Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane

• Published in: PloS one 2015-04, Vol.10 (4), p.e0120497-e0120497
• Main Authors: Takagishi, Teruhisa, Oda, Masataka, Kabura, Michiko, Kurosawa, Mie, Tominaga, Kaori, Urano, Shiori, Ueda, Yoshibumi, Kobayashi, Keiko, Kobayashi, Toshihide, Sakurai, Jun, Terao, Yutaka, Nagahama, Masahiro
• Format: Article
• Language: English
• Subjects: Accumulation; Activation; Annexin V; Bacterial Toxins - metabolism; Bacterial Toxins - pharmacology; Biological Transport; Calcium-Binding Proteins - metabolism; Calcium-Binding Proteins - pharmacology; Cell Line; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell membranes; Cellular signal transduction; Chemokines; Clostridium perfringens; Clustering; Cytokines; Diacylglycerol; Diglycerides; Diglycerides - metabolism; Enzymatic activity; Fluorescence; Fluorescence microscopy; G(M1) Ganglioside - metabolism; Gangrene; Gas gangrene; Host-Pathogen Interactions; Humans; Hydrophobicity; Immune response; Inhibition; Interleukin 8; Interleukin-8 - metabolism; Lipid metabolism; Lipids; Metabolism; Models, Biological; Pathogenesis; Phosphatidylserine; Phospholipase; Phospholipase C; Phospholipase C gamma - genetics; Phospholipase C gamma - metabolism; Phosphorylation; Phosphorylation - drug effects; Protein kinase C; Receptor, trkA - metabolism; RNA Interference; siRNA; Superoxide; Superoxides; Translocation; TrkA protein; Type C Phospholipases - metabolism; Type C Phospholipases - pharmacology; Yellow fluorescent protein; α-Toxin
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0120497

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.