Molecular and Cellular Basis of Microvascular Perfusion Deficits Induced by Clostridium perfringens and Clostridium septicum

Reduced tissue perfusion leading to tissue ischemia is a central component of the pathogenesis of myonecrosis caused by Clostridium perfringens. The C. perfringens α-toxin has been shown capable of inducing these changes, but its potential synergy with perfringolysin O (θ-toxin) is less well understood. Similarly, Clostridium septicum is a highly virulent causative agent of spontaneous gas gangrene, but its effect on the microcirculation has not been examined. Therefore, the aim of this study was to use intravital microscopy to examine the effects of C. perfringens and C. septicum on the functional microcirculation, coupled with the use of isogenic toxin mutants to elucidate the role of particular toxins in the resultant microvascular perfusion deficits. This study represents the first time this integrated approach has been used in the analysis of the pathological response to clostridial toxins. Culture supernatants from wild-type C. perfringens induced extensive cell death within 30 min, as assessed by in vivo uptake of propidium iodide. Furthermore, significant reductions in capillary perfusion were observed within 60 min. Depletion of either platelets or neutrophils reduced the alteration in perfusion, consistent with a role for these blood-borne cells in obstructing perfusion. In addition, mutation of either the α-toxin or perfringolysin O structural genes attenuated the reduction in perfusion, a process that was reversed by genetic complementation. C. septicum also induced a marked reduction in perfusion, with the degree of microvascular compromise correlating with the level of the C. septicum α-toxin. Together, these data indicate that as a result of its ability to produce α-toxin and perfringolysin O, C. perfringens rapidly induces irreversible cellular injury and a marked reduction in microvascular perfusion. Since C. septicum induces a similar reduction in microvascular perfusion, it is postulated that this function is central to the pathogenesis of clostridial myonecrosis, irrespective of the causative bacterium

Comparison of a nontoxic variant of Clostridium perfringens alpha-toxin with the toxic wild-type strain

The alpha-toxin produced by Clostridium perfringens is one of the best-studied examples of a toxic phospholipase C. In this study, a nontoxic mutant protein from C. perfringens strain NCTC8237 in which Thr74 is substituted by isoleucine (T74I) has been characterized and is compared with the toxic wild-type protein. Thr74 is part of an exposed loop at the proposed membrane-interfacing surface of the toxin. The mutant protein had markedly reduced cytotoxic and myotoxic activities. However, this substitution did not significantly affect the catalytic activity towards water-soluble substrate or the overall three-dimensional structure of the protein. The data support the proposed role of the 70-90 loop in the recognition of membrane phospholipids. These findings also provide key evidence in support of the hypothesis that the hydrolysis of both phosphatidylcholine and sphingomyelin are required for the cytolytic and toxic activity of phospholipases

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated front the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C164I165M166, which characterize the "metzincin" superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four a-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single a-helix and several loops. The catalytic zinc ion is coordinated by the N-epsilon2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix