Metabolic network analysis of Pseudomonas aeruginosa persister cells

Anna S. BlazierBiomedical Engineering

University of VirginiaOctober 19th, 2018

Persister cells are transient phenotypic variants that tolerate antimicrobial treatment

Collins(2013)CellHostandMicrobe

Persistercellsareimplicatedin:•  therecalcitrantnatureofchronic

infections•  theresistancetodisinfectionby

industrialantimicrobialsPersistercellmetabolismispoorlyunderstood

Pseudomonas aeruginosa is a model organism for the study of persister cell metabolism

•  Metabolically versatile, allowing it to thrive in diverse environments

•  Clinically relevant: –  Multi-drug resistant, Gram-

negative opportunistic pathogen

•  Industrially relevant:–  Exhibits resistance to

disinfection and preservative treatment

soil catheters

humantissues shampoo

Pseudomonasaeruginosa

High-throughput datasets and modeling will be used to study persister metabolism

Time-kill curve

RNA-seq Metabolomics BiologGenerate condition-specific genome-scale metabolic network reconstructions

Validation P. aeruginosa PA14 grown on LB agar

Isolate colonies in LB broth

Inoculate fresh LB Expose to PBS (diluent) and

BIT (antimicrobial)

BIT

PBSΔt = 5hT = 37C

Collect samples

Simulate pertubations to the networks to generate

testable predictions

xx

Δt = 24hT = 37C

Δt = 12hT = 37C

Δt = 12hT = 37C

Δt ~ 24hT = 37C

Δt = 0hT = 37C

Persister cells enable P. aeruginosa to tolerate treatment with the antimicrobial BIT

0

4

8

0 5 10 15 20 25Time (hours)

log1

0(C

FU/m

L) 0% BIT - untreated0.02% BIT - 10X MIC0.2% BIT - 100X MIC2% BIT - 1000X MIC

0

4

8

0 5 10 15 20 25Time (hours)

log1

0(C

FU/m

L) 0% BIT - untreated0.02% BIT - 10X MIC0.2% BIT - 100X MIC2% BIT - 1000X MIC

Persister cells enable P. aeruginosa to tolerate treatment with the antimicrobial BIT

SamplesforRNA-sequencingdatacollection

U5

U24

P24

P5

P0,U0

P:persistersamplesU:untreatedsamples

Persister cells have a distinct transcriptional state compared to untreated cells

Numbersofdifferentiallyexpressedgenes(DEGs)(adjustedp<0.01)

Comparison DEGsP5vs.P0 925P24vs.P0 805U5vs.U0 693U24vs.U0 92

P5 vs.

P0

P24 vs

. P0

U5 vs.

U0

U24 vs

. U0

−2−1012log2(Fold-change)

Genes up-regulated in persister conditions are significantly enriched for metabolic pathways

valine, leucine and isoleucine biosynthesisribosome

pyruvate metabolismpentose phosphate pathway

microbial metabolism in diverse environmentsmetabolic pathways

fructose and mannose metabolismflagellar assembly

fatty acid metabolismcyanoamino acid metabolism

carbon metabolismbiosynthesis of secondary metabolites

biosynthesis of antibioticsbiosynthesis of amino acids

bacterial chemotaxisaminoacyl−trna biosynthesis

alpha−linolenic acid metabolism2−oxocarboxylic acid metabolism

20 10 0 10 20% of genes

U5 vs. U0U24 vs. U0P5 vs. P0P24 vs. P0

**

**

**

**

**

**

**

*

*** *

*

**

**

**

***

*

*

0

4

8

0 5 10 15 20 25Time (hours)

log1

0(C

FU/m

L) 0% BIT - untreated0.02% BIT - 10X MIC0.2% BIT - 100X MIC2% BIT - 1000X MIC

Persister cells enable P. aeruginosa to tolerate treatment with the antimicrobial BIT

Samplesformetabolomicsdatacollection

U5

U24

P24

P5

P0,U0,D0

D5

D24

P:persistersamplesU:untreatedsamples

D:deadsamples

Persister cell metabolite footprints are distinct from untreated and dead conditions

untreated T24dead T5dead T24persister T5persister T24untreated T5persister T0untreated T0dead T0

