Abstract
Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.
Highlights
Clostridium perfringens type A is a Gram positive, spore forming anaerobic bacterium and is the causative agent of several human diseases, including gas gangrene or clostridial myonecrosis [1,2,3,4,5]
Wild-type. (p.0.05 using log-rank Mantel-Cox test comparing wild type with mutant and complemented strains) (B) Histopathological analysis of muscle tissue isolated from mice infected with the strains indicated
It was previously shown that when purified a-clostripain from C. perfringens was injected intradermally into mice there was an increase in vascular permeability [16], thereby implying that aclostripain may play a role in the pathogenesis of clostridial myonecrosis
Summary
Clostridium perfringens type A is a Gram positive, spore forming anaerobic bacterium and is the causative agent of several human diseases, including gas gangrene or clostridial myonecrosis [1,2,3,4,5]. The extracellular cysteine protease a-clostripain was first discovered in Clostridium histolyticum [8,9,10] and a homologue was later identified in C. perfringens [11,12,13,14] Both the C. perfringens and C. histolyticum a-clostripain proteins exist as heterodimeric polypeptides, consisting of a heavy chain and a light chain, which are held together by strong, non-covalent forces [8,9,15]. They are classified as members of the C11 peptidase superfamily, which includes gingipains and legumains [12], and are grouped based on their structural and functional similarity rather than their sequence similarity
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