ProjectSummary UropathogenicEscherichiacoli(UPEC)arethecausativeagentinover80%ofurinarytractinfections(UTIs). Onaverage,8-9millionpeoplearetreatedforuncomplicatedUTIsandover1millionpeoplearediagnosed withhospital-acquiredUTIsannuallyintheUS.ComplicationsofUPECinfectionsincluderecurrentUTIs, ascendinginfectionintotheuretersandkidneysresultinginpyelonephritis,andinseverecases,urosepsisand death.IdentifyingandcharacterizingthemechanisticfunctionsofUPECvirulencefactorswillleadtoimproved preventionprotocolsandtreatmentforcomplicatedUTIs.Thehemolysinexotoxin(HlyA)isanimportantUPEC virulencefactor.HlyAisexpressedin31-48%ofE.colirecoveredfromuncomplicatedUTIs,whileinstrains isolatedfromcasesofpyelonephritisorurosepsis,50-78%ofstrainsexpressHlyA.Athighdoses,HlyAis cytotoxictoawiderangeofcelltypesandspecies,althoughthemechanismforinitiatingcelldeathremains unclear.Theproposedresearchwilltestthehypothesisthatahostreceptororsignalingpathwayissubverted byHlyAtocausecelldeath.Agenome-wideselectionofaCRISPR/Cas9mutatedlibraryofeukaryoticcells wasperformedtoidentifyhostfactorsthatarerequiredforHlyA-mediatedcelldeath.Twohitswereidentified, theintegrin?2subunitandsignalpeptidepeptidase-likeprotein3(SPPL3),aproteaseinvolvedinregulationof cellularglycosylation.ThisresearchplanisdesignedtovalidatethetopcandidatesbydiscreteCRISPR/Cas9 mediatedgenometargetingtodisruptgenesofinterest.Preliminarydataincelllineswiththesubunitsof?2 familyintegrinstargetedfordisruptionindividuallyrevealsthatnoneoftheindividualalphasubunitsof?2 integrinsarenecessaryforHlyAcytotoxicity.Thesufficiencyofthefourpossible?2familymembersforHlyA cytotoxicitywillbeassessinthisproposal.BasedonthereceptorrangeforHlyA,theinteractionofHlyAwith?2 familyintegrinswillbecharacterizedusingsite-directedmutagenesistargetingregionsofinterest.Theroleof SPPL3inHlyAmediatedcytotoxicitywillbevalidatedbygeneticdisruptionusingCRISPR/Cas9.Aprotease- deficientformofSPPL3,whichcannotregulateglycosylation,willbeusedtocomplementSPPL3-deficient cellstoinvestigateadditionalrolesforSPPL3inHlyAcytotoxicity.Theglycosylationstateofb?2familyintegrins willbeexaminedinSPPL3deficientandoverexpressingcelllines.Finally,thereceptor-mediatedactivityof HlyAwillbeexaminedinaspeciesspecificmanner.Humancellsexpressing?2receptorsare100-foldmore sensitivetoHlyAcytotoxicactivitythanmousecellsexpressing?2receptors.Iftheinteractionisspecies specific,invivoworkinthemousemodelcannotaccuratelyassesstheroleofHlyAinhumanUPECinfections andthegenerationofahumanizedmousemodelwillbewarranted.Thisworkwillprovideimportantinsightinto themechanisticfunctionofHlyA-mediatedcytotoxicityandprovidethegroundworkfortherapeuticintervention incomplicatedUTIs.