NationalScienceFoundationResearchProposal,Example1

Objectives:Theobjectiveofthisstudyistouseinsertionaltransposonmutagenesistoidentifygenesin
HelicobacterpyloriwhichwheninactivatedconferresistancetotheantibioticMetronidazole.Thesegenes
willthenbeanalyzedinstrainsisolatedfrompatientswhereantimicrobialtherapyfails.Finally,these
geneswillbecharacterizedusingbioinformaticstoidentifyhomologousgeneswithknownfunctionand
thegeneproductswillbebiochemicallytestedfortheirabilitytometabolizeMetronidazole.
Background:HelicobacterpyloriisaGramnegative,microaerobic,spiralbacteriumthatcolonizesthe
humanstomach.Approximately50%oftheworldspopulationcarriesH.pylori.Whilemostcasesare
asymptomatic,H.pyloriinfectionisthemajorcausativeagentofstomachulcersandisaleadingriskfactor
forgastriccancer(1).

ThecurrenttreatmentforH.pyloriinfectionsinvolveatripletherapywhichconsistsofoneofa
varietyofdrugsthatdecreasetheacidityofthestomach,andtwoantibioticsincludingMetronidazole(Mtz)
(2).Mtz,usedfortreatmentofmanybacterialandparasiticpathogens,isadministeredasaninactive
prodrug.Theprodrugisconvertedtoacytotoxicformwithinthelowintracellularredoxpotential
environmentofanaerobicandmicroaerobicorganisms,butisunchangedinaerobicorganisms,whichisthe
basisforitsselectivetoxicity.TheactiveformofMtzgeneratesmutagenicoxygenradicalsandnitroradicals
leadingtocelldeath.

Thehighuseofantibiotics,includingMtz,bothinhumansaswellasfordomesticanimals,hasled
toanincreasingrateofresistance,ashighas80%insomeareasoftheworld(3).InH.pylori,RdxA
protein,aputativeoxygeninsensitiveNADPHnitroreductase,isthoughttoactivateMtzbyreducingthe
nitroaromaticgroupofMtztohydroxylamine,formingapotentmutagen.Previously,ithasbeenshown
thatinactivationoftherdxAgeneisassociatedwithMtzresistantH.pylori(4).However,notallcasesof
MtzresistancecorrelatetolossofRdxAprotein(5).Furthermore,theseresistantstrainsarestillsusceptible
toMtzifgiveninhighenoughdoses.Thissuggeststhattheremaybeothergenesresponsiblefor
activatingMtz.Metabolicenzymessuchaspyruvateoxidoreductase(POR)andketoglutarate
oxidoreductase(KOR),forexample,havebeenshowntoactivateMtzinotheranaerobes(6).PORand
KORactivitieshavealsobeenshowntoberepressedinMtzresistantstrainsofH.pyloriculturedinthe
presence,butnotintheabsence,ofMtz(7).Thefocusofthisstudyistodeterminewhatgenes,when
inactivatedinH.pylori,giverisetoMtzresistance.
Hypothesis:MetronidazoleresistanceofHelicobacterpyloriismediatedthroughlossofspecificgenes.
Aim1:ScreenforH.pylorimutantsthatconferMtzresistanceanddeterminewhichgenesweremutated.

AtransposoninsertionlibraryofH.pylorihasbeenconstructedintheSalamaLab(unpublished
data)withaminiTn7basedtransposonconferringresistancetochloramphenicol(Cm).Sincescreening
forMtzresistanceinwildtypebackgroundgivesrisetomanycoloniesthatareresistantduetotransposon
insertionintherdxAgene(SalamaLab,unpublished),H.pylorithatarerdxAduetoadeletionwillbe
transformedwithgenomicDNAfromthetransposonlibraryandtransformantsselectedonCmmedia.
ThesetransformedcellswillthenbegrownonbloodplatesthatcontainhigherconcentrationsofMtzthan
rdxAmutantscantolerate,thusselectingforstrainsthathaveincreasedMtzresistantduetoinactivationof
othergenes.

ThepositionofchromosomalinsertionwillbedeterminedusingasemirandomPCRmethodto
amplifytheDNAflankingthetransposon(8).Thisproductwillthenbesequencedtodeterminewhich
genewasinterruptedbytheinsertionofthetransposon.

InordertodeterminethatinactivationofthegeneidentifiedbythescreencausesMtzresistance,
anindependentmutantwillbeconstructedusingsitedirectedmutagenesisonH.pylorithatisrdxA+.This
mutantwillthenbegrownonplatescontainingMtztoretestforMtzresistance.IfMtzresistanceisfound,
furtherconfirmationwillbeobtainedbyintroducingawildtypecopyofthegeneonashuttlevector
plasmidintothemutantstraintoseeifMtzsensitivityisrestored.Additionally,awildtypecopyofthe
genewillbeclonedintoE.coli,whichisnaturallyMtzresistant,totestforMtzsensitivityinorderto
determinethemechanismofactionofthegeneproduct.

