title: Binding and internalization of Helicobacter pylori VacA via cellular lipid rafts in epithelial cells.
abstract: In this study we investigated the roles of lipid rafts and glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the process of VacA binding and internalization into epithelial cells. Vacuolating activity analysis in AGS, CHO cells, and a CHO-derived line that highly expresses GPI-linked fasI proteins indicated the significance of cholesterol and GPI-APs for VacA activity. Flow cytometric analysis along with VacA-cholesterol co-extraction experiments showed a cholesterol-dependent manner for VacA cell-binding activity, while GPI-APs were not related to it. Differential detergent extraction and fractionation in sucrose density gradient showed co-association of VacA and fasI with rafts on cell membranes. Subcellular distribution of fasI visualized by confocal microscope suggested that fasI trafficked via a newly defined endocytic pathway for GPI-APs in the derived line. Upon VacA intoxication, VacA was visualized to co-migrate along with fasI and finally induced vacuolation coupled with dramatic redistribution of fasI molecules. These results suggest that VacA exploits rafts for docking and entering the cell via the endocytic pathway of GPI-APs.
authors.affiliation: Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Chun-Hsien
authors.initials: CH
authors.lastname: Kuo
authors.forename: Wen-Ching
authors.initials: WC
authors.lastname: Wang
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000493:pharmacokinetics*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003603:Cytotoxins / Q000737:chemistry / Q000493:pharmacokinetics; D004847:Epithelial Cells / Q000382:microbiology* / Q000648:ultrastructure; D016480:Helicobacter pylori / Q000472:pathogenicity / Q000502:physiology*; D021962:Membrane Microdomains / Q000382:microbiology* / Q000502:physiology; D011485:Protein Binding; D021381:Protein Transport; D014617:Vacuoles / Q000382:microbiology / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D003603:Cytotoxins; C091100:GacA protein, Bacteria