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

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.

Clostridium perfringens alpha-toxin recognizes the GM1a-TrkA complex.

Clostridium perfringens alpha-toxin is the major virulence factor in the pathogenesis of gas gangrene. Alpha-toxin is a 43-kDa protein with two structural domains; the N-domain contains the catalytic site and coordinates the divalent metal ions, and the C-domain is a membrane-binding site. The role of the exposed loop region (72-93 residues) in the N-domain, however, has been unclear. Here we show that this loop contains a ganglioside binding motif (H … SXWY … G) that is the same motif seen in botulinum neurotoxin and directly binds to a specific conformation of the ganglioside Neu5Acα2-3(Galß1-3GalNAcß1-4)Galß1-4Glcß1Cer (GM1a) through a carbohydrate moiety. Confocal microscopy analysis using fluorescently labeled BODIPY-GM1a revealed that the toxin colocalized with GM1a and induced clustering of GM1a on the cell membranes. Alpha-toxin was only slightly toxic in ß1,4-N-acetylgalactosaminyltransferase knock-out mice, which lack the a-series gangliosides that contain GM1a, but was highly toxic in α2,8-sialyltransferase knock-out mice, which lack both b-series and c-series gangliosides, similar to the control mice. Moreover, experiments with site-directed mutants indicated that Trp-84 and Tyr-85 in the exposed alpha-toxin loop play an important role in the interaction with GM1a and subsequent activation of TrkA. These results suggest that binding of alpha-toxin to GM1a facilitates the activation of the TrkA receptor and induces a signal transduction cascade that promotes the release of chemokines. Therefore, we conclude that GM1a is the primary cellular receptor for alpha-toxin, which can be a potential target for drug developed against this pathogen.