Relationship between Localization on Cellular Membranes and Cytotoxicity of Vibrio vulnificus Hemolysin 英文参考文献.docVIP

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Relationship between Localization on Cellular Membranes and Cytotoxicity of Vibrio vulnificus Hemolysin 英文参考文献.doc

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Relationship between Localization on Cellular Membranes and Cytotoxicity of Vibrio vulnificus Hemolysin 英文参考文献

RelationshipbetweenLocalizationonCellular MembranesandCytotoxicityofVibriovulnificus Hemolysin HiroyukiSugiyama1,TakashigeKashimoto1*,ShunjiUeno1,HayatoEhara1,ToshioKodama2 ,Tetsuya Iida3,NobuyukiSusa1 1Laboratory of Veterinary Public Health, School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan, 2Department of Bacterial Infections, Research InstituteforMicrobialDiseases,OsakaUniversity,Suita,Osaka,Japan,3InternationalResearchCenterforInfectiousDiseases,ResearchInstituteforMicrobialDiseases, OsakaUniversity,Suita,Osaka,Japan Abstract Vibriovulnificussecretesahemolysin/cytolysin(VVH)thatinducescytolysisintargetcells.Adetergentresistantmembrane domain(DRM)fractionofthecellsaftersucrosegradientcentrifugationincludescholesterol-richmembranemicrodomains whichhavebeencalled‘‘lipidrafts’’.Itwasreportedthatsomepore-formingtoxinsrequireassociationwithDRMand/or lipidraftstoexerttheircytotoxicity.IthasalsobeenthoughtthatcellularcholesterolisinvolvedinVVHcytotoxicitybecause VVHcytotoxicitywasinhibitedbypre-incubationwithcholesterol.However,bothcellularlocalizationandmodeofactionof VVHcytotoxicity remainunclear.Inthisstudy, weinvestigatedtherelationshipbetween VVHlocalizationonthecellular membraneanditscytotoxicity.OligomersofVVHweredetectedfromDRMfractionsbysucrosegradientultracentrifugation butalloftheseoligomersshiftedfromDRMfractionstonon-DRMfractionsaftertreatmentwithmethyl-beta-cyclodextrin (MbCD),acholesterolsequesteringagent.Ontheotherhand,immunofluorescenceanalysisshowedthatVVHdidnotco- localize with major lipid raft markers on cellular membrane of CHO cells. These data suggested that VVH localized at membraneregionswhicharerelativelyabundantincholesterolbutwhicharenotidenticalwithlipidrafts.Todeterminethe linkage between localization and cytotoxicity of VVH, cytotoxicity was evaluated in MbCD-treated CHO cells. The cytotoxicityofVVHwasnotdecreasedbytheMbCDtreatment.Inaddition,theamountofVVHoligomerdidnotdecreasein MbCD-treatedCHOcells.Thus,wef