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EngineeringMicrobialPhysiologywithSyntheticPolymers:CationicPolymersInduceBiofilmFormationinVibriocholeraeandDownregulatetheExpressionofVirulenceGenes.NicolasPerez-Sotoa,b,LaurenMoulea,b,DanielN.Crisanb,c,IgnacioInsuab,c,LeanneM.Taylor-Smitha,b,KerstinVoelza,b,Fran-ciscoFernandez-Trillo*b,candAnne-MarieKrachler.*a,b,daSchoolofBiosciences,bInstituteofMicrobiologyandInfection,cSchoolofChemistry,UniversityofBirmingham,Edgbaston,B152TTBirmingham,UK,E-mail:f.fernandez-trillo@bham.ac.ukdDepartmentofMicrobiologyandMolecularGenetics,UniversityofTexasMcGovernMedicalSchoolatHouston,Hou-ston,TX,77030,USA.E-mail:anne.marie.krachler@uth.tmc.edu.

TableofContents

1.MATERIALS 2

2.MEDIA 2

3.INSTRUMENTATION 2

4.SYNTHESISOFPOLYMERS 3

4.1.SYNTHESISANDCHARACTERISATIONOFP(DMAPMAM)(P1). 34.2.SYNTHESISANDCHARACTERISATIONOFP(APMAM)(P2) 3

5.BACTERIALSTRAINSANDCULTURECONDITIONS 4

6.PLASMIDCONSTRUCTION 5

7.CLUSTERING 5

7.1.IMAGINGOFBACTERIACLUSTERS 5

8.MOTILITYASSAY 6

9.EFFECTOFPOLYMERSONMICROBIALGROWTH 6

9.1.GFPEXPRESSION 69.2.MICROBIALMEMBRANEINTEGRITY 7

10.VIRULENCE 7

10.1.QUANTIFICATIONOFBIOFILMFORMATION 710.2.IMAGINGOFBIOFILMFORMATIONANDQUANTIFICATIONOFEXTRACELLULARDNA 710.3.TRANSCRIPTIONALASSAYS:Β-GALACTOSIDASEACTIVITY 8

11.COLONISATIONANDTOXICITYOFCACO-2CELLS 9

11.1.COLONYFORMINGUNITS 911.2.CYTOTOXICITYINCACO-2CELLS:LDHASSAY 911.3.CYTOTOXICITYINCACO-2CELLS:CAMPASSAY 1011.4.IMAGINGOFCACO-2CELLS 11

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12.COLONISATIONOFZEBRAFISH 11

12.1.ZEBRAFISHCAREANDMAINTENANCE 1112.2.COLONYFORMINGUNITS 1112.3.IMAGINGOFZEBRAFISHLARVAE 12

13.REFERENCES 12

1. MaterialsN-(3-aminopropyl)methacrylamide hydrochloride (APMAm), N-[3-(dimethylamino)propyl]methacrylamide

(DMAPMAm),2,2’-azobis(2-methylpropionitrile)(AIBN)andDulbecco’sModifiedEagle’sMedium(DMEM)werepur-

chasedfromSigma-Aldrich.4,4’-azobis(4-cyanovalericacid)(ACVA)and2-mercaptoethanolwereboughtfromAlfa

Aesar.PolyethyleneglycolstandardswerepurchasedfromAgilentTechnologies.Allotherchemicalswerepurchased

fromFisherScientificandVWRandwereusedwithout furtherpurification.Polymerswere synthesisedby radical

polymerisationasdescribedbelow.

2. Media• LB:10g/Ltryptone,10g/LNaCland5g/Lyeastextract• DMEM:0.265g/LCaCl2,0.0001g/LFe(NO3)3,0.09767g/LMgSO4,0.4g/LKCl,3.7g/LNaHCO3,6.4g/LNaCl,

0.109g/LNaH2PO4,0.084g/LL-Arg,0.0626g/LL-Cys,0.03g/LGly,0.042g/LL-His,0.105g/LL-Iso,0.105g/L

L-Leu, 0.146 g/L L-Lys, 0.03 g/L L-Met, 0.066 g/L L-Phe, 0.042 g/L L-Ser, 0.095 g/L L-Thr, 0.016 g/L L-Trp,

0.12037g/LL-Tyr,0.094g/LL-Val,0.004g/Lcholinechloride,0.004g/Lfolicacid,0.0072myo-inositol,0.004g/Lniacinamide,0.00404g/Lpyridoxine,0.004g/LD-pantothenicacid,0.0004g/Lriboflavin,0.004g/Lthia-

mine,4.5g/Lglucoseand0.584g/LL-Glu

• RedDMEM:Sameformulationplus0.0159g/Lphenolred.

