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UCL INSTITUTE OF CHILD HEALTH

MRes in Biomedicine

Mini Project

COVER SHEET

Due midnight 20th March 2011

CANDIDATE NUMBER SBDH0

Word count 7,488



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Identifying Enteropathogenic

Escherichia coli effector proteins

responsible for blocking host pro-

inflammatory responses.

Supervisor:Dr.MarkLucas



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Abstract

Enteropathogenic Escherichia coli (EPEC) establish long-term infections in the

gastrointestinaltractbyinjectingspecificeffectorproteinsintohostepithelialcells.The

bacteriauseaproteincomplexknownasatypeIIIsecretionsystem(T3SS)toformpores

inthehostplasmamembrane,throughwhichbacterialproteinscanbetranslocatedinto

hostcells,wheretheyeffectcellsignalling.EPECinfectionleadstothemodificationof

host cell cytoskeleton resulting in the effacementofmicrovilli and loss ofabsorptive

surfacearea,oneofthemaincausesofthediarrheagenicphenotypeseenasaresultof

EPECinfections.RecentresearchhasrevealedthatEPECisalsocapableofblockinghost

inflammatory signalling,which results in a depleted host immune response allowing

long-term infection. This project focuses on investigating several different EPEC

effectors and their inhibitory effects on the activation of mitogen activated protein

kinases (MAPKs), which play significant roles in various cellular activities and are

activated during inflammatory responses. Human cells were infected in vitro with

variousEPECstrains,whereindividualgeneislandshadbeenknockedout,toidentify

which genes were responsible for the inhbition of MAPK activation. Once potential

geneshadbeenidentified,cellsweretransfectedwithmammalianexpressionvectors

carrying the selected genes to confirm if these individual geneswere causingMAPK

inhibition. Thisproject identifiestwoEPEC effectorproteins,NleE1 andNleB1,which

potentially play a role in the inhibition of intracellular p38 and JNK phosphorylation

duringEPECinfection.



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Acknowledgements

IwouldliketothankmysupervisorsDr.MLucasandDr.M

Bajaj-Elliott for their guidance, time and feedbackduring

thisproject.



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TableofContents: Abstract.......................................................................................................................................3

Acknowledgements.................................................................................................................4

Abbreviations ...........................................................................................................................6

Introduction..............................................................................................................................7

MaterialandMethods ......................................................................................................... 15

Results...................................................................................................................................... 24

EffectsofT3SSonactivationofMAPKsduringEPECinfection....................................... 24

EffectsofΔPP4EPECmutantonMAPKphosphorylation .................................................27

IL-1βandTNFαinducedMAPKphosphorylationpostEPECinfections ......................30

ImmunofluorescentimagingoftheeffectsofEPECinfections ....................................... 33

CloningofEPECeffectorgenes...................................................................................................38

TransfectionofEPECeffectorvectorsintoHEp-2cells .....................................................43

Discussion............................................................................................................................... 48

References .............................................................................................................................. 52



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Abbreviations

EPEC – EnteropathogenicEscherichiacoli

E.Coli – Escherichiacoli

DEC – DiarrheagenicE.coli

BFP – Bundle-formingPilus

LEE – LocusofEnterocyteEffacement

T3SS – TypeIIISecretionSystem

Esp – EPECSecretedProteins

Nle – Non-LEE

MAPK – MitogenActivatedProteinKinases

(P-)p38 – (Phospho-)p38MAPK

(P-)JNK – (Phospho-)c-JunN-terminalKinase

(P-)ERK1/2 – (Phospho-)ExtracellularSignalRegulatedKinases1/2

NF-κB – NuclearfactorkB

PCR – PolymeraseChainReaction

WT – WildTypeEPEC2348/69

MOI – MultiplicityofInfection

IL-1β – Interleukin-1β

TNFα – TumourNecrosisFactorα



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Introduction

Escherichia coli is a commensal gram-negative bacillus which is the predominant

facultativeanaerobefoundinthe intestinal tractofmammalian species (Sack, 1975).

Whilst the majority ofE. coli strains are non-pathogenic, a minority of strains have

acquiredvirulencefactorswhichcanresultinsignificantpathologicaleffects,leadingto

infections of thegastrointestinal tract,urinary tractormeninges(Kaperet al .,2004).

This project focuses on enteropathogenic Escherichia coli (EPEC), which can cause

severeinfantilediarrhoea.Diarrhoealdiseasesarecurrentlythesecondlargestcauseof

deathtochildrenundertheageof5andarethoughttokillupto1.5millionchildren

everyyear,themajorityofwhichoccurindevelopingcountries(UNICEF/WHO,2009).

SixpathotypesofdiarrheagenicE.coli(DEC)havebeencharacterised;EnterotoxigenicE.

Coli , Enteroinvasive E. Coli , Enteroaggretative E. Coli , Enteropathogenic E. Coli ,

EnterohaemorhagicE.Coli andDiffuselyadherentE.Coli.ThestagesofDECinfectionare

comparable to infectionswith other entericbacterialpathogens, including adherence

andcolonization,evasionormodificationofhostdefencesandmultiplication,leadingto

eventual damage of host tissue (Nataro and Kaper, 1998). Enteropathogenic E. coli

(EPEC)werethefirstpathotypeof DECtobeidentifiedbutunlikemostotherDEC,EPEC

donotutiliseenterotoxins astheirmainmethodofpathogenesis (Jerseetal .,1990).

EPECstrainsutiliseaspecificmechanismofpathogenesisinthehumangastrointestinal

tract, where the bacteria attach directly to the host enterocyte apical membrane,



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causing morphological and functional changes (Hodges and Gill, 2010). Initially the

bacterial ‘bundle-forming pilus’ (BFP) allows interactions betweenEPEC and thehost

cell,afterwhichtheproductsofthegenomicpathogenicityisland‘Locusofenterocyte

effacement’ (LEE) allows intimatebacterialadherenceandcausesmodificationof the

hostcellcytoskeleton(NataroandKaper,1998).Thisprocessisknownasattachingand

effacement(A/E)lesionformation,whichleadstotheeffacementofmicrovillionthe

host cell membrane and the accumulation of actin beneath adherent bacteria. The

effacementofmicrovilli isconsidered tobeone of the main causes ofdiarrheagenic

symptomsduetothelossinabsorptivesurfacearea(Hecht,2001).

The LEE encodes a Type III secretion system (T3SS), a complex found inmany other

gram-negative bacteria, consisting of ~20 different structural proteins which form a

needlecomplexwhichfacilitatestheinjectionofEPECproteinsintohostepithelialcells

(Hecht,2001)(Figure1).TheinjectedproteinsareknownasEPECeffectorproteinsand

alter signallingtransductionpathwaysin thehost cell,allowingsuccessful adherence,

disruptionoftheintestinalbarrierandblockingofspecifichostinflammatoryresponses

(Deanetal .,2005).Theseinteractionscausethechangesinthehostcellcytoskeleton,

whichleadstotheformationofactinpedestalsorcupsatthesiteofbacterialadherence

(Nougayredeetal .,2003).ThefirstEPECeffectorproteinswerefoundtobeencodedon

theLEEalongsidetheT3SSstructuralgenes(McDanielandKaper,1997).Sequencingof

the protypic EPEC strain E2348/69 has revealed many other non-LEE (Nle) effector

genes,theproductsofwhichareinjectedalongwithLEEeffectorsintohostepithelial



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cells,viatheT3SS(Tobeetal .,2006).

Figure1:ModelofEPECLEE-encodedtypeIIIsecretionsystem.T3SSspansbothEPECandhost

membranewhereEPECsecretedproteinsEspBandEspDformapore,whichallowstheinjection

ofEPEC effector proteins into host cytoplasm.Model also shows interactions between EPEC

encodedTirreceptor,whichbindstobacterialintimin.ThisallowsintimateattachmentofEPEC

on host cell and causes the formation of actin pedestals leading to microvilli effacement.

AdaptedfromCellietal.(2000).

Two essential EPEC effector proteins are EspB and Tir, both coded on the LEE

pathogenicitygeneisland.EPECsecretedproteinB(EspB)wasinitiallythoughttoonly

createthetipoftheT3SS,whichformsporesinthehostcellmembrane(Wachteretal .,

1999),however recent studieshavealso suggested that itmay alsohaveaneffector



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role, due to its intracellular presenceand human cells transfectedwith an inducible

plasmidcontainingtheespBgenecausedasignificantchangeinhostcellmorphology

(Tayloretal .,1999).EPECmutantslackingexpressionofthe espBgeneareunableto

undergo attaching and effacing activity due to the lack of a functional T3SS.

