1

FolicacidinstabilityandbacterialmetabolismcombinetoimpactC.elegansdevelopmentandageing

ClaireMaynard1,IanCummins1,JacalynGreen2andDavidWeinkove1*

1DepartmentofBiosciences,DurhamUniversity,SouthRoad,Durham,UK.DH13LE

2MidwesternUniversity,Illinois,Downers Grove, IL 60515, USA

*Correspondingauthor:david.weinkove@durham.ac.uk,+441913341303

ABSTRACT

Folicacidsupplementationisusedtopreventfolatedeficiency,butitisalsoassociatedwithnegative

effectsonhumanhealth.Littleisknownabouthowgutbacteriainteractwiththeuptakeofsynthetic

supplements,suchasfolicacid,andtheconsequencesforhosthealth.UsingthesimplifiedC.elegans-E.

colihost-microbemodelsystem,weexaminehowfolicacidimpactsE.colifolatesynthesis,andinturn,C.

eleganshealth.WefindthatfolicacidsupplementscontainabreakdownproductthatistakenupbytheE.

colitransporterAbgT,leadingtoincreasedbacterialfolatelevels.Weshowthatthisisthemainrouteby

whichfolicacidrescuesaC.elegansdevelopmentalfolatedeficiency,butisalsotheroutebywhichfolic

acidshortensadultlifespan.Together,thisworkshowshowfolicacidinstabilityandbacterialuptakecan

combinetoinfluencehosthealth.

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2

KEYWORDS

Folicacid;Microbiota;Ageing;C.elegans;bacterialmetabolism

INTRODUCTION

Folicacidsupplementationhasbeenamajorpublichealthsuccess,particularlyinthepreventionneural

tubedefects(Imbardetal.,2013),buttherearenumeroushealthconcernsrelatedtoover-

supplementation(ButterworthandTamura,1989;Coleetal.,2007;Kim,2007;Mareanetal.,2011;Milne

etal.,1984;Pickelletal.,2011;Portal-CelhayandBlaser,2012;Selhubetal.,2009).Folicacidisasynthetic

oxidizedfolate.Itcanbeabsorbedinthegutinthisform(Patanwalaetal.,2014)butunlikebacterialand

dietaryfolates,itmustbeconvertedintoareducedtetrahydrofolate(THF),beforeitcanbetakenupin

peripheralcellsbyreducedfolatecarriersandusedinmetabolism(DuckerandRabinowitz,2017;Visentin

etal.,2014).Theroutesoffolicaciduptakeandmetabolismarenotcompletelycharacterizedandits

interactionwithgutmicrobeshasnotbeenwellstudied(Visentinetal.,2014).

Somebacteriacantakeupfolatesdirectlybutmanycannot.Insteadtheyeithermakefolatedenovoor

takeupfolateprecursorssuchaspara-aminobenzoicacid(PABA,Figure1,(Carteretal.,2007;LeBlancet

al.,2013;Magnusdottiretal.,2015).FolicacidandTHFsareinherentlyunstablemolecules,comprisingofa

centralPABAmoietylinkedbyamethylenebridgetoapteridineringtoformpteroicacidandbyits

carboxylgrouptoaL-glutamicacidresiduebyapeptidebond(GreenandMatthews,2007).Themethylene

bridgeispronetodisassociationunderseveralparameters,includinglowpH,togeneratepteridineand

PABA-glutamicacid(PABA-glu)(DeBrouweretal.,2007;Gazzalietal.,2016;Gregory,1989;Hansonand

Gregory,2011;Maruyamaetal.,1978),wherePABA-glucanfurtherdissociatetogeneratePABA(Der-

Petrossianetal.,2007;Thiavilleetal.,2016).Thus,theacidicmicroenvironmentsofthestomachandupper

smallintestinemaycausefolatestodegradetoPABA-gluandPABA(SeyoumandSelhub,1998).Indeed,

PABAhasbeendetectedasasignificantfaecalexcretoryproductfollowingfolicacidsupplementation

(DenkoCwFau-Grundyetal.).MammalianstudieshaveshownthatinjectionoflabelledPABAintothe

cecumofrats(Rongetal.,1991)andpiglets(AsrarandO'Connor,2005)resultsintheincorporationof

bacteriallysynthesizedfolateintohosttissues.Humanstudieshaveshownthatbacterialfolatesynthesisin

boththehumansmall(Camiloetal,1996)andlargeintestine(Kimetal.,2004)canbeincorporatedinto

hosttissues.Itisnotknown,however,ifthisbacterialfolatesynthesisisenhancedbyfolicacid

supplementationandifitis,whetherthereareanyconsequencesformicrobiomefunctionandhosthealth.

Thisstudyusesasimplifiedhost-microbemodelsystemtoexaminehowfolicacidaffectsbacterialfolate

synthesisandhosthealth.Themodelorganism,thenematodeCaenorhabditiselegansismaintainedonlab

strainsofE.coliforwhichitdependsonforfolateaswellasforothervitamins(YilmazandWalhout,2014).

.CC-BY 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted December 6, 2017. . https://doi.org/10.1101/230227doi: bioRxiv preprint

3

LikeseveralotherProteobacteria,E.coliiscapableofdenovofolatesynthesisandthesalvageofPABAand

PABA-glutomakefolate(Carteretal.,2007),butcannottakeupintactfolates(NickersonandWebb,1956;

Webb,1955)Figure1).Here,wefindthatthemajorrouteoffolicacidsupplementationisanindirectroute

thatthisisdependentonthebreakdownoffolicacidintoPABA-gluandsubsequentuptakeviatheE.coli

transporter,AbgT.TheresultantincreaseinbacterialfolatesynthesisisbeneficialforC.elegansduring

development,buthasadetrimentalimpactonC.elegansageing.Thisisconsistentwithourpreviouswork,

whichhasdemonstratedthatE.colifolatesynthesisshortensC.eleganslifespan(Virketal.,2012)most

likelyduetoabacterial-dependentchronictoxicity(Virketal.,2016).Importantly,wealsodetectPABA-glu

andPABAinthreefolicacidsources,withthehighestquantitiesinacommercialfolicacidsupplement.