• E3media:0.292g/LNaCl,0.013g/LKCl,0.044g/LCaCl2,0.081g/LMgSO4,0.477g/LHEPESpH7.0

3. InstrumentationPolymerswerecharacterisedbyNuclearMagneticResonance (NMR)andGelPermeationChromatography (GPC).

NMRspectrawererecordedonaBrukerAvanceIIIspectrometeroperatingat300MHzandfittedwitha5mmBBFO

probe.Chemicalshiftsarereportedinppm(δ)referencedtothecorrespondingsolventsignals:DMSO-d6(δ=2.50)andD2O(δ=4.79).GPCwasrecordedonaShimadzuProminenceLC-20A,fittedwithShodexAsaphipakGF-510HQ

(300×7.5mm,5μm)andGF-310HQ(300×7.5mm,5μm)columnsinseries,andequippedwithaThermoFisher

Refractomax521detector.GPCanalysiswascarriedoutusing100mMacetatebufferatpH2.9aseluentat40°Cand

aflowrateof0.6mL·min-1.Molecularweightswerecalculatedbasedonastandardcalibrationmethodusingpoly-

ethyleneglycol.OpticalmicroscopyofV.choleraeclusterswasmeasuredonaNikon-EclipseTE2000-Umicroscope

andPlanApo60x/1.40NAoilDICobjective(Nikon)andcapturedwithQICAMFast1394camera(Qimaging).Absorb-

anceat402nmor595nmwasrecordedonaFLUOstarOmegaplatereader.Absorbanceat420nmwasrecorded

usinga Jenway6305/UVVisiblespectrophotometer.GFP fluorescenceofGFPexpressingV.cholerae cultureswasrecordedaFLUOstarOmegaplate reader.Fluorescent-activatedcell sorting (FACS)wasperformedonanAttune®

AcousticFocusingCytometer(AppliedBiosystemsTM).AgarplateswereimagedusingaChemiDocMPSystem(Bio-

rad).BiofilmswereimagedusingaNikonTE2000-Umicroscopeusinga100xPlanApoobjective(Nikon)andcaptured

withDigitalSightDS-Qi1MCcamera(Nikon).StainedV.choleraeclusterswereimagedusingaZeissAxioObserver.Z1

microscope,Zeiss40x/1.4PlanApochromatobjective,ORCA-Flash4.0camera(Hamamatsu)andZEN2.0.0.10soft-

ware. Stained Caco-2 cultures and Zebrafish larvaewere imaged using a Zeiss AxioObserver.Z1microscopewith

63x/1.4PlanApochromatobjective,ORCA-Flash4.0camera(Hamamatsu)andZEN2.0.0.10software.Spinningdisk

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wasperformedonaNikonEclipse-TimicroscopewithCSU-X1spinningDiskConfocal,PlanApoVC60x1.4NAOil

objective,AndorNeoCMOScameraand488nmexcitationlaserusingNikonElementssoftwareforimageacquisition.

