title: Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane.
abstract: Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima, 770-8514, Japan.
authors.forename: Teruhisa
authors.initials: T
authors.lastname: Takagishi
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima, 770-8514, Japan; Division of Microbiology and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Oda
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima, 770-8514, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kabura
authors.affiliation: Division of Microbiology and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan.
authors.forename: Mie
authors.initials: M
authors.lastname: Kurosawa
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima, 770-8514, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Tominaga
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima, 770-8514, Japan.
authors.forename: Shiori
authors.initials: S
authors.lastname: Urano
authors.affiliation: Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada-cho, Kahoku-gun, Ishikawa, 920-0293, Japan.
authors.forename: Yoshibumi
authors.initials: Y
authors.lastname: Ueda
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima, 770-8514, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Lipid Biology Laboratory, Rikagaku Kenkyujo (RIKEN), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Toshihide
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima, 770-8514, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Sakurai
authors.affiliation: Division of Microbiology and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Terao
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho 180, Tokushima, 770-8514, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagahama
mesh_terms: D001427:Bacterial Toxins / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport; D002135:Calcium-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D008954:Models, Biological; D051966:Phospholipase C gamma / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D034622:RNA Interference; D020917:Receptor, trkA / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C011439:1,2-diacylglycerol; D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D004075:Diglycerides; D016209:Interleukin-8; D005677:G(M1) Ganglioside; D020917:Receptor, trkA; D010738:Type C Phospholipases; C522157:PLCG1 protein, human; D051966:Phospholipase C gamma; C040649:alpha toxin, Clostridium perfringens