Together,thisworkhighlightsanunappreciatedbacterial-dependentrouteviawhichfolicacidcanhave

bothpositiveandnegativeeffectsonhosthealth,andthushaswiderreachingimplicationsforthe

importanceofthemicrobiotaindetermininghealthoutcomesfollowingvitaminsupplementation.

Figure1.SchematicoftheindirectuptakeoffolicacidbyE.coli.FolicacidiscomposedofacentralPABAmoietylinkedbyamethylenebridgetoapteridineandtoasingleglutamicacidresidueviaapeptidebond.FolicacidisunstableanddisassociatestogeneratePABA-glutamate(PABA-glu),whichcanfurtherdisassociatetoPABA.E.coliisabletosalvagePABA-gluviatransportbytheinnermembraneprotein,AbgT.PABA-gluisthencleavedintracellularlybytheheterodimericcarboxypeptidaseAbgA/BintoPABA,whereitcanbeusedasaprecursorforfolatesynthesistogeneratetheactiveformoffolate,tetrahydrofolate(THF)whichisusedinthefolatecycle.PABAcanalsodiffuseacrossbiologicalmembranes.E.coliisalsocapableofdenovofolatesynthesisbygeneratingPABAfromchorismateandglutaminethroughtheactionofPabA,PabBandPabC.E.coliisunabletoimportfolicaciddirectly.

2 O H

PABA

Chorismate +glutamine

AbgT

PabA FOLATECYCLE

PABA-glu

PABA

AbgA AbgB

THF

2 PABA-glu

E.COLI

FOLATESYNTHESIS

FOLICACID

PABA pteridine

glutamicacid

4

RESULTS

FolicacidsupplementationrescuesC.elegansdevelopmentalfolatedeficiencyviaE.coliabgT

Inordertotestwhetherfolicacidinteractswithbacterialfolatesynthesistosupplementhostfolateinour

modelsystem,weusedthepreviouslycharacterizedC.elegansfolateuptakedeletionmutant,gcp-2.1

(ok1004)(Virketal.,2016).Thegcp-2.1mutantstrainlacksGCP-2.1,theC.eleganshomologueoftheGCPII

glutamatecarboxypeptidaseenzymethatremovesglutamatesfrompolyglutamatedfolates,aprerequisite

forfolateabsorption(Halstedetal.,1998).Thisstrainexhibitsaseveredevelopmentalfolate-deficiency

whenmaintainedonE.colitreatedwiththesulfonamidedrugsulfamethoxazole(SMX)(Virketal.,2016).

Thisgrowthdefectisrescuedwithhighconcentrationsoffolicacid(Virketal.,2016).Itisnotclear,

however,whetherthissupplementationisdirect(C.elegansuptake)orindirect(restorationoffolate

synthesisinE.coli).HereweshowthattheC.elegansgcp-2.1mutantgrownontheE.colipabAmutanton

definedmedia(DM)hasthesamegrowthdefectasthesewormsgrownonwildtypeE.colitreatedwith

128µg/mlSMX(Figure2a).Folicacidwasfoundtoincreasegcp-2.1mutantbodylengthonthepabA

mutantinadose-dependentmanner(Figure2b).AsE.colicannotuptakeintactfolicacid,weexaminedthe

dependenceoffolicacidrescueontheexpressionoftheE.coliabgTgene,whichisresponsibleforthe

uptakeofPABA-glu.Aweakerresponseofthegcp-2.1mutanttofolicacidwasobservedontheabgTpabA

doublemutantcomparedtowormsonthepabAsinglemutant.Incontrast,gcp-2.1mutantsmaintainedon

pabAwithaplasmidoverexpressingabgT(Carteretal.,2007)(pabA(abgTOE))weremoreresponsiveto

folicacid(Figure2b).Analysingtheexperimentbytwo-wayANOVA,wefindthatthereisasignificant

interactioneffectofstraintype(F=102.67,p<0.0001)andfolicacidconcentration(F=123.55,p<0.0001)on

C.elegansgcp-2.1bodylength.MutationofabgTalonedidnotinfluencegrowthofthegcp-2.1

(SupplementaryFigure1a).Rescueofgcp-2.1developmentalfolatedeficiencywasachievedata10-fold

lowerconcentrationandindependentlyofabgTexpression,withtherelativelystableTHF,folinicacid

(SupplementaryFigure2),suggestingthatC.eleganstakesupfolinicaciddirectly.Theseresultssuggest

thatthemajorrouteoffolicaciduptakebythewormisthroughE.coliviauptakeofthefolicacid

degradationproduct,PABA-glubytheAbgTtransporter.