ribosesulfate*

S−adenosylhomocystein5−oxoproline

histidinolarginine

8−hydroxyguanineN−acetylarginine

thioproline2−methylserine

methylsuccinateornithine

gamma−glutamylglutamateN−acetylglutamine

trehalosegamma−glutamyltyrosine

4−methyl−2−oxopentanoatecysteine−glutathione disulfide

gamma−glutamylphenylalanineadenosine−2',3'−cyclic monophosphate

butyrylglutamine/isobutyrylglutaminecarnitine

cadaverine5−(2−Hydroxyethyl)−4−methylthiazole

phenylalanylglycinealanylleucine

maleatevalylglutamine

catechol sulfateleucylglycine

prolinehistidylalanine

adenosine 2'−monophosphateN−formylphenylalanine

glycylleucineglycylvaline

alpha−ketoglutarategamma−glutamylmethionine

N6−carboxymethyllysineisoleucylglycinepyridoxamine

trehalose 6−phosphatephosphate

2'−deoxyuridinegulonate*

trans−nonadecenoate (tr 19:1)*ribonate

2,4−diaminobutyrateN−acetylhistidine

3−(4−hydroxyphenyl)lactate4−imidazoleacetate

N−acetylmethionine sulfoxideadenosine 5'−monophosphate (AMP)

N6−acetyllysine2'−deoxyadenosine 5'−monophosphate

4−hydroxyphenylacetate1−palmitoyl−2−oleoyl−GPE (16:0/18:1)

o−TyrosineO−acetylhomoserine

pipecolate

−1.5

−1

−0.5

0

0.5

1

1.5

Relative Intensity

T0 T5 T24

Untreated

T0 T5 T24

PersisterT0 T5 T24

Dead

ribosesulfate*

S−adenosylhomocystein5−oxoproline

histidinolarginine

8−hydroxyguanineN−acetylarginine

thioproline2−methylserine

methylsuccinateornithine

gamma−glutamylglutamateN−acetylglutamine

trehalosegamma−glutamyltyrosine

4−methyl−2−oxopentanoatecysteine−glutathione disulfide

gamma−glutamylphenylalanineadenosine−2',3'−cyclic monophosphate

butyrylglutamine/isobutyrylglutaminecarnitine

cadaverine5−(2−Hydroxyethyl)−4−methylthiazole

phenylalanylglycinealanylleucine

maleatevalylglutamine

catechol sulfateleucylglycine

prolinehistidylalanine

adenosine 2'−monophosphateN−formylphenylalanine

glycylleucineglycylvaline

alpha−ketoglutarategamma−glutamylmethionine

N6−carboxymethyllysineisoleucylglycinepyridoxamine

trehalose 6−phosphatephosphate

2'−deoxyuridinegulonate*

trans−nonadecenoate (tr 19:1)*ribonate

2,4−diaminobutyrateN−acetylhistidine

3−(4−hydroxyphenyl)lactate4−imidazoleacetate

N−acetylmethionine sulfoxideadenosine 5'−monophosphate (AMP)

N6−acetyllysine2'−deoxyadenosine 5'−monophosphate

4−hydroxyphenylacetate1−palmitoyl−2−oleoyl−GPE (16:0/18:1)

o−TyrosineO−acetylhomoserine

pipecolate

−1.5−1−0.500.511.5R

elative Intensity

T0 T5 T24

Untreated

T0 T5 T24

Persister

T0 T5 T24Dead

Persister cell metabolite footprints are distinct from untreated and dead conditions

untreated T24dead T5dead T24persister T5persister T24untreated T5persister T0untreated T0dead T0

Persister cell metabolite footprints are distinct from untreated and dead conditions

untreated T24dead T5dead T24persister T5persister T24untreated T5persister T0untreated T0dead T0

ribosesulfate*

S−adenosylhomocystein5−oxoproline

histidinolarginine

8−hydroxyguanineN−acetylarginine

thioproline2−methylserine

methylsuccinateornithine

gamma−glutamylglutamateN−acetylglutamine

trehalosegamma−glutamyltyrosine

4−methyl−2−oxopentanoatecysteine−glutathione disulfide

gamma−glutamylphenylalanineadenosine−2',3'−cyclic monophosphate

butyrylglutamine/isobutyrylglutaminecarnitine

cadaverine5−(2−Hydroxyethyl)−4−methylthiazole

phenylalanylglycinealanylleucine

maleatevalylglutamine

catechol sulfateleucylglycine

prolinehistidylalanine

adenosine 2'−monophosphateN−formylphenylalanine

glycylleucineglycylvaline

alpha−ketoglutarategamma−glutamylmethionine

N6−carboxymethyllysineisoleucylglycinepyridoxamine

trehalose 6−phosphatephosphate

2'−deoxyuridinegulonate*

trans−nonadecenoate (tr 19:1)*ribonate

2,4−diaminobutyrateN−acetylhistidine

3−(4−hydroxyphenyl)lactate4−imidazoleacetate

N−acetylmethionine sulfoxideadenosine 5'−monophosphate (AMP)