If,however,theH.pylorimutantisnotresistanttoMtz,thelossofboththisgeneandrdxAmight
benecessarytoconferMtzresistance.Inthiscase,anindependentmutantwillbemadeinrdxAH.pylori.
Alternatively,theinsertionofthetransposoncouldcauseapolareffect,disruptinggenesdownstreamfrom
thegeneinquestion.Independentmutationswillbemadetothesedownstreamgenes,andthenretestedfor
Mtzresistance.

NationalScienceFoundationResearchProposal,Example1
Aim2:DetermineifalterationsofgenesidentifiedintheMtzresistancescreenarefoundinnaturalisolates
ofH.pylori.
Totestwhethermutationofthesegenesarefoundclinically,H.pylorisampleswillbetakenfrominfected
patientswhofailtoshoweradicationupontreatmentwithMtz.Thegenesofinterestwillbesequenced
fromthesestrainsandanalyzedforalterationsincludingstopcodons,deletionsandotherdefects.
Aim3:Determinethefunctionofthesegenesusingsequencehomologyandelectronspinresonance
spectroscopy.
Thesequencesofthesegeneswillbecomparedtootherorganismstosearchforhomologueswithknown
function.Thegenecouldsharehomologywithknownreductases,includingrdxA.Ontheotherhand,it
couldbeinvolvedinregulatingreductaseactivitythroughtranscriptionalcontrolorposttranslational
modifications.Ineithercase,thegeneseffectonreductaseactivitywillbedeterminedusinginvivo
electronspinresonance(ESR)spectroscopy.ESRdetectselectronswithunpairedspins,orfreeradicals,
whicharefoundintheactivated,reducedformofMtz(6).H.pylori,bothwildtypeandmutantinthe
gene,willbetreatedwithMtzandtestedindependentlyforfreeradicalformation.Detectionoffree
radicalsinthepresence,butnottheabsenceofthegenewillverifythegenesroleinreductaseactivation
onMtz,andexplainhowlossofthisgenewillgiverisetoresistantstrains.
Relevance:Antibioticresistancecanbebroughtaboutthroughthegainofgenes,asineffluxchannels
whichpumpouttheantibiotic,oreffectorproteinswhichinactivatetheantibiotic,aswellasthroughlossof
genes,asinthecaseofrdxAwhichcodesforaseeminglynonessentialmetabolicenzyme.Identificationof
theseresistanceconferringgenesallowformethodsofrapiddetectionofresistantpathogenstoensure
propertreatment,aswellasidentifyingtargetsforthedevelopmentofnewantibiotics.H.pyloriprovides
anexcellentmodelforstudyingthegeneticbasisofantibioticresistance,asitwasthefirstorganismin
whichtwodifferentstrainswerefullysequenced(9,10).Thisinvaluabletool,alongwithrecent
advancementsingenomictoolsdevelopedforH.pylori,allowgeneticmanipulationsandcharacterizations
ofgenesofinterest.
References:
1.UemuraN,OkamotoS,YamamotoS.2001.Helicobacterpyloriinfectionandthedevelopmentof
gastriccancer.NEnglJMed345:784789.
2.MalfertheinerP,NegraudF,OMorainC,HunginAP,JonesR,AxonA,GrahamDY,TytgatG.2002.
CurrentConceptsinthemanagementofHelicobacterpyloriinfection.AlimentPharmacolTher2:167
180.
3.EltahawyAT.2002.PrevalenceofprimaryHelicobacterpyloriresistancetoseveralantimicrobialsina
SaudiTeachingHospital.MedPrincPract2:6568.
4.GoodwinA,KersulyteD,SissonG,VeldhuyzenZSJO,BergDE,HoffmanPS.1998.Metronidazole
resistanceinHelicobacterpyloriisduetonullmutationsinagene(rdxA)thatencodesanoxygeninsensitive
NADPHnitroreductase.MolMicro28:383393.
5.LathamSR,LabigneA,JenksP.2002.ProductionoftheRdxAproteininmetronidazolesusceptible
andresistantisolatesofHelicobacterpyloriculturedfromtreatedmice.JAntimicrobialChemo49:675
678.
6.MorenoSNJ,MasonRP,MunizRP,CruzRS,DocampoR.1983.Generationoffreeradicalsfrom
metronidazoleandothernitroimidazolesbyTritrichomonasfoetus.JBiolChem258:40514054.
7.HoffmanPS,GoodwinA,JohnsenJ,MageeK,VeldhuyzenZSJO.1996.Metabolicactivitiesof
MetronidazolesusceptibleandresistantstrainsofHelicobacterpylorirepressionofpyruvate
oxidoreductaseandexpressionofisocitratelyaseactivitycorrelatewithresistance.JBacteriol178:4822
4829.
8.ManoilC.2000.TaggingexportedproteinsusingEscherichiacolialkalinephosphatasegenefusions.
MethodsinEnzymology326:3547.
9.TombJF,WhiteO,KervalageAR,ClaytonRA,SuttonGG,FleishcmannRD.1997.Thecomplete
genomesequenceofthegastricpathogenHelicobacterpylori.Nature388:539547.
10.AlmRA,LingLS,MoirDT,KingBL.1999.Genomicsequencecomparisonoftwounrelatedisolates
ofthehumangastricpathogenHelicobacterpylori.Nature397:176180.