FolicacidsupportsE.coligrowthviaAbgT-dependentuptakeofPABA-glu

TheabgT-dependenceoffolicacidtorescuethegcp-2.1developmentalfolate-deficiencystronglysuggests

thatPABA-gluisavailabletoE.colifollowingfolicacidsupplementation.TotesttherelativeabilityofE.coli

totakeupPABA-gluandfolicacid,weaddedthesecompoundstopabA,abgTpabAandpabA(abgTOE)E.

coliandassessedgrowthonDMagarplatesafter4daysincubationat25°C.Undertheseconditions,we

foundthatallstrainscontainingthepabAmutantshowedslowergrowththanwildtypeandabgT

expressiondeterminedtheresponsetofolicacidandPABA-glu;10µMfolicacidrescuedgrowthofthe

5

Figure2.FolicacidsupplementsC.elegansviaanE.coliabgT-dependentrouteduringdevelopmenta)bodylengthofwild-typeandgcp-2.1mutantC.elegansatL4stageraisedonDMagarplatesseededwithWTE.coli(control),pabAmutantorWTE.colitreatedwith128μg/mlSMXb)bodylengthofgcp-2.1mutantC.elegansatL4stageraisedonDMagarplatesseededwithpabAmutant,abgTpabAdoublemutantorpabAmutantover-expressingabgTwithincreasingconcentrationsoffolicacid.Bytwo-wayANOVAanalyses,wefindthatthereisasignificantinteractioneffectofstraintype(F=102.67,p<0.0001)andfolicacidconcentration(F=123.55,p<0.0001)onC.elegansgcp-2.1bodylength.Over-expressionisconferredbytransformationwithahighcopynumberplasmid,pJ128.pabAandabgTpabAstrainaretransformedwiththeemptyvector,pUC19.ErrorbarsrepresentstandarddeviationofC.elegansbodylength;n≥40.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Control pabA SMX

Celeg

ansb

odylength(m

m)

WT

gcp-2.1

pabA

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 16 32 64 128

gcp-2.1bo

dylength(m

m)

[Folicacid](µM)

pabA

abgTpabA

pabA(abgTOE)

b)

2a)

6

pabAmutant,whereas100µMwasneededtoachieveanequivalentrescueintheabgTpabAdouble

mutant(Figure3ai).10µMfolicacidincreasedthegrowthofthepabAstrainover-expressingAbgTabove

thatofthepabAmutantaloneorthewildtypestrain.SupplementationbyPABA-gluhadasimilareffect

butata10-foldlowerconcentrationthanfolicacid(Figure3aii).PABA,whichcanfreelydiffuseacross

biologicalmembranes(TranandNichols,1991),rescuedbacterialgrowthwasindependentofabgT

expressionandachievedatnanomolarconcentrations(Figure3aiii).Overall,rescueofbacterialgrowthby

folicacidatlowconcentrationscanbeexplainedbyPABA-gluuptakebyAbgTwhilelowconcentrationsof

PABApresentinfolicacidpreparationsmayexplaintheabilityof100µMfolicacidtorescueE.coligrowth

andC.elegansgcp-2.1developmentalfolate-deficiencyindependentlyofabgT(Figure2).

FolicacidincreasesE.colifolatelevelsinanAbgT-dependentmechanism

ItordertoverifythatE.coligrowthfollowingfolicacidsupplementationisattributabletorestoredbacterial

folatesynthesis,weusedLC-MS/MStodetectE.colifolatelevelsundertheconditionsusedintheabove

experiment.LevelsofthemostdetectableandthuslikelythemostabundantTHFspecies,5-methylTHF-

glu3,arepresentedinFigure3b.Intheabsenceoffolicacid,folatelevelsinthepabAmutantstrainswere

significantlylowerthaninWTextracts(Figure3b,SupplementaryFigure3).Additionoffolicacidincreased

folatelevels,wherethescaleofincreasewasdependentonabgTexpression;with100µMfolicacid,folate

specieswerehighestinpabA(abgTOE)followedbyWT,pabA,andfinallylowestintheabgTpabAdouble

mutant.OtherTHFspeciesfollowedthesametrendas5-methylTHF-glu3andarepresentedin

SupplementaryFigure3.Insummary,folicacidsupplementationincreasesE.colifolatelevelsinanabgT-

dependentmechanism.

FolicacidpreparationscontainPABA-gluandPABA

Together,thedatapresentedhereindicatethatthemainrouteofC.elegansfolicacidsupplementationis

indirectviaE.coliuptakeofPABA-gluandPABA.WeusedLC-MS/MStotestfortheexpectedpresenceof

thesebreakdownproductsinfolicacidpreparationsfromSchircks(usedinallotherexperimentsinthis

study),SigmaAldrichandBoots,aUKretailer.Wealsotestedtheimpactoftheexperimentalconditions

usedhereonfolicacidbreakdownbyanalysingextractsfromagarmediasupplementedwithSchircksfolic

acidandincubatedat25°Cfor4days.WedetectedPABA-gluinallthreefolicacidsourcesatbetween0.3%

(Schircks)and4%(Boots)(Figure3ci).UndertheconditionsusedforC.elegansexperimentsPABA-glu

increasedto1.18%,suggestingfurtherbreakdown.PABAwasfoundatbetween0.01%(Schircks)and0.06%

(Boots)(Figure3cii).

7

Figure3.FolicacidbreakdownsupportsbacterialgrowthandincreasesfolatelevelsviaAbgT-uptakeofPABA-glua)growthofE.coliWT,pabA,abgTpabAandpabAover-expressingabgT(abgTOE)onagarplatessupplementedwithi)folicacidii)PABA-gluandiii)PABAasmeasuredbyOD600after4daysgrowthat25℃(seemethods).Eachdatapointistheaverageof8plates.Errorbarsrepresentstandarddeviation.AsterisksdenotetheteststatisticfromStudent’sttestcomparisonofmeans,where*=P<0.05comparedtoWTgrowthonthesameconditionb)5-methylTHF-glu3levelsinextractsofE.coliWT,pabA,abgTpabA,pabA(abgTOE)mutantssupplementedwith10µMand100µMfolicacid.FolatecountsfromtheLC-MS/MSarenormalisedbydividingbycountsofaninternalMTX-glu6spike.Extractsweremadeafter4daysofbacterialgrowthat25°Consolidagarplates.SeeSupplementaryFigure3forfullfolateanalysis.c)Levelsofi)PABA-gluandii)PABAdetectedbyLC-MS/MSinfolicacidpreparationsfromSchircks,Sigma,BootsandSchircksfolicacidafteradditiontotheagarmediaandincubationfor4daysat25˚C.Errorbarsrepresentstandarddeviationovertriplicateindependentpreparations.