Translocatedintiminreceptor(Tir)issecretedbytheT3SSduringEPECinfectionsinto

theepithelialcell,whereitismodifiedbythehostcellandconsequentlymovedintothe

cellularmembraneandactsasafunctionalreceptor( Kennyetal .,1997).ThisallowsTir

tointeractwithintimin,aproteinexpressedonthesurfaceofEPECallowingintimate

adhesionandactinnucleationinthehostcell(Kennyetal .,1997).

TheadditionaleffectorproteinsencodedbyEPEChavespecifictargetsandfunctionsin

thehostcellandcollaboratetogethertoallowlong-termadherenceandcolonisation.

Interestingly, EPEC infections result in a relatively low inflammatory response when

comparedtoothersimilarvirulententericbacteria.HoweverstudieswithEPECmutants

revealed that the flagellin of EPEC can cause the release of interleukin8 (IL-8) from

epithelial cells in vitro (Zhou et al ., 2003). However this response is only seen with

specificgene islandmutantsand atasigficantly lower levelwith thewild typestrain

(WT)(Zhouetal .,2003).

SubsequentlyaparticulargroupofEPECeffectorproteinswereshowntointerferewith

host inflammatory signalling pathways (Ruchaud-Sparagano et al ., 2007), including

proteinsoftheNF-κBpathwayandmitogenactivatedproteinkinases(MAPKs)(Pearson



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etal .,2011),specificallyp38MAPK,extracellularsignalregulatedkinases(ERK)andc-

Jun N-terminal kinases (JNKs), all of which play a role in regulating IL-8 production

(Hoffmannetal .,2002).MAPkinasesarealargegroupofproteinsvitaltotheregulation

ofcellgrowth,survival,differentiation,migrationandmitogenesis.Theycanbegrouped

into5mainfamilies:MAPKerk1/2

,MAPKp38

,MAPK jnk

,MAPKerk3/4

andMAPKerk5,

eachwith

their own signalling cascades and cellular functions (Widmann et al ., 1999). These

proteinkinasesareusuallyfoundinthecytoplasmandareactivatedbyotherkinases

known as MAPK kinase (MKK), which phosphorylate specific serine, threonine or

tyrosineresiduesandallowtheMAPKs in turn tophosphorylate theirown particular

substrate.ItisthoughtthatEPECseffectorproteinseitherblockthephosphorylationof

theseMAPKsoractually cleave specifickinases (Baruchet al .,2011),resulting inthe

depleted innate immune response and changes in cell morphology leading to the

effacementofmicrovilliinthegastrointestinaltract.

The4non-LEEEPECeffectorproteinscoveredinthisprojectareNleB,NleD,NleEand

NleF,andarecodedonseveraldifferentpathogenicityislands.Previousresearchinto

non-LEE EPEC effector proteins has revealed that they can have various affects on

specifichostcellsignallingpathways,howevermanyoftheirfunctionsarestillunknown

(DeanandKenny,2009).Forexamplethemechanismsbywhichtheseeffectorsaffect

the NF-κB pathwayhave been identified.Two EPECeffectorproteinsNleEand NleB,

foundon the gene island IE6 ofthe EPECgenomehavebeenshownto decreasethe

levelofNF-κBmediatedIL-8expressionindifferentmanners.NleEstabilisestheNF-κB



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inhibitorIκBbypreventing itsphosphorylationandsubsequentdegradation(Nadleret

al .,2010,Newton etal .,2010),whereasNleBblocksTNFαmediatedNF-κBactivation

(Newtonetal .,2010).TheeffectorNleCfromthegeneislandPP4hasalsobeenshown

tointerferewithNF-κBsignallingbycleavingthep65subunitoftheNF-κBdimer(Baruch

etal .,2011,Muhlenetal .,2011,Pearsonetal .,2011).HoweverinterferenceoftheNF-

κBpathwayisnotsolelyaccountableforthelowinnateimmuneresponseandlowIL-8

products.It isthoughtthatthecombinationofvariouseffectorproteinsaffectingboth

NF-κBandMAPKsignallingtogetherareresponsible.However,thewaythesespecific

effectorsaffectMAPKsignallingisstillnotfullyunderstood,butcurrentlystudieshave

startedto focusonunderstandingthe targetsandmechanismsoftheseeffectors.An

exampleistherecentidentificationoftargetsforNleDfromthegeneisland PP4,which

causes the cleavageof the MAP kinases JNK and p38 ata conserved activation loop

(Baruchetal .,2011).

This project aims to investigate the effects of several different EPEC effector and

secretoryproteins,includingEspB,Tir,NleB,NleD,NleEandNleFandwhataffectthey

haveonhost cytoskeletonstructure andphosphorylationof thethreeMAPK families

p38, ERK1/2 and JNK, as a result ofbacterial infection. The ERK1/2 MAP kinasesare

activated mostly by external factors such as growth factors, phorbol esters and

cytokinesbutalsobyosmoticorcytoskeletalstress(Lewisetal .,1998)andareinvolved

in theregulation ofcellproliferation,differentiation,mitogenesisandapoptosis. JNKs

areactivatedbycytokines,DNAdamagingagents(e.g.UVlight),cellstressandalackof



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cellulargrowthfactorstimulation(KyriakisandAvruch,2001).JNKshavebeenlinkedto

roles incell survival, apoptosis and reactions tocell stress.p38 isa family ofMAPKs

involvedinthenormalfunctionofimmuneandinflammatoryresponsesbyregulatingT

celldifferentiation,apoptosisandinterferongammaproduction(IFNγ)andisactivated

byspecificcellsof the immune systemviaextracellularmediators, such ascytokines,

chemokinesandPAMPs(OnoandHan,2000).

Infecting human cell lines in vitrowith different EPECmutants, where the genes for

these differenteffectorsare inactive,allows theanalysis their effectson intracellular

MAPK levels. Itwas expectedthatdifferent gene islandmutantswould showvarying

levels of inhibition on phosphorylation of the different MAP kinases. These findings

could then be used to determine which potential EPEC effectors were causing the

resulting phenotype. With the aim of eliminating problems of redundancy between

different effector pathways and confirming which individual effector proteins were

causinginhibition,thespecificgenesforthesedifferentproteinswereinsertedintothe

shuttlevectorpCMV-Script(Figure2)toallowexpressionineukaryoticcelllines.Once

suitable plasmids containing the correct insert could be identified, they would be

extracted and transfected into human HEp-2 cells and their effects on MAPK

phosphorylationanalysed.



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pCMV-Script

Figure 2: Physical map of plasmid

pCMV-Script (Agilent Technologies).

EPEC effector genes were inserted

intomultiplecloningsite(MCS)and

expressed in HEp-2 cells via the

human cytomegalovirus (CMV)

promoter.



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MaterialandMethods

BacterialStrains,CellCultureandInfections

HEp2 (HEl-R) and Caco-2 cells were grown in Dulbecco’s modified Eagle’s medium

(DMEM) (Invitrogen)with GlutaMax(synthetic L-glutamine, Invitrogen) and10%heat

inactivated foetal calfserum (FCS, Sigma), 1%non-essentialamino acids (Sigma),100

mg/mlpenicillin(Invitrogen)and100mg/mlstreptomycin(Invitrogen)at37°Cin5%CO2

inside T75 (filtered) flasks.Cellsused inthisstudywerebetweenpassage41and46.

Once the cells had grown to 90-100% confluence they were detachedusingTrypsin

(Invitrogen) and EDTA (Ethylenediaminetetraacetic acid, Invitrogen) and were

resuspended(either1:5 or1:10dilution) innewmedia. For infectionwithEPECcells

were aliquoted into 12-well plates and grown to 100% confluence, reaching a final

densityof~1x106cellsperwell.Priortoinfectioncellswerewashedtwicewithsterile

PBSbefore1mlofantibioticandserumfreemedia(DMEM+GlutaMax,1%Non-essential

AAs)wasadded.Cellswereincubatedfor2hourspriortoinfection.

EPECwild-type (E2348-69) and isogenicmutant strainswere grownonLuria–Bertani

plates(LB,Oxoid).Forinfectionsbacteriawereinoculatedinto3mlLBbrothandgrown

overnight37°C,5%CO2withoutshakingtoafinaldensityof ~5x108CFUml

-1.Priorto

infection25μlofovernightculturewasdiluted1:40inantibioticandserumfreeDMEM

andincubatedfor2hoursat37°Cin5%CO2,toafinalconcentrationof~5x107CFUml

-1.



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Infectionsweremaintainedfor3-4hoursat37°Cin5%CO2untilsignificantamountsof

thebacteriahadadheredtothecells.

Table1:BacterialStrainsandMammalianExpressionvectorusedinthisstudy.