4. SynthesisofPolymers

4.1. Synthesisandcharacterisationofp(DMAPMAm)(P1).

N-[3-(dimethylamino)propyl]methacrylamide(DMAPMAm)(2.2mL,12.025mmol),2,2'-azobis(2-methylpropionitrile)

(AIBN)(19.6mg,0.117mmol)and2-mercaptoethanol(4.0µL,0.056mmol)weredissolvedintoluene(9.5mL).This

solutionwasdegassedunderargonfor10minutesandthenheatedat70°Cunderstirringfor18hours.Afterthis

time,thereactionflaskwasopenedtotheairandthecrudewasprecipitatedtwice:firstintodiethylether(200mL)

andthenintoadiethylether/hexanemixture(1:1)(100mL).Theprecipitatewasfreeze-driedandacrystallinewhite

solidwasobtained(1.66g,87%yield).1H-NMR(300MHz,DMSO-d6)�(ppm):7.51(br,1H,CO-NH),2.96(br,2H,CO-

NH-CH2),2.19(br,5H,CH2-N-(CH3)2+CH3backbone),2.11(br,6H,N-(CH3)2),1.50(br,2H,CH2-CH2-N),0.78(br,2H,CH2

backbone).Mn(bufferGPC)46331,ĐM(bufferGPC)1.14.

Fig.S1.1H-NMR(300MHz,dmso-d6)spectrumofP1.

4.2. Synthesisandcharacterisationofp(APMAm)(P2)

N-(3-aminopropyl)methacrylamide(APMAm)hydrochloride(505.0mg,2.770mmol),4,4’-azobis(4-cyanovalericacid)

(ACVA)(12.4mg,0.033mmol)and2-mercaptoethanol(1.0µL,0.014mmol)weredissolvedinMilliQwater(2.2mL).

Thissolutionwasdegassedunderargonfor10minutesandthenheatedat70°Cunderstirringfor17hours.After

thistime,thereactionflaskwasopenedtotheairandthecrudewasprecipitatedthreetimesintodiethylether(50

mL).Theprecipitatewasfreeze-driedandacrystallinewhitesolidwasobtained(70.0mg,14%yield).1H-NMR(300

MHz,D2O)�(ppm):3.11(br,3H,CH3backbone),2.93(brt,J=7.0Hz,2H,CO-NH-CH2),1.78(br,2H,-CH2-NH2),1.63(br,

2H,-CH2-CH2-NH2),0.98(br,1H,CH2backbone),0.83(br,1H,CH2backbone).Mn(GPC)46997,ĐM(GPC)1.16.

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Fig.S2.1H-NMR(300MHz,D2O)spectrumofP2.

5. BacterialstrainsandcultureconditionsVibriocholeraestrainsusedinthisstudy(TableS1)werederivedfromtheElTorstrainN16961usedasparentalstrain.

Strainswerepropagatedat37oCinLuria-Bertani(LB)brothsupplementedwith50μg/μLspectinomycin,30μg/μL

kanamycinor10μg/μLtetracyclinewhereselectionwasrequired.PlasmidswereintroducedintoV.choleraebycon-jugation,aspreviouslydescribed.1Briefly,aliquotsofovernightculturesofV.choleraeN16961,E.coliDH5acarryingthedesiredplasmid(donor)andanE.coliSM10helperstraincarryingpRK2013weremixedatavolumetricratioof

1:2:2andspottedontobrain-heartinfusion(BHI)agar.Followingovernightincubation,spotsofbacterialgrowthwere

dislodgedandsuspendedin3mLofPBS.100μLofbacterialsuspensionwereplatedontoM9mediacontaining50

μg/μLof spectinomycin.Resulting colonieswere checkedbyPCRand sequencing in the caseofpRW50-oriT con-

structs,andbyscreeningforgreenfluorescenceinthecaseofpMW-gfptransformants.

TableS1.V.choleraestrainsusedinthisstudy.

Strain Description Source or reference N16961 Wild-type; O1 biovar El Tor. Heidelberg et al.2

NP5001 N16961 carrying promoterless pRW50-oriT plas-mid; TetR. This study

NP5002 N16961 carrying pRW50-oriT plasmid containing the upstream region of toxT promoter; TetR. This study

NP5003 N16961 carrying pRW50-oriT plasmid containing the upstream region of ctxAB promoter; TetR. This study

NP5004 N16961 carrying pRW50-oriT plasmid containing the upstream region of tcpA promoter; TetR. This study

NP5005 N16961 carrying pRW50-oriT plasmid containing the upstream region of aphA promoter; TetR. This study

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NPMW1 N16961 carrying pMW-gfp plasmid; SpectR. Ritchie et al.3 BH1651 luxOD47E Ng et al.4

