Signal Transduction Mechanism Involved in Clostridium perfringens Alpha-Toxin-Induced Superoxide Anion Generation in Rabbit Neutrophils

Abstract

ABSTRACT Clostridium perfringens alpha-toxin induces the generation of superoxide anion (O 2 − ) via production of 1,2-diacylglycerol (DG) in rabbit neutrophils. The mechanism of the generation, however, remains poorly understood. Here we report a novel mechanism for the toxin-induced production of O 2 − in rabbit neutrophils. Treatment of the cells with the toxin resulted in tyrosine phosphorylation of a protein of about 140 kDa. The protein reacted with anti-TrkA (nerve growth factor high-affinity receptor) antibody and bound nerve growth factor. Anti-TrkA antibody inhibited the production of O 2 − and binding of the toxin to the protein. The toxin induced phosphorylation of 3-phosphoinositide-dependent protein kinase 1 (PDK1). K252a, an inhibitor of TrkA receptor, and LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), reduced the toxin-induced production of O 2 − and phosphorylation of PDK1, but not the formation of DG. These inhibitors inhibited the toxin-induced phosphorylation of protein kinase C θ (PKCθ). U73122, a phospholipase C (PLC) inhibitor, and pertussis toxin inhibited the toxin-induced generation of O 2 − and formation of DG, but not the phosphorylation of PDK1. These observations show that the toxin independently induces production of DG through activation of endogenous PLC and phosphorylation of PDK1 via the TrkA receptor signaling pathway and that these events synergistically activate PKCθ in stimulating an increase in O 2 − . In addition, we show the participation of mitogen-activated protein kinase-associated signaling events via activation of PKCθ in the toxin-induced generation of O 2 − .