Strain/Plasmid RelevantCharacteristics Reference

Enteropathogenic

Escherichiacoli

E2348/69

EPECWildtypestrain,BFPpositive,LEEpositive Kindgiftof

BrendonKenny

Universityof

Newcastle

EPEC/ΔespB E2348/69mutantwithespBgeneislandknockout(T3SS

deficient)

(Newtonetal .,

2010)

EPEC/ΔIE6 E2348/69mutantwithIE6geneislandknockout,lacking

nleE1,nleB1andespLeffectorgenes

(Newtonetal .,

2010)

EPEC/ΔPP4 E2348/69mutantwithPP4knockout,lackingnleB2,nleC ,

nleDandnleGeffectorgenes

(Newtonetal .,

2010)

XL10-GoldCam

ultracompetent

cells

Htephenotype;hightransformationefficiencieswithligated

DNAandlargesupercoiledDNAmolecules;tetracycline-and

chloramphenicol-resistant;endonucleasedeficient(endA1)andrecombinationdeficient(recA)

Agilent

Technologies

pCMV-Script Derivedfromahighcopy-numberpUC-basedplasmid;allows

proteinexpressionineukaryoticsystemsviaCMVimmediate

earlypromoter;Kanamycinresistant

Agilent

Technologies



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Proteinanalysis

ProteinswereseparatedbySDS-PAGEandanalysedbyWesternblotting.Between120-

175µlof1Xloadingbuffer(62.5mMTris-HClpH6.8,,2%w/vSDS,10%glycerol,50mM

DTT,0.01%w/vbromophenolblue) wasaddedtoeachwell,andincubatedonicefor3-

5min.Sampleswerethenscrappedandtransferredtotubesbeforebeingstoredat-

20˚Corheatedto95°Cfor3-5minutes.Sampleswerethenvortexedtosheargenomic

DNA and spun down for 5 minutes at 15,000 rpm (Cabnet Spectrafuge 24D). The

sampleswererunon12%acrylamidegels(40%of30%Acrylamide/BisMix25:1-Sigma,

25%0.25MRunninggelbuffer(1.5MTRIZ-Baseand0.4%SDS,pH8.8),10%glycerol–

BDH Supplies, 0.33% Ammonium persulfate (APS) - Sigma, 0.07%

Tetramethylethelenediamine (TEMED - Sigma)) with a stacking gel (15% of 30%

Acrylamide/BisMix25:1,25%0.25MStackinggelbuffer(0.5MTRIZ-Baseand0.4%SDS,

pH 6.8), 0.5% APS, 0.1% TEMED)). 0.25M Running Buffer (0.25mM TRIS base, 0.2M

Glycineand1% SDS) wasaddedand the gels were run for ~2hours at 90~120Vand

~75mA (Consort E831). Proteins were transferred onto PDVF or nitrocellulose

membranes(Hybond)usingelectroblottingwith0.25MBlottingbuffer(20%methanol,

0.2MGlycineand0.25mMTRISbase)for1 hourand10minutesat~50Vand ~325mA

(ConsortE831).Membranesweresoakedinwashingbuffer(Phosphatebufferedsaline-

Sigma,0.1%TWEEN20-SigmaUltra)with3%albumin(BSA,Sigma)or5%skimmedmilk

for 1 hour, to block any free membrane binding spots. Blocked membranes were

incubated withwashingbuffer containing either 3%BSA or5%mild and the specific

primaryantibodyat4°Covernightbeforebeingwashedtwicewithwashingbuffer(5min



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and 40min) and then incubated at room temperature for 2 hours with the specific

secondaryantibody.Membraneswerewashedtwice(5minand40min)anddeveloped

by chemiluminescence with X-ray film (CL X-posure, Thermo) using enhanced

chemiluminescence (ECL) Western Blotting Analysis System (Amersham) as per

manufacturersprotocol.



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ImmunofluorescentMicroscopy

HEp-2orCaco-2cellswerecountedusingahaemocytometerandsubsequentlygrown

to4x105onsterileglasscoverslipswithcompletemediain24wellplates.Thesecells

weretheninfectedwithdifferentEPECstrainsusingthestandardprotocolfor4hours.

After infection the cellswerewashed2-3 timeswith sterile PBS toremove any non-

adherentbacteriaand0.5mlof4%formaldehydewasaddedtoeachwellfor15minutes

tofixthecells.StaticcellswerethenwashedwithsterilePBSand0.5mlof0.1%TritonX-

100wasaddedfor10minutes.Thecoverslipswerethenwashedafurther3timeswith

the finalwash lasting5minutes.Coverslipswereblockedbyadding 2%normal Goat

serum in0.5%BSA was added toeachwell and incubated at room temperature for

30min. The specific primary antibodies were added at a 1:50 (anti P-p38) or 1:600

dilution(antiHAepitope)andincubatedovernightat4°C.Thecellswerewashedtwice

andsecondaryantibodieswiththeaconjugatedflurophorewerethenaddedtoeach

sampledependingonthedesiredstainingatadilutionof1:400andincubatedatroom

temperature for 1 hour. Samples were then analysed and pictured using a UV

microscopeunderUVlight.



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Table2:Antibodiesusedinthisstudy.

Antibody Source ManufacturerDilutionwith

PBS+0.1%TWEEN

Phosphorylated

Residue(s)ConjugatedMolecule

P-ERK1/2

(P-p44/42)RabbitIgG CellSignalling 1:2,000 T202,Y204 -

P-JNK

(P-SAPK/JNK)MouseIgG CellSignalling 1:500/1:1,000 T183,Y185 -

P-p38

(P-p38MAPK)RabbitIgG CellSignalling 1:500/1:1,000 T180,Y182 -

P-STAT1 RabbitIgG CellSignalling 1:1,000 T701 -

ERK

(p44/42)RabbitIgG CellSignalling 1:1,000 - -

JNK

(SAPK/JNK)RabbitIgG CellSignalling 1:1,000 - -

p38

(p38MAPK)RabbitIgG CellSignalling 1:1,000 - -

Anti-RabbitIgG MouseIgG Sigma 1:2,000/1:5,000 - HorseradishPeroxidase

Anti-MouseIgG RabbitIgG Sigma 1:2,000/1:5,000 - HorseradishPeroxidase



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MammalianExpressionVectorCloning

ThemammalianexpressionvectorpCMV-Script(AgilentTechnologies)wasusedin this

project,whereEPECeffectorproteinDNAsequenceswereamplifiedusingpolymerase

chain reaction (PCR) with their specific forward and reverse primers and the

proofreading Phusion DNA polymerase (Pfu, Finnzymes). Optimal annealing

temperaturesforthespecificEPECprimershadfirstbeendeterminedbyusingvarying

annealing temperatures (48-56°C), where the products were then separated by gel

electrophoresis (1.5% agarose gels) and analysed under UV light. The Phusion PCR

productswerethenpurified(StrataPrepPurificationKit)andligatedintopCMV-Scriptat

theMCSaspermanufacturersprotocol.Ligatedvectorsweretransformed intoXL10-

GoldCamultracompetent cells (Agilent Technologies) as permanufacturers protocol

and the resultingbacteriawereplated onto LB-Agar+Kanamycin(50μg/ml) plates and

incubated at 37°C overnight to select for colonies with successful vector uptake.

Determiningtheorientationoftheinsertwasachievedbyscreeningrandomlychosen

coloniesviaPCRusingthevectorsT3primerbindingsiteandthereverseprimerofthe

specificinsert.PCRproductswerethenseparatedbygelelectrophoresisandanalysed

underUV light. PCR productswith the correctly orientated insertwere sequenced to

determine if the insert had the accurate DNA sequence. The suitable vectors were

extracted and purified from the selected EPEC colonies using MINIPrep columns

(Qiagen)aspermanufacturersinstructionsfortransfectionintoHEp-2cells.