6. PlasmidconstructionPrimersusedforcloningandsequencingtheconstructsusedinthisstudyarelistedinTableS2andweredesignedbasedonthesequenceofV.choleraeN16961.2Primerswerepairedasappropriatetoamplifyupstreamregionsof

toxT,ctxAB,tcpA,andaphA(Fig.4A).AmplifiedDNAwasdigestedusingEcoR1andHindIII,andligatedintodigested

pRW50-oriTvector(giftfromtheGraingerlab,UniversityofBirminghamhttp://graingerlab.com).Thevectorisade-

rivativeofpRW50,5whichhasbeenmodifiedby inserting theoriT sequence fromthevectorpRK2.6Theresulting

reporterconstructswereusedtotransformV.choleraeandtheresultingstrainsarelistedinTableS1.

TableS2Primersusedinthisstudy,withrestrictionsitesunderlined.

Primer Sequence (5´-3´) pRW50 F GTTCTCGCAAGGACGAGAATTTC pRW50 R AATCTTCACGCTTGAGATAC aphApF1 TGCAGAATTCCTGGTTAACAAATCGCTAAATGTCAG aphApR1 ATTCAAGCTTGTGTGGTAATGACATGTCTTCAATC toxTpF1 TGTAGAATTCGATAAGATAACAGCCATATTCGTGG toxTpR1 GATCAAGCTTTCCCAATCATTGCGTTCTACTC ctxABpF1 GCTTGAATTCCTGTGGGTAGAAGTGAAACGG ctxABpR1 TCATAAGCTTTATCTTTACCATATAATGCTCCCTTTG tcpApF1 CTTAGAATTCGGTCTTATCATGAGCCGCC tcpApR1 TGATAAGCTTTGCATATTTATATAACTCCACCATTTGTG

7. Clustering

7.1. Imagingofbacteriaclusters

AnovernightcultureofV.cholerae(asdescribedinsection5)wasdilutedtoanOD600of1.0inDMEMwithoutphenol

redandpolymerswereadded to final concentrations ranging from0-500μg/mL (Fig.1 andFig.S3).For imaging,

aliquotsoftheseculturesweretakenafter15and60minofincubationandmountedwithProLong®AntifadeGold

solution(LifeTechnologies).CuredsampleswerevisualisedusingaNikon-EclipseTE2000-UmicroscopeandPlanApo

60x/1.40NAoilDICobjective(Nikon)andcapturedwithQICAMFast1394camera(Qimaging).Representativeimages

weretakenusingNikonNIS-ElementssoftwareandpreparedwithImageJandCorelDrawX5software.

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Fig.S3.SpinningdiskfluorescentmicrographofGFP-V.choleraeN16967incubatedinPBS(pH7.4)for15mininthepresence500μg/mLofP1(a)orP2(b).Maximumintensityprojectionsareshown,withcolor-codedZ-depth.Totaldepthoftheclustersshownhereare17and27µm,respectively.

8. MotilityassayMotilityofV.choleraewasassessedaspreviouslydescribed.7Briefly,overnightculturesofV.choleraeN16961(asdescribedinsection5)weredilutedtoanOD600of1.0andthenincubatedintheabsenceandpresenceofpolymers

overnight.Analiquotof1μLwasthenplacedintosoftagarLBplates(0.4%agar)andincubatedovernightatroom

temperature.Forcontrolsinthepresenceofhighconcentrationsofsalt,followingincubationwithpolymers,samples

weretreatedwith1mLofPBSbuffercontaining200mMNaCl.Whenthesolutionturnedcloudyanaliquotof1μL

wasplacedintosoftagarplatesandincubatedovernightatroomtemperature.Afterincubation,picturesoftheplates

weretakenusingaChemiDocMPSystem(Bio-rad).

Fig.S4.MotilityofV.choleraeN16961insoftagarplatesintheabsenceorpresenceofP1,P2and200mMNaCl.

