Abstract
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(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer (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 β1,4-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.
Highlights
We reported that alpha-toxin simultaneously induced the formation of diacylglycerol through the activation of endogenous phospholipase Cs (PLCs) and phosphorylation of ERK1/2, NFB, and p38MAPK via activation of tyrosine kinase A (TrkA) in human lung adenocarcinoma epithelial cell line (A549) cells and that these events induced the release of interleukin-8 (IL-8) [19]
To investigate the effect of gangliosides and sialic acid on the binding of alpha-toxin to A549 cells and on the release of IL-8, the cells were treated with various concentrations of PPMP, which is a ganglioside synthase inhibitor, or neuraminidase from C. perfringens, which catalyzes the hydrolysis of terminal sialic acid residues, at 37 °C for 4 days or 60 min, respectively
In this study we have shown that the pretreatment of A549 cells with PPMP resulted in attenuation of alpha-toxin binding
Summary
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 alphatoxin to GM1a facilitates the activation of the TrkA receptor and induces a signal transduction cascade that promotes the release of chemokines. We previously reported that alpha-toxin-induced activation of endogenous PLC and sphingomyelinase via pertussis toxinsensitive GTP-binding protein plays an important role in hemolysis of rabbit and sheep erythrocytes [8, 11, 18]. We have investigated the role of gangliosides in the pathogenicity of alpha-toxin, and analyzed the binding region with which the toxin associates with gangliosides
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