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Table3:Primersusedinthisstudy.HA(humaninfluenzahemagglutinin)epitopetag

Primers Sequence Source

T3 Forward:AATTAACCCTCACTAAAGGGMWG

Operon

IE6Forward:CTGATTGTTAAAATCTAACCTGTA

Reverse:GCCTGCGTTTTCAGGAAAAT

MWG

Operon

TirForward:ATGTACCCATACGACGTCCCAGACTACGCTATGCCTATTGGTAACCTTGG

Reverse:TTAAACGAAACGTACTGGTC

MWG

Operon

EspJForward:ATGTACCCATACGACGTCCCAGACTACGCTATGCCAATCATAAAGAACTG

Reverse:TCATTTTTTGAGTGGGTGGA

MWG

Operon

NleB-1Forward:ATGTACCCATACGACGTCCCAGACTACGCTATGTTATCTTCATTAAATGT

Reverse:TTACCATGAACTGCTGGTAT

MWG

Operon

NleDForward:ATGTACCCATACGACGTCCCAGACTACGCTATGCGCCCTACGTCCCTC

Reverse:CTAAAGCAATGGATGCAGTC

MWG

Operon

NleE-1Forward:ATGTACCCATACGACGTCCCAGACTACGCTATGATTAATCCTGTTACTAA

Rerverse:CTACTCAATTTTAGAAAGTT

MWG

Operon

NleFForward:ATGTACCCATACGACGTCCCAGACTACGCTATGTTACCAACAAGTGGTTC

Reverse:TCATCCACATTGTAAAGATC

MWG

Operon



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TransfectionofpCMV-ScriptvectorintoHEp-2cells

PurifiedvectorsweretransfectedintoconfluentHEp-2cellsviausingLipofectamineLTX

reagent(Invitrogen).ThismethodutiliseslipofectiontotransfectplasmidDNAintoHEp-

2 cellswith relatively low cytotoxicity. HEp-2cellsseeded into in24well or12-well

plates at a density of ~4x104

or ~8x104 cells per well respectively. Cells were grown

overnightto60-80%confluencebeforebeingtransfected.Cellswerethentransfected

with250ng(24well)or500ng(12well)plasmidDNAusingthemanufacturersprotocol.

Transfectedcellswereincubatedat37°Cwith5%CO2for24hoursbeforebeingusedfor

immunofluorescentstainingorWesternblottingusingthestandardprotocols.



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Results

EffectsofT3SSonactivationofMAPKsduringEPECinfection

Itwasimportanttostartbyconfirming,whichMAPkinaseswerebeingtargetedbyWT

EPEC, and towhat extent the phosphorylation of these kinases was being affected.

Caco-2cellswereinfectedwithWTEPECandthemutantΔespB,toallowacomparison

betweenaneffectiveandnon-functional T3SS,whichmeansnoeffectorproteinsare

injectedintothehostcellsbyΔespB.Samplesweretakenat0.5,1,2and4hourspost

infection with 30 minute IL-1β stimulation in both WT and ΔespB, resulting in an

infectiontimecourseshowingthelevelofERK1/2,JNKandp38phosphorylation(Figure

3).

CellsinfectedwitheitherWTorΔespBactivatedthephosphorylationofp38andERK1/2

MAPKs. Activation of JNK could not be reproducibly seen after infection with either

strain.CellsinfectedwiththeΔespBmutantshowedarelativelyconsistentlevelofP-

p38 throughoutthewholetimecourseofinfection.ThislevelofP-p38 fromΔespB is

considerably higher than in the control of unstimulated cells, which suggests that

PAMPs are being recognised on the ΔespB mutants causing activation of cellular

inflammatorypathwaysincludingp38.TheWTinitiallycausedasignificantlylowerlevel

ofP-p38comparedtoΔespBandsimilartothecontrolofnon-infectedandunstimulated

cells. Howeverwith increasing time of infection the level of P-p38 increased in cells

infectedwiththeWTandat4hourstheamountofP-p38presentwasapproximately



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thesameaswiththeΔespBmutant.ThisindicatesthattheWTisusingitsfunctional

T3SSandeffectorproteinstoinitiallydecreasethelevelofp38phosphorylation.These

findingscorresponded topreviousresearch, whichhas shownthat the EPECeffector

NleDiscapableofcleavingbothJNKandp38(Baruchetal .,2011).Toidentifyifany

other effectors were responsible for this effect, cells needed to be infected with

bacteriawithoutfunctionalNleD.SinceNleDisfoundonthePP4geneisland,cellscould

be infected with the EPEC ΔPP4 mutant and theirMAPK levels analysedbywestern

blottingtodetermineifanyotherproteinsarecausingtheseeffects.

Figure 3: Images of developed Western blot membranes showing time course (0.5-4h) of

intracellularlevelsofphosphorylatedp38,JNKandERK1/2duringEPECWTandΔ espBinfection

ofIFNγstimulatedCaco-2cells.Atindicatedtimepointscellswerewashed,lysedandsubjected

towesternblottingusinganti-P-ERK1/2,anti-P-JNKoranti-P-p38rabbitantibodies.Thecontrol

C-indicatesCaco-2cellsthatwereuninfectedandnotstimulatedbyIL-1β.C+showswhatlevel

ofresponseiscapableincellsthathavebeenstimulatedfor30minuteswithIL-1β.Resultsare

representativeof2-3independentexperiments.



Page26of54

The results for phosphorylation of JNK were not completely conclusive for the time

course infections, due to a combination of technical problemswith protein transfer

from gel to PVDF membrane and significant amounts of background from the

immunostainingevenafterseveraltrials.OverallthelevelsofJNKphosphorylationdid

notseemtovarybetweenWT,ΔespBmutantandtheunstimulatedcontrol.IntheWT

sample of4hourspost infection adecrease inthe levelofphosphorylation couldbe

seen, indicating possible inhibition, however the results cannot be entirely trusted

because differences in the intensity of the bands may be the result of incomplete

transfer.

Similar trends aswithp38 were seenwithERK1/2,wherean increase in the timeof

infectionwithWTresultedinanincreaseofphosphorylatedERK1/2.Thisincreasecould

alsobeseenwiththeΔespBmutant,howeveroverallthelevelsofP-ERK1/2appeared

lowerintheWTat2-4hourscomparedtothemutant,indicatingthattheWTmayagain

beaffectingthephosphorylation ofERK1/2.These findingscorrespondedtoprevious

research,whichstatedthatEPECiscapableofinhibitingthephosphorylationofERK1/2

howevertheexactproteinsareunknown(Ruchaud-Sparaganoetal .,2007).

The results from the time course infections meant that other EPECmutants lacking

specificgeneislandsneededtobeusedtotryanddeterminewhicheffectorproteins

playaroleinthedifferentp38andERKinhibitorypathwayandalsoinvestigatewhat

effectstheyhadonJNKphosphorylation.



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EffectsofΔPP4EPECmutantonMAPKphosphorylation

ThefirstmutantutilisedwasΔPP4,whichlackstheEPECeffectorsNleD,NleG,NleB2,

and NleC. The functions of these effectors are relatively unknown, however NleD is

thought toaffect intracellular levelsofp38and JNK.Todetermine theeffectsofPP4

effectorsonMAPK,thelevelsofphosphorylatedandtotalMAPkinaseswerecompared

4hourspostinfectionwithWT,ΔespBandΔPP4strains.RatherthanexaminingMAPK

phosphorylation immediately post infection, the ability of each strain to block IL-1β

inducedMAPKphosphorylationwasexamined(Figure4).Levelsof totalMAPkinases

wereanalysedasaloadingcontroltoconfirmthatanydifferencesinphosphorylation

didnotresultsfromunevenloadinggelsorasaresultofproteindegradation.

Asinthetimecourse(Figure3)levelsofP-p38inWTandunstimulatedcells(C-)were

similar, whereas the uninfected and IL-1β stimulated cells (C+) showed very strong

phosphorylation of p38 alongwith ΔespB, whichwas expected due to the lack of a

functional T3SS (Figure 4A). Cells infected with the ΔPP4 mutant showed absent or

levels ofp38 phosphorylation,whencomparedtotheWT infectedand unstimulated

cells(C-),howevertheresponsewasnotasstrongaswithΔespB.SincethelevelofP-

p38wassignificantlyhigherinΔPP4thanwiththeWTitiscertainthatPP4effectorsare

involvedresponsibleforinhibitingp38phosphorylation,howevertheresponsewasnot

as strong as with ΔespB. This indicated that other EPEC effectors could possibly be

involvedinthisprocess.ComparedtoP-p38,thelevelsoftotalp38incellsinfectedwith

the same bacterial strains remained constant. Itmay bepossible thatWTEPECwas



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causinga smalldecrease intotalp38,howeverthismaynotbesignificantdue tothe

inaccuraciesofWesternblotting.ThisindicatesthatEPECdoesnotaffectlevelsoftotal

p38asaresultofinfection,butcaninhibititsphosphorylationandactivation.

Figure4:Levelsofphosphorylatedandtotalp38(A),JNK(B)andERK1/2(C)inIL-1βstimulated

Caco-2cells4hourspostinfectionwithEPECWT,Δ espBandΔPP4mutants.UnstimulatedCaco-

2cells(C-)andIL-1βstimulated(C+)Caco-2cellsusedascontrols.Cellswerewashed,lysedand

subjectedtowestern blottingusing anti-P-ERK1/2,anti-P-JNKoranti-P-p38rabbit antibodies.