9. Effectofpolymersonmicrobialgrowth

9.1. GFPexpressionOvernightculturesofGFP-expressingV.cholerae(asdescribedinsection5)weredilutedintoDMEMwithoutphenol

redcontaining50μg/μLofspectinomycintoanOD600of0.02asstartingdensity.PolymersaliquotsinDMEMwere

thenaddedtogivethedesiredfinalconcentrationsin200μLcultureusinga96-wellplate.Theplatewascovered

withaBEM-1breatheeasygaspermeablemembrane toavoidevaporationand incubatedat37 oCwithconstant

shakingat200rpm.GFPfluorescencewasrecordedevery30minutesover24hoursusingaFLUOstarOmegaplate

reader.

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Fig.S5.RelativeFluorescentUnits (RFU) forGFPexpressingV.choleraeN16961 intheabsenceandpresenceof increasingamountsofP2.InitialOD600=0.02.Resultsaremeans±s.e.m.ofthreeindependentexperiments.

9.2. Microbialmembraneintegrity

MembraneintegrityofV.choleraecellswasassessedbyFACSusingtheLIVE/DEADBacLightTMkit(LifeTechnologies).Overnightcultures(asdescribedinsection5)weredilutedtoanOD600of1.0in1mLofDMEMwithoutphenolred,

andcontainingpolymersatfinalconcentrationsrangingfrom0to500μg/mLandincubatedfor20hours.Following

incubation,sampleswerestainedaccordingtothemanufacturer’sinstructions.ReadingsweretakenonanAttune

FlowCytometer,ataflowof100μL/mincountingto10,000events.Samplescontainingnopolymerwereusedas

“non-permeable/viable”controls.Inthecaseof“highpermeability/dead”controls,DMEMwasreplacedby70%2-

propanolandincubatedfor1h.Priortostaining,2-propanolwasremovedandcellswerewashedonceandresus-

pendedinfreshDMEMtocarryoutthestainingprocedure.

10. Virulence

10.1. Quantificationofbiofilmformation

Theamountofbiofilmwasdeterminedbycrystalvioletstaining,aspreviouslydescribed.8Briefly,anovernightculture

ofV.cholerae(asdescribedinsection5)wasdilutedtoanOD600of0.2infreshDMEMmediaandincubatedina96-

wellplateovernight(i.e.20h)at37oCwithshakingat200r.p.m.,inthepresenceandabsenceofpolymers.Cultures

werethenremovedandbiofilmwasrinsedwithPBSandstainedwith200μLof1%crystalvioletsolutioninwater.

After30minutes, thecrystalviolet solutionwas removedand thewellwas rinsedagainwithPBS.200μLof95%

ethanolwasthenaddedtoeachwelltodetachthedye.Theamountofbiofilmwasdeterminedbymeasuringat595

nmusingaFLUOstarOmegaplatereader.

10.2. ImagingofbiofilmformationandquantificationofextracellularDNA

BiofilmsofGFP-V.choleraeweregrown,asdescribedabove,for20hoursat37°Cin96-wellglass-bottomplatescon-

tainingbacteriaataninitialOD600of0.2inDMEMonly,orDMEMcontainingP1orP2atconcentrationsrangingfrom

5to500μg/mL,inatotalvolumeof200μL.InthecaseofDNAaseItreatedsamples,2unitsofDNAseI(NEB)were

addedandculturesincubatedforanother2hoursat37oC.Followingincubation,plateswererinsedwithPBS,samples

fixedwith4%formaldehydeinPBSfor15minutesandthenwashedwithPBS.DNAwasstainedwith10μg/mLHoechst

inPBSfor10minutes.SampleswereimagedusingaZeissAxioObserver.Z1microscope,Zeiss40x/1.4PlanApochro-

mat,objective,ORCA-Flash4.0camera(Hamamatsu)andZEN2.0.0.10software.ImageswereprocessedusingImage

JandCorelDrawX5.ExtracellularDNAwasdeterminedfromtherelativepixelintensityofbluechannelfrompictures

takenwithaNikonTE2000-Umicroscopeusinga100xPlanApoobjective(Nikon)andcapturedwithDigitalSightDS-

Qi1MCcamera(Nikon).Fromeachpicture,theintensityofpixelswasmeasuredfromthebluechannelusingImageJ.

