relationship between localization on cellular membranes and cytotoxicity of vibrio vulnificus hemolysin关系定位在细胞膜和细胞毒性的创伤弧菌溶血素.pdfVIP

Relationship between Localization on Cellular Membranes and Cytotoxicity of Vibrio vulnificus Hemolysin 1 1 1 1 2 Hiroyuki Sugiyama , Takashige Kashimoto *, Shunji Ueno , Hayato Ehara , Toshio Kodama , Tetsuya Iida3, Nobuyuki Susa1 1 Laboratory of Veterinary Public Health, School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan, 2 Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan, 3 International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan Abstract Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis in target cells. A detergent resistant membrane domain (DRM) fraction of the cells after sucrose gradient centrifugation includes cholesterol-rich membrane microdomains which have been called ‘‘lipid rafts’’. It was reported that some pore-forming toxins require association with DRM and/or lipid rafts to exert their cytotoxicity. It has also been thought that cellular cholesterol is involved in VVH cytotoxicity because VVH cytotoxicity was inhibited by pre-incubation with cholesterol. However, both cellular localization and mode of action of VVH cytotoxicity remain unclear. In this study, we investigated the relationship between VVH localization on the cellular membrane and its cytotoxicity. Oligomers of VVH were detected from DRM fractions by sucrose gradient ultracentrifugation but all of these oligomers shifted from DRM fractions to non-DRM fractions after treatment with methyl-beta-cyclodextrin (MbCD), a cholesterol sequestering agent. On the other hand, immunofluorescence analysis showed that VVH did not co- localize with major lipid raft markers