Resultsarerepresentativeof3independentexperiments.



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Unlikeinthetimecourse,theeffectsofWTandΔespBEPECinfectionaredemonstrated

inFigure4B.WTEPECshowedverylowlevelsofJNKphosphorylationwhencompared

tothe stimulated control (C+) andΔespB. These findingswereexpectedasEPEChas

beenshowntocleavephosphorylatedJNK.InterestinglytheΔPP4mutantalsoshowed

extremely low levels of P-JNK. This was surprising, since this mutant lacks the EPEC

effectorNleD,whichhaspreviouslybeenlinkedtoJNKdegradation.Thisindicatesthat

otherEPECeffectorsmustalsoberesponsiblefortheinhibitionofJNKphosphorylation

inhostcellsduringEPECinfections.Aswiththetimecoursetherewereproblemswith

theresultsoftheeffectsofEPECinfectionsontotalJNKlevels.Inseveraltrialscellsthat

had been infectedwithEPEC ingeneral seemed tohave significantly higher levelsof

totalJNKcomparedtothetwouninfectedcontrols(C-/C+).Inadditiontherewasagaina

highlevelofbackgroundfromtheimmunostaining.Itwasexpectedthatthelevelsof

totalJNKwouldstayrelativelyconstantforbothallthreemutantsandthetwocontrols.

Relativetonon-infectedpositivecontrolsamples,noneoftheEPECstrainscouldinhibit

phosphorylationofERK1/2.LevelsofP-ERK1/2wereslightlylowerincellsinfectedby

WTEPEC comparedtothe ΔespB mutant and theΔPP4 mutant also showed a slight

decreaseinP-ERK1/2levelswhencomparedtotheΔ espBmutant(Figure4C).Overall

thisdatasuggeststhatEPECeffectorsarenotdirectlyinvolvedintheinhibitionofIL-1β

induced ERK1/2 activation or cleavageof ERK1/2. These findings are consistent with

previousresearchintotheΔPP4effectorNleD,whichcausesthedegradationofp38and

JNKbutnotERK1/2.ERK1/2lackstheconservedactivationloopfoundinp38andJNK,



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whichactsasacleavagesite.TheintracellularlevelsoftotalERK1/2remainedrelatively

constantinallthreebacterialstrains.



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IL-1βandTNFαinducedMAPKphosphorylationpostEPECinfections

ToinvestigatetheeffectsofEPECeffectorsNleE1andNleB1ontheMAPkinasesp38

andERK1/2 an ΔIE6mutant was used. This gene island codes3mainEPECeffectors

NleE1,NleB1andEspL2,andpreviousresearchhasshownthatbothNleE1andNleB1

caninhibitdifferentpartsoftheNF-κBsignallingcascade,howevertheireffectsonMAP

kinasesareunknown.ForthismutantinfectionswerecarriedoutonHEp2cellsinstead

of Caco-2 cells, due to previous experiments indicating that ΔIE6 strains can have

difficultyinformingtightadhesionstohostcellsandarenotcapableofformingactin

pedestals, without which the EPEC effectors would not be injected. To increase the

probabilityofsignificantamountsofΔIE6attachment,increasingamountsofmultiplicity

ofinfection(MOI)wereused.MOIistheratioofbacteriatothehostcellsinasample

andcanbemodifiedbyknowingtheamountofcellsinasampleandtheamountof

bacteria.Cellsamplesarecountedusingahaemocytometerandthenumberofbacteria

thathavebeengrownovernightinLB-brothcanbeestimated.Byincreasingtheratioof

bacteriatohostcellstheprobabilityofsuccessfulinfectionincreases.HEp-2cellswere

infectedfor4hourswithWT,ΔespB,andΔIE6strainsandweresubsequentlystimulated

witheitherIL-1βorTNFαfor30minutesandanalysedbyWesternblottingtodetermine

their effects on intracellular P-p38 and P-ERK1/2 levels (Figure 5). IL-1β and TNFα

stimulationwas used to try and distinguishwhich part of the intracellular signalling

pathwaysarebeingtargetedbytheeffectorproteins.

ConsistentwiththepreviousinfectionsinCaco-2cells,theWTbacteriawerecapableof



Page32of54

inhibitingthephosphorylationofp38incellsstimulatedbothbyIL-1βandTNFα(Figure

5A).ΔespBinfectionswerealsounabletoproducethisinhibitoryeffectandhadsimilar

levelsofintracellularP-p38asthecontrolstimulatedcells(C+).Theselevelswerealso

seeninΔIE6infectedcells,whichshowedstrongimmuno-reactivityforphosphorylated

p38suggestingthatIE6effectorsdoplayamajorroleintheinhibitionofP-p38.Atvery

high multiplicity of infections (75-100) there was a small decrease in P-p38 when

comparedtothelowerMOI,possiblyduetoredundancy.Similarresultswerefoundfor

cellsstimulatedbyTNFα,showinghighlevelsofP-p38incellsinfectedwithΔespBand

ΔIE6,withasmalldecreaseinp38phosphorylationwithincreasingMOI

Figure 5: Comparison of levels of phosphorylated p38 (A) and ERK1/2 (B) in IFNγ+IL-1β or

IFNγ+TNFαstimulatedHEp-2cells,4hourspostinfectionwithEPECWT,Δ espBandΔIE6with

increasingmultiplicityofinfection(MOI).UnstimulatedHEp-2cells(C-)andIFNγ+IL-1β/TNFα

stimulatedcells(C+)wereusedascontrols.Cellswerewashed,lysedandsubjectedtowestern

blottingusinganti-P-ERK1/2oranti-P-p38rabbitantibodies.



Page33of54

As with the previous infections, WT EPEC showed limited inhibition of ERK1/2

phosphorylationcomparedtoΔespBand(C+),indicatingthatitwaslargelycapableof

inhibiting P-ERK1/2phosphorylation in cells stimulated by IL-1βor TNFα (Figure 5B).

CellsinfectedwiththeΔIE6mutantandstimulatedcells(C+)forbothIL-1βandTNFα,

resultedinP-ERK1/2levelssimilartothoseshownbyΔespB.

These resultssuggestthat the IE6effectorsdoplayamajorroleintheinhibitingthe

phosphorylationp38.ThedifferentcytokinesIL-1βandTNFαusedtostimulatethecells

resultedinnomajordifferencesinMAPKlevels,whichimpliesthatEPECcaninhibitthe

phosphorylationoftheseMAPkinasesviabothpathways.



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ImmunofluorescentimagingoftheeffectsofEPECinfections

Toinvestigatesomeofthepropertiesofthebacterialstrainsusedinthisstudyandtheir

effectsoninfectedcells,HEp-2andCaco-2sampleswereinfectedwithEPECstrainsWT,

ΔespBandeitherΔIE6orΔPP4 respectively. Immunofluorescentstainingwasusedto

visualise theeffectsof these bacterial infectionson intracellularactin structure. Post

infections,cellsweretreatedwithformaldehyde,whichcausestheirfixationtoprevent

cellfunctions,movement,deathanddecay.Byusingdifferentfluorophoreswithnon-

overlappingexcitationandemissionspectrums,thesamecellsamplecouldbestained

for F-actinandDNA.Actin stainingwasachievedbyusingthe toxinphalloidin,which

binds to F-actin and prevents its depolymerisation.Phalloidinwas conjugated to the

fluorophorefluoresceinisothiocyanate(FITC),whichemitsaredlightwhenstimulated.

ThenucleusofthecellsandtheDNAofbacteriawerestainedbyusing4',6-diamidino-2-

phenylindole(DAPI),whichbindstoA-TrichregionswithinDNA.DAPIemitsbluelight

whenboundtoDNAandstimulatedbyUVlight.



Page35of54

Figure6:RepresentativeimmunofluorescencefieldsshowingF-actinstaining(red)andnucleic

acidwithDAPI (blue) inuninfectedHEp-2cells(C-)andHEp-2cellsinfected for 4hourswith

EPECWT,ΔespBandΔIE6.Arrowsindicateactinpedestalformation(A/Elesions).



Page36of54

HEp-2cellsampleswereinfectedfor4hourswithWT,ΔespBorΔIE6tovisualisethe

formationofactinpedestals(Figure6)anduninfectedcells(C-)wereusedasacontrol.

Cells infected with WT EPEC showed clear actin pedestals, where the bacteria had

attachedtothehostcellmembraneandusedtheirT3SSandeffectorproteinstomodify

thecytoskeleton,whencomparedtouninfectedcells.TheΔ espBmutantwasunableto

form these A/E lesions on the host cells because intimate attachment requires a

functional T3SS and showed similar actin structure as with the uninfected cells.