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Fig.S6.IntensityofpixelsfromthebluechannelfromconfocalmicrographsofGFPexpressingV.choleraeN16961culturesintheabsence(●)andpresenceofP1(A)orP2(B)(○).Polymer-inducedclusterswerealsotreatedwithDNAseI(●).Dataareindividualmeasurementsfromatleastfourrepresentativeimages,meansandstdev.Analysisofvariance(ANOVA),followedbyTukey’sposthoctest,wasusedtotestforsignificance.Statisticalsignificancewasdefinedasp<0.05(*).

10.3. Transcriptionalassays:β-galactosidaseactivityOvernightculturesofV.choleraereporterstrainsweregrowninLBat37oC,asdescribedinsection5.Priortoinfec-tion,cultureswereadjustedtoanMOIof10andincubatedat30oCfor1hourin1mLofDMEMwithoutphenolred,

intheabsenceandpresenceofpolymers(500μg/mLP1and50μg/mLP2).Caco-2cellswerewashedwithPBStoremovemediacontainingantibioticsandinfectionswerestartedbytransferringtheaboveculturesofbacteriainto

thewells.Plateswerecentrifugedat1000xgfor5minat20oCtosynchronisetheinfections.After7hoursofincuba-

tionat37oCunder5%CO2,Caco-2werewashedwithPBSandhomogenizedwith1mLof0.5%tritonX100inPBSfor

5minutes. β-galactosidase activity was assayed as previously described.9 Briefly, ortho-nitrophenyl-β-galactoside

(ONPG)wasdissolvedatafinalconcentrationof0.2mMinbufferZwith0.27%of2-mercaptoethanol.Bacterialcells

weredisruptedaddingadropoftolueneandadropof1%sodiumdeoxycholatelettingthetubeopenaftervortexing

forabout30minutestoletthetolueneevaporatesatroomtemperature.100μLofthelysissolutionwereaddedwith

2.5mLofONPGsolutionandtimewasrecorded.Whenthesolutionturnsyellow,1mLof1MNa2CO3wasaddedto

stopthereaction.Absorbanceat420nmwasrecordedusingaJenway6305/UVVisiblespectrophotometer.Tran-

scriptionalactivity(inMillerUnits)wascalculatedaspreviouslydescribed.9Percentagereductionwascalculatedas

thepercentagedifferencebetweenmillerunitsintheabsenceandpresenceofthepolymer.

Fig.S7.PromoteractivitiesofaphA-andtcpA-lacZfusionsfollowinginfectionofCaco-2cellsfor7hoursintheabsence(black)orpresenceof50μg/mLP2(hollow).Student’spairedt-testwasusedtotestforsignificance.Statisticalsignificancewasde-finedasp<0.05(*)orp<0.005(***).Forconsistency,therangeofmillerunitsisthesameastheoneusedintheothertran-scriptionalassays(Fig.4BandFig.5H).

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11. ColonisationandtoxicityofCaco-2cells

11.1. Colonyformingunits

OvernightculturesofV.choleraeN16961weregrown inLBat37 oC,asdescribed in section5.Prior to infection,cultureswereadjustedtoanMOIof10andincubatedat30oCfor1hourin1mLofDMEMwithoutphenolred,inthe

absenceandpresenceofpolymers.Caco-2cellswerewashedwithPBStoremovemediacontainingantibioticsand

infectionswerestartedbytransferringtheaboveculturesofbacteriaintothewells.Plateswerecentrifugedat1000x

gfor5minat20oCtosynchronisetheinfections.After7hoursofincubationat37oCunder5%CO2,Caco-2were

washedwithPBSandhomogenizedwith1mLof0.5%tritonX100inPBSfor5minutes.Oncethedebriswasdislodged

fromthewell,sampleswereserialdilutedandplatedoutontoTCBSagar.Plateswereincubatedat37oCovernight

forCFUcounting.

