Article

Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative

Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Victoria 3800, Australia.
Journal of Bacteriology (Impact Factor: 2.69). 12/2007; 189(21):7531-8. DOI: 10.1128/JB.00767-07
Source: PubMed

ABSTRACT Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.

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    • "For C. perfringens, reverse genetic approaches have been, until recently, applied only to study the pathogenesis of the relatively simple type A isolates, where they clearly identified the enterotoxin as a key contributor to the GI pathogenesis of enterotoxin-positive type A isolates (Sarker et al., 1999) and established roles for both alpha toxin and PFO in the pathogenesis of gas gangrene caused by enterotoxin-negative type A isolates (Awad et al., 1995; 2001). However, we recently used classical allelic exchange approaches to inactivate the epsilon toxin gene (etx) in two type D disease isolates (Hughes et al., 2007). Trypsin-activated culture supernatants prepared from either type D isolate were highly cytotoxic for MDCK cells. "
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