong dependency of cellular lipid metabolism for flavivirus infection

Biomarkers of infection/ disease progression

Lipid-based antiviral candidates(broad spectrum potential)

Novel therapeutics Repositioning drugs