Fig.S8.NumberofcolonyformingunitspermL(CFU/mL)ofGFPexpressingV.choleraeN16961(A)orBH1651mutant(B)fromCaco-2cells incubatedintheabsenceandpresenceofpolymertreatedV.choleraecultures,followingwashingandlysisofCaco-2cells.Resultsaremeans±s.e.m.ofthreeindependentexperiments.Analysisofvariance(ANOVA),followedbyTukey’sposthtest,wasusedtotestforsignificance.Statisticalsignificancewasdefinedasp<0.05(*),p<0.01(**)orp<0.0001(****).

11.2. CytotoxicityinCaco-2cells:LDHassayOvernightculturesoftheGFP-expressingV.choleraeweregrowninLBat37oC,asdescribedinsection5.Priortoinfection,cultureswereadjustedtoanMOIof10andincubatedat30oCfor1hourin1mLofDMEMwithoutphenol

red,intheabsenceandpresenceofpolymers.Caco-2cellswerewashedwithPBStoremovemediacontaininganti-

bioticsandinfectionswerestartedbytransferringtheaboveculturesofbacteriaintothewells.Plateswerecentri-

fugedat1000xgfor5minat20oCtosynchronisetheinfections.After7hoursofincubationat37oCunder5%CO2,

supernatantswereusedtomeasurelactatedehydrogenase(LDH)activityusingLDHCytotoxicityDetectionKit(Takara

Clonetech)accordingtothemanufacturer’sinstructions.ThedatawasexpressedaspercentageofExtracellularLDH,

normalised tountreatedcells (0%)and0.1%TritonX-100 lysedcells (100% lysis)andwascalculatedaccording to

Equation1:

Equation1:%"#$%&'())*)&%,-. = 01234501234 6789:;8:<01234 =9>8?7 501234 6789:;8:<

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Fig.S9.PercentageofextracellularLDHinCaco-2culturesincubatedintheabsenceandpresenceofP1andP2.ResultswerenormalisedtountreatedCaco-2cells(0%)andcellslysedwithTritonX-100(100%).Resultsaremeans±s.e.m.ofthreeinde-pendentexperiments.Analysisofvariance(ANOVA),followedbyTukey’sposthoctest,wasusedtotestforsignificance.Sta-tisticalsignificancewasdefinedasp<0.01(**),p<0.001(***)orp<0.0001(****).

Fig.S10.PercentageofextracellularLDHinCaco-2culturesincubatedintheabsenceandpresenceofpolymertreatedV.chol-eraeN16961cultures(left)orBH1651mutant(right).ResultswerenormalisedtountreatedCaco-2cells(0%)andcellslysedwithTritonX-100(100%).Resultsaremeans±s.e.m.ofthreeindependentexperiments.Analysisofvariance(ANOVA),followedbyTukey’sposthoctest,wasusedtotestforsignificance.Statisticalsignificancewasdefinedasp<0.05(*),p<0.01(**)orp<0.0001(****).

11.3. CytotoxicityinCaco-2cells:cAMPassay

OvernightculturesofV.choleraeweregrowninLBat37oC,asdescribedinsection5.Priortoinfection,cultureswereadjustedtoanMOIof10andincubatedat30oCfor1hourin1mLofDMEMwithoutphenolred,intheabsenceand

presenceofpolymers.Caco-2cellswerewashedwithPBStoremovemediacontainingantibioticsandinfectionswere

startedbytransferringtheaboveculturesofbacteriaintothewells.Plateswerecentrifugedat1000xgfor5minat

20oCtosynchronisetheinfections.After7hoursofincubationat37oCunder5%CO2,supernatantswereusedto

measuresampleusingCyclicAMPCompetitiveELISAkit(Invitrogen)followingmanufacturer´sinstructions.Concen-

trationofcAMP,expressedinpmol/mL,wasestimatedfromacalibrationcurvemadewithpurecAMP.

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Fig.S11.LevelsofcAMPproductioninCaco-2cellsintheabsenceandpresenceofpolymertreatedV.choleraeN16961(A)orBH1651mutant(B)cultures.V.choleraecultureswereadjustedtoanMOIof10,andincubatedintheabsenceorpresenceofpolymersfor1hpriortoinfectionofculturedCaco-2intestinalepithelialcellsfor7hrs.Resultsaremeans±s.e.m.ofthreeindependentexperiments.Analysisofvariance(ANOVA),followedbyTukey’sposthoctest,wasusedtotestforsignificance.Statisticalsignificancewasdefinedasp<0.05(*),p<0.01(**)orp<0.0001(****).

