Abstract

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.

Public Health Relevance

UrinarypathogenicEscherichiacoli(UPEC)areamongthemostcommoncausesofbacterialinfection, resultinginover9milliondoctorvisitsannuallyinthiscountryandmorethan150millioninfectionsworld-wide. ThisprojectwillcharacterizeatoxinassociatedwithsevereUPECinfectionsincludingkidneyorbloodstream infectionsanddeath.Understandingtheactivityofthistoxininsevereinfectionwillprovidedetailstodevelop therapeuticsforpreventionofandtreatmentsforcomplexurinarytractinfections.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32AI126900-02
Application #
9562877
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Ernst, Nancy L
Project Start
2017-09-01
Project End
2019-08-31
Budget Start
2018-09-01
Budget End
2019-08-31
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost

  Institution

Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715