Control

1000.0

0.1

0.2

0.3

5-methyl-T

HF-glu

3:MTX-glu

6

100µM

10µM

0.0

1.0

2.0

3.0

4.0

5.0

10 100

[PAB

A-glu](µ

M)

[Folicacid](µM)

Schircks

Sigma

Boots

Schircksinmedia

0.00

0.02

0.04

0.06

0.08

10 100

[PAB

A](µ

M)

[Folicacid](µM)

0.10

0.15

0.20

0.25

0.30

0 1 10

Bacterialgrowth( O

D60

0)

[PABA-glu](µM)

WT

pabA

abgTpabA

pabA(abgTOE)

0.10

0.15

0.20

0.25

0.30

0 10 100

Bacterialgrowth(O

D600)

[Folicacid](µM)

*

*

*

0.10

0.15

0.20

0.25

0.30

0 0.1 1

Bacterialgrowth(O

D600)

[PABA](µM)

pabA

(abgTOE)

3ai)

aii)

aiii)

b)

ci)

cii)

*

8

FolicacidshortensC.eleganslifespanviaAbgT-dependentuptakeofPABA-gluduringadulthood

Inhibitingbacterialfolatesynthesis,withoutaffectingbacterialgrowth,isknowntoincreaseC.elegans

lifespan(Virketal.,2012;Virketal.,2016).Itwasthereforehypothesizedthatfactorsthatincrease

bacterialfolatesynthesis,suchasfolicacid(asshownhere),mayshortenC.eleganslifespan.Consistent

withourpreviousfindings(Virketal.,2016),wefindthatC.elegansmaintainedonanyE.colipabAmutant

arelong-livedcomparedtoC.elegansfedwildtypeE.coli(Figure4,SupplementaryTable1),whereasthe

abgTmutationalonehadnoimpactonC.eleganslifespan(P=0.4312,SupplementaryFigure1b).10µM

folicacidwasfoundtodecreaseC.eleganslifespanonpabAby9.4%(P=0.0052),anddecreaselifespanon

thepabAmutantover-expressingabgTfurther(by16.3%,P<0.0001,Figure4a).Incontrast,10µMfolicacid

hadnoeffectonlifespanontheabgTpabAdoublemutant(P=0.1901,Figure3a).100µMfolicacid

decreasedlifespanonpabAE.coliby23.9%(P<0.0001),whereasthisconcentrationonlyshortenedlifespan

ontheabgTpabAdoublemutantby4.7%(P=0.0467,Figure4a).Lifespansonmediasupplementedwith

PABA-glushowedanabgT-dependentresponsesimilartothatobservedwithfolicacidsupplementation,

butata10-foldlowerconcentration(Figure4b).Incontrast,PABAsupplementationshortenedC.elegans

lifespaninallcasesindependentlyofabgTexpression,consistentwithitsabilitytodiffuseacrossbiological

membranes(Figure4c).FolicacidhadnoeffectonthelifespanofC.elegansmaintainedonWTE.coli

(SupplementaryFigure4).Together,theseresultssuggestthatfolicacidshortensC.eleganslifespanon

folate-deficientE.coliviaAbgT-dependentuptakeofPABA-gluduringadulthood.

9

Figure 4. Folic acid shortensC. elegans lifespan via an E. coli abgT-dependent route during adulthood.MeanlifespanofWTC.elegansmaintainedfromday1ofadulthoodonpabA,abgTpabA,orpabA(abgTOE)E.coliwithsupplementationof(a)folicacid(b)PABA-gluand(c)PABA.Errorbarsrepresentstandarderror.Asterisksdenotethe Log-rank non-parametric statistical test of survival, where: *P<0.05; **P<0.01; ***P<0.005 compared tolifespanonthenon-supplementedconditionofthesamestrain.FulllifespandatainSupplementaryTable1.

14

16

18

20

22

0 0.1 1

Meanlifespan(days)

[PABA](µM)

14

16

18

20

22

0 10 100

Meanlifespan(days)

[Folicacid](µM)

***

***

**

*

***

14

16

18

20

22

0 1 10

Meanlifespan(days)

[PABA-Glu](µM)

pabA

abgTpabA

pabA(AbgTOE)***

***

***

**

pabA(abgTOE)

4a)

b)

c)

10

DISCUSSION

Folicacidsupplementationgeneratesasupplyoffolateinadditiontothatprovidedbythedietandgut

microbiota.Itisnotclear,however,exactlyhowfolicacidsupplementshostfolate.Consideringthe

conflictingreportsofthesafetyoffolicacidsupplementationonhosthealth,itisimportanttounderstand

allpossibleroutesofuptake.UsingtheestablishedC.elegans-E.colihost-microbemodelinwhichbacterial

folatesynthesishasbeenshowntoinfluencehosthealth(Chaudharietal.,2016;Hanetal.,2017;Virket

al.,2016),thisstudyshowsthatfolicacidcanincreasebacterialfolatesynthesis(Figure3b)inaroutethatis

dependentonitsbreakdownintoPABA-glu(Figure3c)anduptakebythebacterialtransporterAbgT.We

haveshownthatthisroutecanhavebeneficialconsequencesinthecaseofC.elegansdevelopmental

folate-deficiency(Figure2)butitcanalsohaveanegativeimpactonlong-termhosthealth,byshortening

lifespan(Figure4).Ourpreviousworkindicatesthatlifespandecreaseisduetoabacterialfolate-

dependenttoxicity.Together,wehaveuncoveredaroutebywhichfolicacidinteractswithbacterial

metabolismtoimpacthosthealth(Figure5).