InterestinglytheΔIE6bacteriadidcausetheformationofactinpedestalsontheinfected

HEp-2 cells, indicating that intimateattachment had occurredand the EPEC effector

proteinswere able tobe introduced into the host cells. Previous infectionswith this

mutantonCaco-2cellshadshownthatΔIE6canhavetroublewithachievinglong-term

attachment (personal communication from Dr Mark Lucas), however in HEp-2 cells

similarlevelsofA/ElesionswereseenaswiththeWT.TheseresultsmeantthattheΔIE6

mutant could be used as a viable strain for infections investigating the IE6 effector

proteinsNleEandNleB(Figure5),andalsothatthe IE6effectorsarenotessentialfor

actinpedestalformation.

Caco-2cellswere infectedwiththeEPECstrainsWT, ΔespBandΔPP4 for4hours,to

investigateandvisualisetheeffectsofPP4mutantonhostcellactinstructure(Figure7).

AswiththeHEp-2cellsWTEPECcausedthemodificationofhostcellactinstructureat

thesiteofbacterialattachmentandtheformationofA/Elesions.Asexpectedinfection

withΔespBstrainhadnosignificantaffectsonthehostcellscomparedtotheuninfected



Page37of54

cells and noactinpedestals were formed. The ΔPP4mutant was capable of forming

largeamountsofactinpedestalsoninfectedCaco-2cells,indicatingsuggestingthatthe

effectorsfrompathogenicityislandPP4donotplayasignificantroleinthemodification

ofthehostcytoskeletonandthatitboundwithcomparableefficiencyastheWTstrain.

Figure7:RepresentativeimmunofluorescencefieldsshowingF-actinstaining(red)andnucleic

acidwithDAPI(blue)inuninfectedCaco-2cells(C-)andCaco-2cellsinfectedfor4hourswith

EPECWT,ΔespBandΔPP4.Arrows indicateactin pedestalformation(A/E lesions).ΔPP4field

onlyshowsF-actinstaining(white).



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CloningofEPECeffectorgenes

ToinvestigatetheeffectsofsingleEPECeffectorsitwasnecessarytoutilizeasystem

thatallowedthe specificeffectorgenetobe expressed either bya bacteria,whichis

thanintroducedintohumancellsviainfectionoruseamammalianexpressionvector.

ThemajorityofpreviousstudiesusedEPECstrainswherethespecificeffectorsortheir

correspondinggeneislandswereknockedoutanditseffectswerecomparedtoother

strains.Thegeneofinterestwassubsequentlyinsertedbackintotheknockoutmutant

inaplasmid toconfirm if the genewas causing the cellulareffects.Howeverdue to

multiple problems concerning different mutants, such as ineffective attachment and

redundancy issues from other EPEC effectors this method was not chosen. Using a

shuttle vector,whichallowed the gene inserts tobeexpressed inbothbacterial and

eukaryoticcellswouldguaranteethattheresultingeffectsonMAPkinasesweredueto

theselectedeffectorproteinandnototherEPECproteins.

Thefirststepwastoconstructthedifferentexpressionvectorswiththespecificeffector

genesandtransformthesevectorsintocompetentE.Colitoallowtheiramplification.6

EPECproteinswerechosen,3basedonthepreviousinfectionresults(NleD,NleEand

NleB)and 3 otherproteins to investigate theirpotential effects (Tir, EspJ andNleF).

Before amplifying their corresponding genes itwas necessary to first determine the

optimal annealing temperature for the different primers. A gradient of annealing

temperature was used for all 6 effector gene ranging from 48°C to 56°C. The PCR

productswereseparatedbygelelectrophoresisandanalysedunderUVlight(Figure8).



Page39of54

Sampleswith a relatively high concentration, single band and relatively littleprimer-

dimercomplexeswerechosenastheoptimalannealingtemperatureforeacheffector.

Figure 8:Optimisation ofEPEC geneprimers (IE6, tir , espJ, nleD, nleE and nleF ). Each set of

primersusedarangeof5differentannealingtemperatures(48,50,52,54,55and56°C)during

amplification of WT EPEC template. PCR products separated by gel electrophoresis and

photographedunderUVlight.HyperladdersIIIandIV(Bioline)usedtodeterminebandsize.

It was essential that the effector genes were amplified with as little mutations as

possible for the ligation into vectors.Mutations couldcauseproblemssuchasframe

shifts,whichwouldresultinnon-functionaleffectorproteinsbeingproduced.Forthis

reasontheproofreadingDNApolymerasePhusion(Pfu)(Finnzymes)wasusedinsteadof

conventionalTaq(Bioline)polymerase,whichhas50-foldhighererrorrate.Additionally

Taq DNA polymerase unlike Pfu is known to exhibit terminal

deoxynucleotidyltransferase activity, where nucleotides are added to the end ofPCR

productsto create a3’overhang,which can alsoaffect cloningand causeframshifts.

SubsequentlytheeffectorgeneswereamplifiedasecondtimefromEPECWTgenome,

exceptfornleBandnleE ,wheretheIE6geneislandwasusedasatemplate.Thisisdue



Page40of54

to different nleB and nleE isoforms being present on different gene islands. These

isoformsaretruncatedformsofnleB1andnleE1andasaresultcouldalsobecopiedby

theNleB1andNleE1primersandinsertedintothevectors.ByusingtheIE6geneisland

asatemplateonly nleB1andnleE1wereamplified.Theseproductswerepurifiedand

analysed by gel electrophoresis (Figure 9). For unknown technical problems the

amplificationoftheeffectorgenenleBwasunsuccessfulafterseveralattemptsandwas

consequentlyabandoned.

Figure 9: EPEC effector genes (tir , espJ, nleD, nleE and nleF ) amplified using proofreading-

enzyme Phusion DNA polymerase via PCR and polished for insertion into pCMV-Script. PCR

products separatedbygel electrophoresis andphotographedunderUV light. Amplifiedgene

sizesweredeterminedbyusingHyperLadderIV(Bioline).

OncetheEPECeffectorgenestir ,espJ,nleD,nleE andnleF weresuccessfullyamplifiedto

reasonableconcentrations,apolishingstepwasusedtomakesurethatthepurifiedPCR

products had blunt ends to assist ligation. The restriction enzyme Srf I was used to

cleave theMCSonthepCMV-Scriptvectorandtheeffectorgeneswere insertedinto

thissiteandligatedviaT4DNAligase.EPECeffectorgeneswerecheckedbeforecloning



Page41of54

tonot contain any Srf I sites,whichwouldcausesevere problemssuchasthe insert

beingcleavedinhalfandligatedbacktogetherinlargechainsresultinginnon-functional

effectorproteins.

To confirm that the specific effector genes had been successfully inserted into the

vectors,theywereinitiallytransformedintocompetent E.Coli cellsviaheatshock.The

resultingbacteriawereplatedontoLB-Agarplatescontainingtheantibiotickanamycin

(50μg/ml).Thisallowstheselectionofcolonieswithsuccessfulvectoruptake,sincethe

pCMV-Script vector contains the kanamycin resistance gene. Bacteria that have not

acquiredtheplasmidwillnotbekanamycinresistantandwouldthereforebeunableto

grow on the agar plates. This procedure does not however confirm that the EPEC

effector genes have been inserted into the transformed plasmid. Random colony

samples were taken for each EPEC effector from the LB-Agar+Kanamycin(50μg/ml)

plates,whichhadbeenincubatedat37°Covernightandwereusedastemplateforthe

amplificationofthespecificeffector genesviaPCR.Thedirection ofthe insert isalso

essentialfortheexpressionandproductionoffunctionEPECeffectorproteins.Byusing

the forward T3 primer sequence, which is located directly before the MCS and the

reverseeffectorgeneprimer,thedirectionoftheinsertcouldalsobedetermined.This

meant that amplification of the effector geneswould only be successful in colonies

containingthespecificeffectorgeneinthecorrectorientation.



Page42of54

TheproductsfromthesePCRreactions(Figure10)werecomparedtothecorresponding

bandsinFigure9toconfirmthatthecorrectgenehadbeeninsertedandwasinthe

properorientation.TheresultsshowedthatforeachchosenEPECeffectorgenethere

weremultiplecolonies,whichhadsuccessfullytakenuptheplasmidwiththeinsertin

theappropriatedirection. Thecolonieswiththe highestconcentrations of the inserts

werechosenforfurtheruse.

Figure10:AmplificationofplasmidinsertsusingPCRinselectedE.Coli coloniesusingplasmidT3

primer and specific reverse effector primer, where single band verifies correct direction of

insert.PCRproductsseparatedbygelelectrophoresisandphotographedunderUVlight.

