Article

Epsilon-Toxin Plasmids of Clostridium perfringens Type D Are Conjugative

University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Journal of Bacteriology (Impact Factor: 2.81). 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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Available from: Sameera Sayeed, Jul 01, 2014
    • "Clostridium perfringens is a Grampositive pathogen that causes histotoxic and gastrointestinal diseases in both humans and animals through its ability to produce a range of 20 toxins or extracellular enzymes, many of which are plasmid encoded (Uzal et al., 2014). Conjugative transfer of large toxin-encoding plasmids has been demonstrated for plasmids encoding the CPE, ETX, CPB2 and NetB toxins (Brynestad et al., 2001; Hughes et al., 2007; Bannam et al., 2011) and has been postulated for other toxin plasmids (Li et al., 2013). Conjugative plasmids were first identified in C. perfringens by the transfer of tetracycline resistance plasmids (Brefort et al., 1977; Rood et al., 1978). "
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    ABSTRACT: Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) have been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP), encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.
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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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    ABSTRACT: Clostridium perfringens is an anaerobic gram-positive spore-forming bacillus. It is one of the pathogens with larger distribution in the environment; it can be isolated from soil and water samples, which also belongs to the intestinal flora of animals and humans. However, on some occasions it can act as an opportunistic pathogen, causing diseases such as gas gangrene, enterotoxemia in sheep and goats and lamb dysentery, among others. In human beings, it is associated to diseases such as food poisoning, necrotic enterocolitis of the infant and necrotic enteritis or pigbel in Papua-New Guinea tribes. The renewed interest existing nowadays in the study of C. perfringens as a veterinarian and human pathogen, together with the advance of molecular biology, had enabled science to have deeper knowledge of the biology and pathology of these bacteria. In this review, we discuss and update the principal aspects of C. perfringens intestinal pathology, in terms of the toxins with major medical relevance at present.
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