ThedetectionofsignificantquantitiesofPABA-gluandPABAinthreefolicacidsourcesinthisstudy,

includinginacommercialfolicacidsupplement(Figure3c)indicatesthatthisroutemayhaveimplications

forhumanfolatesupplementation.Indeed,investigationsintofolicacidsupplementshavereported

significantfailingsofsupplementstomeetUS(Hoagetal.,1997)orUK(Sculthorpeetal.,2001)standards

fordissolution.Inlightoftheseissueswithfolicacidstability,manufacturershaveadoptedapolicyof

‘overages’inordertoensuresufficientvitaminisreleased(Andrewsetal.2017).AstheE.coliPABA-glu

transporter,AbgT,caninfluenceC.eleganshealthfollowingfolicacidsupplementation,dependingonboth

bacterialandhostgenotype,itishypothesizedthatsupplementstabilityandgutmicrobiotacomposition

maybetwopreviouslyunexploredvariablesinunderstandingthehealthconsequencesoffolicacid

supplementation.

TheAbgTproteinisamemberofaconservedfamilyofover13,000transporters,manyofwhichare

encodedforinthegenomesofseveralpathogenicbacteriaofthehumangutmicrobiome,including

Enterobactercloacae,N.gonorrhoeae,Salmonellaenterica,ShigellaboydiiandStaphylococcusaureusin

additiontoE.coli.Interestingly,severaldiseasesareassociatedwithanincreasedabundanceoffolate-

synthesizinggutbacteria,suchasinflammatoryboweldisease(IBD(Shinetal.,2015)andsmallintestinal

bacterialovergrowth(SIBO,(Camiloetal.,1996).Humantrialsarenecessarytoexaminehowmicrobial

folatesynthesisandmicrobialcompositionalchangesinthehumangutmicrobiotafollowingfolicacid

supplementation.Wehypothesizethattheremaybespecificgroupsofpatientswhereeffectsoffolicacid

11

onmicrobialfolatesynthesisneedtobeconsidered.Itmaybenecessarytodesignalternativestrategiesto

preventfolatedeficiencywithoutincreasingbacterialfolatesynthesis.

Figure5.SchematicoftheimpactoffolicacidsupplementationonC.elegansviaindirectuptakeofbreakdownproductsbyE.coli.FolicacidisnottakenupwellbyC.elegansdirectly.WefindthatthemajoruptakeoffolicacidbyC.elegansisdependentonitsbreakdownintoPABA-gluanduptakebytheE.coliAbgTtransporter.Thisrouteincreasesbacterialfolatesynthesisinbothwild-typeandpabAmutantE.coli.Underconditionsoffolate-deficiency(pabAmutantE.coli),increasingbacterialfolateviathisrouteisbeneficialforC.elegansdevelopment.DuringC.elegansadulthood,thisroutehasanegativeimpactonlongevityasitpromotesabacterialfolate-dependenttoxicity.

ACKNOWLEDGEMENTS

WethanktheC.elegansGeneticsCenter,theC.elegansKnockoutConsortium,andNBRP-E.coliatNIGfor

strainsandwethankSushmitaMaitraandJohnMathersforusefulcommentsonthemanuscript.Thiswork

wassupportedbyaBBSRCDTPstudentship.

2 O H

PABA

Chorismate +glutamine

AbgT

PabA FOLATECYCLE

PABA-glu

PABA

AbgA AbgB

THF

2 PABA-glu

E.COLI

C.ELEGANS

FOLATESYNTHESIS

DEVELOPMENT ü Increased

fitness

FOLICACID

PABA pteridine

glutamicacid

TOXICITY

ADULTHOOD × Decreasedlongevity

12

MATERIALSANDMETHODS

Folatesandrelatedcompounds

Folicacid,Folinicacid,PABA-Glu,5-formylTHF-Glu3,5-methylTHF-Glu3,methotrexate-Glu6wereobtained

fromSchircks,Switzerland.PABA,VitaminB12andfolicacidwerefromSigmaAldrichandfolicacid

supplementfromBoots,UK.

Cultureconditions

Definedmedia(DM)waspreparedasdescribed(Virketal.,2016),exceptthat10nMB12wasadded.B12,

folicacidandantibioticsareaddedpost-autoclavingforagarplates.DMforliquidcultureisfilter-sterilised.

0.1µMPABAaddedtotheliquidDMmediausedtoseedtheplatesinordertomaintainbacterialgrowth

(apartthegrowthexperimentsinFigure3a).30µlof3mlfreshovernightLBcultureisusedtoinoculate

5mlDM(15mlFalcontube).25µg/mlkanamycin(50ug/mlampicillinifnecessary)addedtobothLBand

DMpre-incubation.DMliquidculturesareincubatedfor18hrat37°C,220RPM.