The pCMV-Scriptvectorsfromthe selectedsampleshad tofirstbepurifiedfromthe

appropriatecoloniesbycarryingoutaMiniPrepfortheuseinsubsequenttransfection

intomammalianHEp-2cells.ExtractedandpurifiedMiniPrepproductswererunonan

agarosegel,whichconfirmedthatthevectorshadbeenretainedineachsample(Figure

11),indicatedbypresenceofstrongbands.Twobandswerevisibleonthegelforeach

sample, which represented the different topological structures of the vector due to

supercoiling.



Page43of54

Figure 11: Plasmid preparation (MiniPrep) of selected colonies with vectors containing the

specific EPEC effector genes. Purified products separated by gel electrophoresis and

photographedunderUVlight.



Page44of54

TransfectionofEPECeffectorvectorsintoHEp-2cells

Purified vectors were transfected into HEp-2 cells by using the transfection agent

lipofectamine. This causes changes to the eukaryotic plasma membrane allowing

vectors to enter the host cell via lipofection. The resulting cellswere then used for

immunofluorescent microscopy or Western blotting to determine the effects of the

differenteffectorproteinsonp38phosphorylation.

Todetermine if the specificEPECeffectorgene had been transfectedandwasbeing

successfully expressed, human influenza hemagglutinin (HA) epitope tag sequences

wereaddedtotheendofallforwardeffectorprimers.ThiswouldcauseHAtagstobe

ligatedintothevectoralongwiththeeffectorgeneandexpressedintheHEp-2cellsas

part of the effectorprotein.The resulting effectorproteinscouldthenbeselectively

stained and visualised by using anti-HA monoclonal antibodies conjugated to the

fluorophoreFITC.Cellsampleswerestainedfornucleicacids(DAPI)andthepresenceof

EPECeffectorproteinviatheHAtag(GFP),andwerevisualisedusingaUVmicroscope

(Figure12).



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Figure 12: Representative immunofluorescence fields showing staining of nucleic acids with

DAPI (blue) and HA-tag with GFP (green) in HEp-2 cells transfected with the selected EPEC

effectorgenevectors.HEp-2cellsamplestransfectedwithNleEvectorusedasanexample.

All the transfectedsampleswith each EPECgene vector showed noresponse for HA

staining.ThiscouldbeduetothestainingoftheHA-taghadbeenineffective,theHA-tag

notbeingexpressedbyhostthecellsorthatthetransfectionswereunsuccessfuland

therefore the vectors were not present in the HEp-2 cells. Despite these findings

samples transfected with the EPEC effector gene nleE were stained for P-p38 to

investigateanypotentialinhibitoryeffectsofthevectoronp38phosphorylation(Figure

13). Transfected cells were stimulated with 5ng/ml IL-1β and P-p38 levels were

compared to the control of unstimulated transfected HEp-2 cells. Stimulated cells

showed high levels of cytomplasmic p38 phosphorylation, when compared to the

unstimulatedsampleswhichshowednop38activation.Theseresultsagainsuggested

thateitherthevectorhadn’tbeentransfectedintotheHEp-2cellsorthatthenleE gene



Page46of54

wasnotbeingexpressedathighenoughlevelstoseeanyeffectonthephosphorylation

ofp38.

Figure13:

Representative immunofluorescence fields

showing staining of nucleic acids with DAPI

(blue) and phosphorylated p38 with GFP

(green) in HEp-2 cells transfected with the

NleEvector.LevelsofP-p38werecomparedin

unstimulated (C-) and transfected cells

stimulatedwith5ng/mlIL-1β.



Page47of54

To investigate if any levels of the HA-tagged proteins could be detected, Western

blottingwas performed onselectedHEp-2 samples.As a control P-p38 levels in cell

lysateswerealsoexamined.Whilstphosphorylationofp38waseasilydetectedinIL-1β

stimulatedsamples(Figure14),noHAtaggedproteinscouldbedetectedinlysates(data

not shown). Thus it appeared that the transfection of Hep-2 cells had been wholly

ineffective.Duetotimerestraintsrepeattransfectionswerenotcarriedout.

Figure14:Westernblotimageshowinglevelsofphosphorylatedp38inlysatesoftransfected

HEp-2samples,stimulatedbyIL-1β.UnstimulatedtransfectedHEp-2cellsusedascontrol(C-).



Page48of54

Discussion

EnteropathogenicE.Coli infectionsofthehumangastrointestinaltractleadtochanges

in both cell morphology and intracellular signalling. This project investigated the

interactions between specific bacterial proteins and host inflammatory signalling

proteinsasresultofEPECinfection.Theseinteractionsareachievedbybacteriausinga

T3SS,whichiscapableofinjectinghighlyconservedeffectorproteinsintoinfectedcells.

The majority of previous research into the specific functions and targets of these

effectors has focused on the NF-κB pathway and production of IL-8, however their

effectsonMAPkinasesignallingisrelativelyunknown.

WesternblotsofhumancellsinfectedwithEPEC(Figures3,4and5)confirmedthatWT

EPECiscapableofinhibitingtheintracellularaccumulationofhighlevelsofthetwoMAP

kinases p38 and JNK whilst phosphorylation of ERK1/2 is largely unaffected. To

differentiatetheeffectsofthedifferenteffectorproteinsusedbyWTEPEC,infections

with EPEC mutants lacking specific gene islands were carried out. Due to previous

research linking theEPEC effectorNleDto intracellular JNKdegradation,human cells

wereinfectedwithabacterialstrainlackingthepathogenicityisland PP4,inwhichNleD

is coded. Interestingly, cells thatwere infectedwith the ΔPP4 mutant retained their

ability to inhibit the phosphorylation ofboth JNK and partially for p38. Theseresults

indicatethatNleDisonlypartiallyinvolvedintheseinteractionsandotherEPECeffector

proteinsmustalsoberesponsible.AsexpectedtheΔ PP4mutantsshowedsimilarlevels



Page49of54

ofERK1/2phosphorylationcomparedtotheWT,showingthatthePP4effectorsarenot

directlyinvolvedinP-ERK1/2inhibition.TheeffectorsoftheIE6geneislandwerechosen

aspotentialpartsoftheMAPKinhibition,sincetheyhadpreviouslybeenlinkedtothe

blocking of NF-κB. HEp-2 cells were infected with an ΔIE6mutant, which therefore

lacked functional NleB1 and NleE1, to investigate the intracellular levels of p38

phosphorylation. The results suggested that the ΔIE6mutant had lost the ability to

inhibitp38phosphorylation(Figure5),thereforeeitherNleB1orNleE1oracombination

ofbothareresponsiblefortheeffectsseentheWT.Slightp38inhibitoryeffectswere

seen by ΔIE6 mutants at high MOIs, which may indicate that other EPEC effector

proteinsmaybepartiallyinvolvedbutnotthemajorcause.Totryanddeterminewhich

partoftheinflammatorysignallingpathwaysarebeingeffectedbytheseEPECeffectors

thecellswerestimulatingwithTNFαorIL-1β.Howevertheresultsshowedthatthere

wasnodifferenceinthelevelofinhibitionofp38,indicatingthatEPECcanblockp38

activationviabothpathways.ItispossiblethatEPECtargetsadownstreamproteinused

in both signalling cascades. One potential example is the protein TNF receptor

associatedfactor6(TRAF6),whichisactivatedbybothTNFαandIL-1βandisinvolvedin

MAPKandNF-κBactivation.

Todeterminetheeffectsofthe3individualeffectorproteinsidentifiedbythesegene

islandresults,EPECeffectorgeneswereclonedintoashuttlevectorandtransformed

intocompetentE.Coli .3additionalEPECproteins(Tir,EspJandNleF)werechosenand

investigated, due previous studies and their effects onMAP kinases beingunknown.

Oncethevectorswiththeindividualeffectorgeneswereconstructedandconfirmedto



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havethecorrectinsertandinsertorientation,thevectorsweretransfectedintohuman

HEp-2cellstoexaminetheireffectsonintracellularMAPKlevels.Howevertheresults

from immunofluorescent stainingof the HA-tag and P-p38 in transfectedHEp-2 cells

suggestedthatthetransfectionswereineffectiveandthevectorshadnotbeeninserted

intothehostcells.ThismayhavebeencausedbytheinadvertentpresenceofFCSinthe

mediathatwasusedtomixtheplasmidDNAwiththelipofectamine.Thetransfection

protocoladvises that FCS-freemedia shouldbeused during transfections, asFCScan

interferewiththeformationofDNA-lipofectaminecomplexes.Anotherpossibilityisthat

thevectorsweren’tbeingexpressedathighenoughlevelsforthestainingoftheHA-

tagged proteins. To investigate if therewas even a low level ofHA-tag expression a

Western blot was carried out, where the HA-tagged effector protein was stained.