11.4. ImagingofCaco-2cells

Caco-2cellswereseededontosterilisedglasscoverslips inserted intothewellsof theplateand incubated inthe

absenceorpresenceofV.choleraeasdescribedabove.ImagingofinfectedCaco-2cellswasthendonebyfixingthe

sampleswith4%formaldehydeinPBSfor15minutesandthenwashingwithPBS.Cellswerepermeabilisedbyadding

0.1%TritonX-100inPBSandincubationatroomtemperaturefor5minutes,andwashedthreetimeswithPBS.Sam-

pleswerethenstainedwith10μg/mLHoechstand66ng/mLofrhodamine-phalloidininthedarkfor10minutesto

visualiseDNAand F-actin, respectively. Stainingwas followedby threewashing stepswithPBS (5minutes each).

Samplesweremountedusingantifadegoldmountingsolution(Life-Technologies)andcuredovernightat22oCprior

tovisualisation.SampleswereviewedunderaZeissAxioObserver.Z1microscopewith63x/1.4PlanApochromatob-

jective.ImagesweretakenusingZenProsoftware(Zeiss)andprocessedusingImageJsoftware.

12. ColonisationofZebrafish

12.1. Zebrafishcareandmaintenance

Zebrafish(DanioreriowildtypestrainAB)werekeptinarecirculatingtanksystemattheUniversityofBirmingham

AquaticFacility.Zebrafishwerekeptundera14h-10hlight-darkcyclewithwatertemperaturemaintainedat28oC.

Zebrafishcare,breedingandexperimentswereperformedinaccordancewiththeAnimalScientificProceduresAct

1986,underHomeOfficeProjectLicense40/3681.Aftercollectionofeggs,larvaewerekeptinadiurnalincubator

undera14h-10hlight-darkcyclewiththetemperaturemaintainedat33oC.Eggsweremaintainedat40eggsper50

ml inE3mediaplus0.00003%methylenebluefor8handE3mediaplus26.6µg/ml1-phenyl-2-thiourea(PTU)to

inhibitmelanisation.Thefishlineusedwaswild-typeABzebrafish.Allzebrafishcareandhusbandryprocedureswere

performedasdescribedpreviously.10

12.2. Colonyformingunits

Prior to infection,106and107CFU/mLofV. choleraewere incubated in3mLofE3medium in thepresenceand

absenceofpolymers.After1hourofincubationtoensureclusterformation,2mLofsupernatantwereremovedfrom

theseculturesandreplacedwith2mLofE3mediumor2mLofE3mediumcontainingzebrafishlarvae(5dayspost

fertilisation–d.p.f).Additionally,the2mLofsupernatantweremixedwith3mLofE3mediumor3mLofE3medium

containingzebrafishlarvae(5dayspostfertilisation–d.p.f),toevaluatetheabilityofunclusteredbacteriatocolonise

thefish.V.choleraecultureswerethenincubatedwithrotationat25oCfor6hours.LarvaeweretheneuthanisedwithanoverdoseofTricaine-S(1600μg/mL)andhomogenisedbywashingthelarvaewithPBSandincubatingthem

in1%TritonX-100for30minutes.Lysateswerepassedseveraltimesthroughaneedletohomogeniseand100μLof

theresultingsolution,aswellasserialdilutions,wereplatedontoTCBSagarandcoloniescountedfollowingovernight

incubationat37oC.

ChemicalScience ElectronicSupplementaryInfo(ESI)

S12

12.3. Imagingofzebrafishlarvae

Visualisationofzebrafishlarvaewasdonebydirectlymountingthelarvaein0.4%lowmeltingpointagarosecontain-

ing160μg/mLofTricaine-S.SampleswereviewedunderaZeissAxioObserver.Z1microscopewith63x/1.4PlanAp-

ochromatobjective.ImagesweretakenusingZenProsoftware(Zeiss)andprocessedusingImageJsoftware.

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