13

AllstrainsderivedfromtheKeiocollection(Babaetal.,2006).Seetable.WT-Kan(Virketal.,2016).

abgTpabAdoublemutantwasmadeusingP1transductionprotocolasdescribedin(Moore,2011).The

abgTover-expressionplasmid(pJ128)(Carteretal.,2007)andemptyvector(puc19)(Yanisch-Perronetal.,

1985)weretransformedintoappropriatestrains.

C.elegansstrainsused

SS104glp-4(bn2),UF208(wild-type),andUF209gcp-2.1(ok1004)(Virketal.,2016).

E.colipreparationandgrowthassay

TableofE.colistrainsusedinthisstudy

Strain Genotype Plasmid Characteristics Source

BW25113/pGreen0029

WT pGreen0029 kanr Virketal2016

JW3323-1 ΔpabA n/a kanr Babaetal2006

JW5822-1 ΔabgT n/akanr

Babaetal2006

CMabgTpabA ΔabgTΔpabA n/a kanr Thisstudy

CM1 WTpUC19‡,pGreen0029

kanr,ampr Thisstudy

CM2 ΔpabA pUC19 kanr,ampr Thisstudy

CM3 ΔabgTΔpabA pUC19 kanr,ampr Thisstudy

CM4 WT(abgTOE) pJ128‡,

pGreen0029

kanr,ampr Thisstudy

CM5 ΔpabA(abgTOE) pJ128 kanr,ampr Thisstudy

CM6 ΔabgTΔpabA(abgTOE)

pJ128 kanr,ampr Thisstudy

14

E.coliwaspreparedasfollowsforallE.coliandC.elegansexperiments.30µlofanovernightLBcultureof

E.coliwastransferredinto5mlDMandincubatedfor18hrat37◦C,220RPM.100µltheDMculturewas

seededontoDMagarplatesand incubatedat25°Cfor4days.E.coliwasremovedbypipetting1mlM9

ontotheplateandaglassspreadertoscrapeoffthebacteriallawn.Thebacterialsuspensionwaspipetted

into a 1.5ml Eppendorf and the volumewas recorded (v). Tubeswere vortexed vigorously to obtain a

homogenisedsolution.100µlwastakenanddilutedwith900µlM9inacuvette.Aspectrophotometerwas

used to read bacterial growth at 600 nm. Bacterial growth was calculated by multiplying OD600by the

volumeofthesample(v).

E.colifolateextraction

BacteriallawnswerescrapedfromplatesintomicrocentrifugetubesusingM9solutionandkeptonice.

Volume(v),multipliedbytheOD600ofthesolution(diluted1:5)givesameasureoftheamountofmaterial.

Sampleswereconcentratedinchilledmicrocentrifugeandpelletsweresnapfrozeninliquidnitrogen.

Pelletswerethawedandresuspendedinavolumeofice-cold90%methanol:10%folateextractionbuffer

(FEB:50mMHEPES,50mMCHES,0.5%w/vascorbicacid,0.2MDTT,pH7.85withNaOH)inproportionto

bacterialcontent(37.5×OD600×v).FEBisspikedwith10nMmethotrexate-Glu6asaninternalstandard.

Sampleswerevortexedvigorouslyandleftonicefor15minbeforecentrifugationinacooled

microcentrifugefor15minatfullspeed.Supernatantswereusedforanalysis.

FolateLC-MS/MSanalysis

Folatesweredetectedbymultiplereactionmonitoring(MRM)analysisusingaSCIEXQTRAP6500

instrument.MRMconditionsforfolicacid,PABA,PABA-Glu,5-Me-H4PteGlu3(5-methylTHF-Glu3)and5/10-

CHO-H4PteGlu3(formylTHF3)wereoptimisedbyinfusionofstandardsintotheinstrument.Theoptimised

conditionsfor–Glu3folateswereappliedtootherhigherfolatesusingMRMtransitionsdescribedbyLuet

al.,2007(Luetal.,2007).Furtherconfirmationofidentityforfolatesofinterestwasachievedby

performingenhancedproductionscansandcomparingthefragmentspectrawithknownstandards.

TheQTRAP6500wasoperatedinESI+modeandwasinterfacedwithaShimadzuNexeraUHPLCsystem.

SampleswereseparatedusingaThermoPA2C18column(2.2µm,2.1x100mm)withagradientof0.1%

formicacidinwater(mobilephaseA)andacetonitrile(mobilephaseB).Samplesweremaintainedat4⁰C

and2µLaliquotswereinjected.Thecolumnwasmaintainedat40⁰Cwithaflowrateof200µL/min,

startingat2%B,heldfor2minutes,withalineargradientto100%Bat7minutes,heldfor1minute,before

a7-minutere-equilibrationstepat2%Bthatwasnecessaryforconsistentretentiontimes.Thecolumn

15

eluateflowtotheMSwascontrolledviatheQTRAPswitchingvalve,allowinganalysisbetween4and8

minutestominimiseinstrumentcontamination.Folateswerequantifiedwithreferencetoexternal

standardsandmatrixeffectswereassessedbyspikingofstandardsintoextractedsamples.

Lifespananalysis

Survivalanalyseswereperformedasdescribed(Virketal.,2012).glp-4(bn2)wormsweremaintainedat

15°Candshiftedto25°CattheL3stage.AttheL4/youngadultstage,animalswereplacedonbacteria

undertheexperimentalconditions.AlllifespandataisinTableS1.Statisticalsignificancewasdetermined

usingLogRankandWilcoxontestsoftheKaplan-Meiersurvivalmodel.