HowevertheresultsshowednodetectablelevelsofHA-taggedproteinsconfirmingthat

thetransfectionshadbeenunsuccessful.

ThisstudyhasshownthatEPECiscapableofinhibitingthephosphorylationofbothp38

andJNKinhumancellsinvitro,howeveritdoesnothavethesamesignificanteffectson

ERK1/2. Infections with various EPEC mutants identified the EPEC effector proteins

NleE1 and NleB1 were as possible causes of this inhibition, however due to time

restrainedthetransfectionsshowingtheactivityoftheindividualeffectorscouldnotbe

carried out. In future the transfection procedure and expression of individual EPEC

effectorgenesinhumancellsneedstobeoptimised,sotheEPECeffectorsresponsible

forinhibitionofp38andJNKphosphorylationcanbeidentified.Thiswouldallowfurther



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insightintothespecificmechanismsutilisedbyenteropathogenic E.Coli toinhibithost

inflammatorypathwaysandachievelong-terminfection.



Page52of54

References

BARUCH,K.,GUR-ARIE,L.,NADLER,C.,KOBY,S.,YERUSHALMI,G.,BEN-NERIAH,Y.,

YOGEV,O.,SHAULIAN,E.,GUTTMAN,C.,ZARIVACH,R.&ROSENSHINE,I.2011.

MetalloproteasetypeIIIeffectorsthatspecificallycleaveJNKandNF-kappaB.

EMBOJ,30,221-31.

CELLI,J.,DENG,W.&FINLAY,B.B.2000.EnteropathogenicEscherichiacoli(EPEC)

attachmenttoepithelialcells:exploitingthehostcellcytoskeletonfromthe

outside.CellMicrobiol,2,1-9.

DEAN,P.&KENNY,B.2009.TheeffectorrepertoireofenteropathogenicE.coli:

ganginguponthehostcell.CurrOpinMicrobiol,12,101-9.

DEAN,P.,MARESCA,M.&KENNY,B.2005.EPEC'sweaponsofmasssubversion.Curr

OpinMicrobiol,8,28-34.

HECHT,G.2001.Microbesandmicrobialtoxins:paradigmsformicrobial-mucosal

interactions.VII.EnteropathogenicEscherichiacoli:physiologicalalterations

fromanextracellularposition. AmJPhysiolGastrointestLiverPhysiol,281,G1-

7.

HODGES,K.&GILL,R.2010.Infectiousdiarrhea:Cellularandmolecularmechanisms.

GutMicrobes,1,4-21.

HOFFMANN,E.,DITTRICH-BREIHOLZ,O.,HOLTMANN,H.&KRACHT,M.2002.Multiple

controlofinterleukin-8geneexpression. JLeukocBiol,72,847-55.

JERSE,A.E.,YU,J.,TALL,B.D.&KAPER,J.B.1990.Ageneticlocusof

enteropathogenicEscherichiacolinecessaryfortheproductionofattaching

andeffacinglesionsontissueculturecells.ProcNatlAcadSciUSA,87,7839-

43.

KAPER,J.B.,NATARO,J.P.&MOBLEY,H.L.2004.PathogenicEscherichiacoli.NatRev

Microbiol,2,123-40.

KENNY,B.,DEVINNEY,R.,STEIN,M.,REINSCHEID,D.J.,FREY,E.A.&FINLAY,B.B.

1997.EnteropathogenicE.coli(EPEC)transfersitsreceptorforintimate

adherenceintomammaliancells.Cell,91,511-20.

KYRIAKIS,J.M.&AVRUCH,J.2001.Mammalianmitogen-activatedproteinkinase

signaltransductionpathwaysactivatedbystressandinflammation.Physiol

Rev,81,807-69.



Page53of54

LEWIS,T.S.,SHAPIRO,P.S.&AHN,N.G.1998.SignaltransductionthroughMAP

kinasecascades. AdvCancerRes,74,49-139.

MCDANIEL,T.K.&KAPER,J.B.1997.Aclonedpathogenicityislandfrom

enteropathogenicEscherichiacoliconferstheattachingandeffacing

phenotypeonE.coliK-12.MolMicrobiol,23,399-407.

MUHLEN,S.,RUCHAUD-SPARAGANO,M.H.&KENNY,B.2011.Proteasome-

independentdegradationofcanonicalNFkappaBcomplexcomponentsbythe

NleCproteinofpathogenicEscherichiacoli. JBiolChem,286,5100-7.

NADLER,C.,BARUCH,K.,KOBI,S.,MILLS,E.,HAVIV,G.,FARAGO,M.,ALKALAY,I.,

BARTFELD,S.,MEYER,T.F.,BEN-NERIAH,Y.&ROSENSHINE,I.2010.ThetypeIII

secretioneffectorNleEinhibitsNF-kappaBactivation.PLoSPathog,6,

e1000743.

NATARO,J.P.&KAPER,J.B.1998.DiarrheagenicEscherichiacoli.ClinMicrobiolRev,11,142-201.

NEWTON,H.J.,PEARSON,J.S.,BADEA,L.,KELLY,M.,LUCAS,M.,HOLLOWAY,G.,

WAGSTAFF,K.M.,DUNSTONE,M.A.,SLOAN,J.,WHISSTOCK,J.C.,KAPER,J.B.,

ROBINS-BROWNE,R.M.,JANS,D.A.,FRANKEL,G.,PHILLIPS,A.D.,COULSON,

B.S.&HARTLAND,E.L.2010.ThetypeIIIeffectorsNleEandNleBfrom

enteropathogenicE.coliandOspZfromShigellablocknucleartranslocationof

NF-kappaBp65.PLoSPathog,6,e1000898.

NOUGAYREDE,J.P.,FERNANDES,P.J.&DONNENBERG,M.S.2003.Adhesionof

enteropathogenicEscherichiacolitohostcells.CellMicrobiol,5,359-72.

ONO,K.&HAN,J.2000.Thep38signaltransductionpathway:activationandfunction.

CellSignal,12,1-13.

PEARSON,J.S.,RIEDMAIER,P.,MARCHES,O.,FRANKEL,G.&HARTLAND,E.L.2011.A

typeIIIeffectorproteaseNleCfromenteropathogenicEscherichiacolitargets

NF-kappaBfordegradation.MolMicrobiol .

RUCHAUD-SPARAGANO,M.H.,MARESCA,M.&KENNY,B.2007.Enteropathogenic

Escherichiacoli(EPEC)inactivateinnateimmuneresponsespriorto

compromisingepithelialbarrierfunction.CellMicrobiol,9,1909-21.

SACK,R.B.1975.HumandiarrhealdiseasecausedbyenterotoxigenicEscherichiacoli.

AnnuRevMicrobiol,29,333-53.

TAYLOR,K.A.,LUTHER,P.W.&DONNENBERG,M.S.1999.ExpressionoftheEspB

proteinofenteropathogenicEscherichiacoliwithinHeLacellsaffectsstress

fibersandcellularmorphology.InfectImmun,67,120-5.



TOBE,T.,BEATSON,S.A.,TANIGUCHI,H.,ABE,H.,BAILEY,C.M.,FIVIAN,A.,YOUNIS,

R.,MATTHEWS,S.,MARCHES,O.,FRANKEL,G.,HAYASHI,T.&PALLEN,M.J.

2006.AnextensiverepertoireoftypeIIIsecretioneffectorsinEscherichiacoli

O157andtheroleoflambdoidphagesintheirdissemination.ProcNatlAcad

SciUSA,103,14941-6.

UNICEF/WHO2009.Diarrhoea:whychildrenarestilldyingandwhatcanbedone.

NewYork:UNICEF/WHO.

WACHTER,C.,BEINKE,C.,MATTES,M.&SCHMIDT,M.A.1999.InsertionofEspDinto

epithelialtargetcellmembranesbyinfectingenteropathogenicEscherichia

coli.MolMicrobiol,31,1695-707.

WIDMANN,C.,GIBSON,S.,JARPE,M.B.&JOHNSON,G.L.1999.Mitogen-activated

proteinkinase:conservationofathree-kinasemodulefromyeasttohuman.

PhysiolRev,79,143-80.

ZHOU,X.,GIRON,J.A.,TORRES,A.G.,CRAWFORD,J.A.,NEGRETE,E.,VOGEL,S.N.&

KAPER,J.B.2003.FlagellinofenteropathogenicEscherichiacolistimulates

interleukin-8productioninT84cells.InfectImmun,71,2120-9.

mini: identifying enteropathogenic escherichia coli effector proteins responsible for blocking host...

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