mini: identifying enteropathogenic escherichia coli effector proteins responsible for blocking host...
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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.
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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
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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.
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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.
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Figure6:RepresentativeimmunofluorescencefieldsshowingF-actinstaining(red)andnucleic
acidwithDAPI (blue) inuninfectedHEp-2cells(C-)andHEp-2cellsinfected for 4hourswith
EPECWT,ΔespBandΔIE6.Arrowsindicateactinpedestalformation(A/Elesions).
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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
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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).
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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
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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
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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.
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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.
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Figure 11: Plasmid preparation (MiniPrep) of selected colonies with vectors containing the
specific EPEC effector genes. Purified products separated by gel electrophoresis and
photographedunderUVlight.
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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
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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.
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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-).
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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
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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.
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