16

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19

SUPPLEMENTARYINFORMATION

SupplementaryFigure1.TheE.coliabgTdeletionhasnoeffectonC.elegansdevelopmentandlifespana)bodylengthofwild-typeandgcp-2.1mutantC.elegansatL4stageraisedonabgTmutantorwild-typeE.coli(errorbarsrepresentstandarddeviation)b)survivalcurvesofwild-type(glp-4)C.elegansonE.coliwild-typeandabgTmutant.SeeSupplementaryTable1forfurtherdetails.

0

0.2

0.4

0.6

0.8

1

WT gcp-2.1

C.elega

nsbod

ylength(m

m)

C.elegansgenotype

WT

abgT

0.0

0.2

0.4

0.6

0.8

1.0

0 5 10 15 20 25 30

Survival

Dayofadulthood

WT

abgT

1a)

b)

20

SupplementaryFigure2.FolinicacidrestoresgrowthofC.elegansgcp-2.1independentlyofE.coliabgT.Bodylengthofgcp-2.1mutantC.elegansatL4stageraisedonDMagarplatesseededwithpabAmutant,abgTpabAdoublemutantorpabAmutantover-expressingabgTwithincreasingconcentrationsoffolinicacid.Over-expressionisconferredbytransformationwithahighcopynumberplasmid,pJ128.pabAandabgTpabAstrainaretransformedwiththeemptyvector,pUC19.ErrorbarsrepresentstandarddeviationofC.elegansbodylength;n≥40.

0.0

0.2

0.4

0.6

0.8

1.0

2 4 8 16 32 64 128

gcp-2.1bo

dylength(m

m)

[Folinicacid](µM)

pabA/puc19

abgTpabA/puc19

pabA/pJ128(abgTOE)

21

SupplementaryFigure3.LC-MS/MSdetectslowerlevelsofTHFsinpabAmutants,whereabgTdeterminesresponsetofolicacid.LevelsofTHFsdetectedinextractsofWT,pabA,abgTpabA,pabA(abgTOE)mutantsdisplayedasaratiowithaninternalMTX-glu6spikefornormalization.Extractsweremadeafter4daysofbacterialgrowthat25°C.AsterisksdenotetheteststatisticfromStudent’sttestcomparisonofmeans,where*=P<0.05.Errorbarsrepresentstandarddeviationoverfourreplicates.

0.00

0.10

0.20

0.30

0 10 100

Folate:M

TX-glu6

5-methyl-THF

*

**

0.00

0.15

0.30

0.45

0 10 100

5/10-formyl-THF

*

*

*

0.00

0.35

0.70

0 10 100

Folate:M

TX-glu6

[Folicacid](µM)

5,10-methenyl-THF

* *

*

0.00

0.10

0.20

0.30

0 10 100[Folicacid](µM)

THF

*

*

*

0.00

0.03

0.05

0 10 100

Folate:M

TX-glu6

[Folicacid](µM)

5,10-methylene-THF

WT

pabA

abgTpabA

pabA(abgTOE)

**

22

SupplementaryFigure4.TheimpactoffolicacidsupplementationonC.eleganslifespanonwild-typeE.coli.SurvivalcurvesofC.elegansmaintainedfromday1ofadulthoodonplatessupplementedwith10µMand100µMfolicacid.

0.0

0.2

0.4

0.6

0.8

1.0

0 5 10 15 20 25 30

Survival

Dayofadulthood

WTcontrol

WT+10µmfolicacid

WT+100µmfolicacid

WTcontrol

WT+10µMfolicacid

WT+100µMfolicacid

SupplementaryTable1:SummaryoflifespandataAlllifespansconductedondefinedmedia(DM)agarplatesincubatedat25ºC

Fig. Groupname n Censor Meanlifespan Std.error %change pvalue(Logrank) pvalue(Wilcoxon) Supplement Bacterialgenotype Plasmid Antibiotics

5 WT 108 0 15.79 0.53 n/a n/a n/a Alkalinecontrol CM1 pGreen0029,pUC19 50µg/mlcarb,25µg/mlkan

pabA 94 3 20.08 0.52 Control n/a n/a Alkalinecontrol CM2 pUC19 50µg/mlcarb,25µg/mlkan

pabA+0.1µMPABA 107 0 19.30 0.46 -3.89% 0.0973 0.3296 0.1µMPABA CM2 pUC19 50µg/mlcarb,25µg/mlkan

pabA+1µMPABA 118 0 15.91 0.45 -20.78% <.0001* <.0001* 1µMPABA CM2 pUC19 50µg/mlcarb,25µg/mlkan

pabA+1µMPABA-Glu 110 2 18.58 0.40 -7.49% 0.0030* 0.0277* 1µMPABA-Glu CM2 pUC19 50µg/mlcarb,25µg/mlkan

pabA+10µMPABA-Glu 125 10 16.43 0.46 -18.16% <.0001* <.0001* 10µMPABA-Glu CM2 pUC19 50µg/mlcarb,25µg/mlkan

pabA+10µMfolicacid 93 5 18.20 0.55 -9.36% 0.0052* 0.0061* 10µMSchircksfolicacid CM2 pUC19 50µg/mlcarb,25µg/mlkan

pabA+100µMfolicacid 112 1 15.28 0.45 -23.93% <.0001* <.0001* 100µMSchircksfolicacid CM2 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA 91 3 20.10 0.44 Control n/a n/a Alkalinecontrol CM3 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA+0.1µMPABA 91 6 18.72 0.49 -6.88% 0.0631 0.0477* 0.1µMpABA CM3 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA+1µMPABA 102 1 16.71 0.45 -16.86% <.0001* <.0001* 1µMpABA CM3 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA+1PABA-Glu 101 11 18.11 0.52 -9.94% 0.1733 0.0076* 1µMPABA-Glu CM3 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA+10µMPABA-Glu 106 4 19.00 0.46 -5.49% 0.1817 0.0512 10µMPABA-Glu CM3 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA+10µMfolicacid 110 1 19.20 0.43 -4.49% 0.1901 0.114 10µMSchircksfolicacid CM3 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA+100µMfolicacid 100 3 19.16 0.40 -4.67% 0.0476* 0.0743 100µMSchircksfolicacid CM3 pUC19 50µg/mlcarb,25µg/mlkan