N6−acetyllysine2'−deoxyadenosine 5'−monophosphate

4−hydroxyphenylacetate1−palmitoyl−2−oleoyl−GPE (16:0/18:1)

o−TyrosineO−acetylhomoserine

pipecolate

−1.5−1−0.500.511.5R

elative Intensity

T0 T5 T24

Untreated

T0 T5 T24

Persister

T0 T5 T24Dead

Persister cell metabolite footprints are distinct from untreated and dead conditions

untreated T24

dead T5

dead T24

persister T5

persister T24

untreated T5

persister T0

untreated T0

dead T0

−3−2−10123R

elative Intensity

ribosesulfate*

S−adenosylhomocystein5−oxoproline

histidinolarginine

8−hydroxyguanineN−acetylarginine

thioproline2−methylserine

methylsuccinateornithine

gamma−glutamylglutamateN−acetylglutamine

trehalosegamma−glutamyltyrosine

4−methyl−2−oxopentanoatecysteine−glutathione disulfide

gamma−glutamylphenylalanineadenosine−2',3'−cyclic monophosphate

butyrylglutamine/isobutyrylglutaminecarnitine

cadaverine5−(2−Hydroxyethyl)−4−methylthiazole

phenylalanylglycinealanylleucine

maleatevalylglutamine

catechol sulfateleucylglycine

prolinehistidylalanine

adenosine 2'−monophosphateN−formylphenylalanine

glycylleucineglycylvaline

alpha−ketoglutarategamma−glutamylmethionine

N6−carboxymethyllysineisoleucylglycinepyridoxamine

trehalose 6−phosphatephosphate

2'−deoxyuridinegulonate*

trans−nonadecenoate (tr 19:1)*ribonate

2,4−diaminobutyrateN−acetylhistidine

3−(4−hydroxyphenyl)lactate4−imidazoleacetate

N−acetylmethionine sulfoxideadenosine 5'−monophosphate (AMP)

N6−acetyllysine2'−deoxyadenosine 5'−monophosphate

4−hydroxyphenylacetate1−palmitoyl−2−oleoyl−GPE (16:0/18:1)

o−TyrosineO−acetylhomoserine

pipecolate

−1.5−1−0.500.511.5R

elative Intensity

T0 T5 T24

Untreated

T0 T5 T24

Persister

T0 T5 T24Dead

Persister cell metabolite footprints are distinct from untreated and dead conditions

untreated T24dead T5dead T24persister T5persister T24untreated T5persister T0untreated T0dead T0

Persister cell metabolite footprints are distinct from untreated and dead conditions

T0 T5 T24

uridine 3'−monophosphate (3'−UMP)cyano−alanine

2−methylcitrate/homocitrate2−aminoadipate

3−hydroxy−2−ethylpropionate3,4−dihydroxy−1,5−cyclohexadiene−1−carboxylic acid

N−acetylserineerythritol

N−delta−acetylornithineurea

N−acetylleucineN−acetyltryptophan

malatethymine

4−acetamidobutanoateN−acetyltyrosine

quinolinateN−acetylmethionine

phenylacetatepyridoxate

3−ureidopropionatethymidine

N−acetylvalineindoleacetate

N−acetylthreoninenicotinate ribonucleoside

N−acetylisoleucinedimethylglycine

1,5−anhydroglucitol (1,5−AG)N2−acetyllysine

arabonate/xylonateS−methylcysteine

salicylateglutamate

pyridoxine (Vitamin B6)lysylleucine

tyrosylglycine4−hydroxybenzoate

tyramine2−keto−3−deoxy−gluconate

pantothenateN−monomethylarginine

3−hydroxyisobutyratethreonine

10−undecenoate (11:1n1)serine

gamma−glutamylisoleucine*2−hydroxyphenylacetate

glycineN−acetyl−cadaverine

homoserinegamma−glutamylglycine

N6,N6,N6−trimethyllysine

T0 T5 T24

succinate1−oleoylglycerol (18:1)

xanthineglutarate (C5−DC)

uracilgluconate

leucylglutamine*alpha−ketoglutaramate*

alanyl−glutamyl−meso−diaminopimelate2−oxoarginine*

2−ketogluconate3−methyl−2−oxobutyrate

3−hydroxyhexanoatesebacate (C10−DC)

