title: Invasion of epithelial cells by Campylobacter jejuni is independent of caveolae.
abstract: Caveolae are 25-100 nm flask-like membrane structures enriched in cholesterol and glycosphingolipids. Researchers have proposed that Campylobacter jejuni require caveolae for cell invasion based on the finding that treatment of cells with the cholesterol-depleting compounds filipin III or methyl-β-cyclodextrin (MβCD) block bacterial internalization in a dose-dependent manner. The purpose of this study was to determine the role of caveolae and caveolin-1, a principal component of caveolae, in C. jejuni internalization. Consistent with previous work, we found that the treatment of HeLa cells with MβCD inhibited C. jejuni internalization. However, we also found that the treatment of HeLa cells with caveolin-1 siRNA, which resulted in greater than a 90% knockdown in caveolin-1 protein levels, had no effect on C. jejuni internalization. Based on this observation we performed a series of experiments that demonstrate that MβCD acts broadly, disrupting host cell lipid rafts and C. jejuni-induced cell signaling. More specifically, we found that MβCD inhibits the cellular events necessary for C. jejuni internalization, including membrane ruffling and Rac1 GTPase activation. We also demonstrate that MβCD disrupted the association of the β1 integrin and EGF receptor, which are required for the maximal invasion of epithelial cells. In agreement with these findings, C. jejuni were able to invade human Caco-2 cells, which are devoid of caveolae, at a level equal to that of HeLa cells. Taken together, the results of our study demonstrate that C. jejuni internalization occurs in a caveolae-independent manner.
authors.affiliation: School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Life Sciences Bldg, Room 302c, Pullman, WA, USA. konkel@vetmed.wsu.edu.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Konkel
authors.forename: Derrick R
authors.initials: DR
authors.lastname: Samuelson
authors.forename: Tyson P
authors.initials: TP
authors.lastname: Eucker
authors.forename: Eric A
authors.initials: EA
authors.lastname: Shelden
authors.forename: Jason L
authors.initials: JL
authors.lastname: O'Loughlin
mesh_terms: D018938:Caco-2 Cells; D016123:Campylobacter jejuni / Q000502:physiology*; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000382:microbiology* / Q000648:ultrastructure; D066246:ErbB Receptors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D047392:beta-Cyclodextrins / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D051242:Caveolin 1; D019012:Integrin beta1; C107953:RAC1 protein, human; D034741:RNA, Small Interfering; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D066246:ErbB Receptors; D020830:rac1 GTP-Binding Protein