pabA(abgTOE) 62 10 19.90 0.52 Contol n/a n/a Alkalinecontrol CM5 pJ128 50µg/mlcarb,25µg/mlkan

pabA(abgTOE)+0.1µMPABA 121 1 18.55 0.45 -6.72% 0.0049* 0.0184* 0.1µMPABA CM5 pJ128 50µg/mlcarb,25µg/mlkan

pabA(abgTOE)+1µMPABA 104 2 16.07 0.50 -19.19% <.0001* <.0001* 1µMPABA CM5 pJ128 50µg/mlcarb,25µg/mlkan

pabA(abgTOE)+1µMPABA-Glu 104 1 17.01 0.43 -14.47% <.0001* <.0001* 1µMPABA-Glu CM5 pJ128 50µg/mlcarb,25µg/mlkan

pabA(abgTOE)+10µMPABA-Glu 117 0 15.05 0.41 -24.33% <.0001* <.0001* 10µMPABA-Glu CM5 pJ128 50µg/mlcarb,25µg/mlkan

pabA(abgTOE)+10µMfolicacid 119 0 16.65 0.42 -16.30% <.0001* <.0001* 10µMSchircksfolicacid CM5 pJ128 50µg/mlcarb,25µg/mlkan

pabA(abgTOE)+100µMfolicacid 117 0 16.19 0.42 -18.61% <.0001* <.0001* 100µMSchircksfolicacid CM5 pJ128 50µg/mlcarb,25µg/mlkan

Sup.1 WT 91 9 13.85 0.44 Control n/a n/a n/a WT pGreen0029 25µg/mlkan

abgT 114 3 12.68 0.35 -8.48% 0.4312 0.0963 n/a JW5822-1 n/a 25µg/mlkan

Sup.2 WT 108 0 14.58 0.40 Control n/a n/a Alkalinecontrol WT pGreen0029 25µg/mlkan

WT+10µMfolicacid 115 0 15.00 0.39 2.86% 0.4448 0.4588 10µMSchircksfolicacid WT pGreen0029 25µg/mlkan

WT+100µMfolicacid 106 1 14.57 0.48 -0.10% 0.566 0.844 100µMSchircksfolicacid WT pGreen0029 25µg/mlkan

Repeatexpt. WT 102 9 15.96 0.48 Control n/a n/a Alkalinecontrol CM1 pGreen0029,pUC19 50µg/mlcarb,25µg/mlkan

(notshown) WT+10µMfolicacid 98 12 15.96 0.58 0.03% 0.8268 0.4283 10µMSchircksfolicacid CM1 pGreen0029,pUC19 50µg/mlcarb,25µg/mlkan

WT+100µMfolicacid 102 18 16.01 0.48 0.36% 0.866 0.7792 100µMSchircksfolicacid CM1 pGreen0029,pUC19 50µg/mlcarb,25µg/mlkan

pabA 113 10 18.35 0.56 Control n/a n/a Alkalinecontrol CM2 pUC19 50µg/mlcarb,25µg/mlkan

pabA+10µMfolicacid 90 11 17.44 0.64 -4.95% 0.2527 0.2605 10µMSchircksfolicacid CM2 pUC19 50µg/mlcarb,25µg/mlkan

pabA+100µMfolicacid 122 3 15.86 0.40 -13.57% <.0001* 0.0019* 100µMSchircksfolicacid CM2 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA 136 14 18.93 0.50 Control n/a n/a Alkalinecontrol CM3 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA+10µMfolicacid 115 26 19.22 0.52 1.51% 0.6416 0.5684 10µMSchircksfolicacid CM3 pUC19 50µg/mlcarb,25µg/mlkan

abgTpabA+100µMfolicacid 93 11 19.26 0.60 1.71% 0.5868 0.4482 100µMSchircksfolicacid CM3 pUC19 50µg/mlcarb,25µg/mlkan

pabA(abgTOE) 101 23 18.42 0.56 Control n/a n/a Alkalinecontrol CM5 pJ128 50µg/mlcarb,25µg/mlkan

pabA(abgTOE)+10µMfolicacid 124 0 15.84 0.47 -14.00% 0.0005* 0.0035* 10µMSchircksfolicacid CM5 pJ128 50µg/mlcarb,25µg/mlkan

pabA(abgTOE)+100µMfolicacid 105 0 15.77 0.50 -14.36% 0.0003* 0.0041* 100µMSchircksfolicacid CM5 pJ128 50µg/mlcarb,25µg/mlkan

WT(abgTOE) 110 9 16.11 0.50 Control n/a n/a Alkalinecontrol CM4 pGreen0029,pJ128 50µg/mlcarb,25µg/mlkan

WT(abgTOE)+10µMfolicacid 99 4 16.05 0.57 -0.37% 0.9479 0.8905 10µMSchircksfolicacid CM4 pGreen0029,pJ128 50µg/mlcarb,25µg/mlkan

WT(abgTOE)+100µMfolicacid 127 4 15.78 0.39 -2.02% 0.3698 0.8168 100µMSchircksfolicacid CM4 pGreen0029,pJ128 50µg/mlcarb,25µg/mlkan