(3'−5')−uridylyladenosine6−hydroxynicotinate

(3'−5')−cytidylyladenosine(3'−5')−cytidylyluridine*

3−indoxyl sulfateisobutyrylglycine

4−ethylphenylsulfatealpha−hydroxyisocaproate

N−acetylglutamateisovalerylglycine

6−oxopiperidine−2−carboxylateadenosine 3'−monophosphate

T0 T5 T24

(3'−5')−adenylyluridineallantoin

alpha−hydroxyisovaleratebeta−guanidinopropanoatehydantoin−5−propionic acid

2−piperidinonemevalonolactone

1−methyl−beta−carboline−3−carboxylic acid3−hydroxy−3−methylglutarate

N−acetylneuraminatephenol sulfate

N−alpha−acetylornithinephenethylamineN−acetylproline

guanosine 3'−monophosphate (3'−GMP)N−carbamoylalanine

N−formylanthranilic acidbetaine

cytidine 2',3'−cyclic monophosphate3−methylhistidine

2'−deoxyguanosine4−hydroxycinnamate

tryptaminecytidine 2' or 3'−monophosphate

malonate3−sulfo−L−alanine

fructosesuccinimide

lactatetyrosine

azelate (C9−DC)harmane

phenylalanylalanineerucate (22:1n9)

oxalate (ethanedioate)

−1.5−1−0.500.511.5R

elative Intensity

Untreated Persister Dead

High-throughput datasets and modeling will be used to study persister metabolism

Time-kill curve

RNA-seq Metabolomics BiologGenerate condition-specific genome-scale metabolic network reconstructions

Validation P. aeruginosa PA14 grown on LB agar

Isolate colonies in LB broth

Inoculate fresh LB Expose to PBS (diluent) and

BIT (antimicrobial)

BIT

PBSΔt = 5hT = 37C

Collect samples

Simulate pertubations to the networks to generate

testable predictions

xx

Δt = 24hT = 37C

Δt = 12hT = 37C

Δt = 12hT = 37C

Δt ~ 24hT = 37C

Δt = 0hT = 37C

GENREs are emerging as powerful tools for antimicrobial target discovery

AdaptedfromSchellenbergeretal.,NatProtoc.,2011

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10A -1 0 0 0 -1 0 0 0 0 0B 0 0 0 0 1 1 -1 0 0 0C 0 -1 0 -1 0 0 0 0 0 0D 0 0 0 1 0 0 -1 -1 0 0E 0 0 0 0 0 0 1 0 -1 0F 0 0 -1 0 0 -1 0 0 0 0G 0 0 0 0 0 0 0 1 0 -1

Metabolite

s

ReactionsGENRE:Genome-scalemetabolicnetworkreconstruction

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10A -1 0 0 0 -1 0 0 0 0 0B 0 0 0 0 1 1 -1 0 0 0C 0 -1 0 -1 0 0 0 0 0 0D 0 0 0 1 0 0 -1 -1 0 0E 0 0 0 0 0 0 1 0 -1 0F 0 0 -1 0 0 -1 0 0 0 0G 0 0 0 0 0 0 0 1 0 -1

Metabolite

s

Reactions

AdaptedfromSchellenbergeretal.,NatProtoc.,2011

GENREs are emerging as powerful tools for antimicrobial target discovery

GENRE:Genome-scalemetabolicnetworkreconstruction

Condition-specific models can be generated by integrating omics data with the PA GENRE

Xenobiotics

Virulence Factor

Transport

Terpenoids & polyketides

Secondary Metabolites

Other amino acids

Other

Nucleotides

Lipids

Glycans

Exchange

Energy

Cofactors & Vitamins

Carbohydrates

Amino acids

0 50 100 150 200Counts

Func

tiona

l Cat

egor

y

U5U24P5P24

fdnH is important for persister cell viability

fdnH-Nitrate-inducibleformatedehydrogenasesubunitbeta•  Involvedinthebreakdownofformate

•  Associatedwithantibioticresistance,ironhomeostasis,andadaptationtochronicallyinfectedhostsites

0.00

0.25

0.50

0.75

1.00

Wild−type

fdnH

Frac

tion

of C

FUs

at 2

4 ho

urs

*

Summary: Metabolism of persister cells is altered compared to untreated cells

•  RNA-sequencinganalysisindicatedthatpersistercellsareinadistincttranscriptionalstate

•  Metabolomicsprofilingrevealedthatpersistercellshaveauniquemetabolicfootprint

•  Computationalmodelingsuggestedmetabolictargetsthatinterruptviabilityofpersistercells

AcknowledgementsPapin LabAdvisor: Jason PapinGlynis KollingMatt JeniorDebi LuzaderMaureen CareyGreg MedlockKris RawlsBonnie DoughertyLaura DunphyTom MoutinhoPatrick GelbachJulia Hiser

Funding SourcesUnileverWagner FellowshipSEAS Teaching FellowshipNIH R01 GM088244NIH 5T32 GM008715-15

UnileverAlejandro AmezquitaAline Metris

Thesis CommitteeChair: Kevin JanesPhil BourneAlison CrissShayn Peirce-Cottler