Abstract
Clostridium perfringens enterotoxin (encoded by the cpe gene) contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne) cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644) found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity.
Highlights
Clostridium perfringens is an important pathogen of humans and domestic animals
Using well-established conditions that allow plasmids to enter a pulsed-field gel and migrate according to their molecular sizes, previous studies [11,21,25,27] had demonstrated that, i) some cpe-positive type A isolates carry a chromosomal cpe gene, while ii) other cpe-positive type A isolates and most, or all, cpe-positive type D isolates carry their cpe genes on large plasmids
Most cpe plasmids of type A isolates were found to be,70 kb or,75 kb in size [11], the cpe plasmids of type D isolates were shown to range in size from,75 kb to,110 kb [25], and the plasmids carrying silent cpe sequences in type E isolates were determined to vary in size from,100 kb to,135 kb [21]
Summary
Clostridium perfringens is an important pathogen of humans and domestic animals The virulence of this organism is largely attributable to its producing at least 16 different potent toxins, individual C. perfringens isolates never express this entire toxin arsenal [1,2]. This characteristic is exploited by a commonlyused classification system assigning C. perfringens isolates to one of five types (A–E) based upon their production of four typing toxins. About 75–80% of all type A food poisoning isolates carry their enterotoxin gene (cpe) on the chromosome [5,6,7,8,9,10]. The chromosomal cpe locus present in most type A food poisoning isolates is highly conserved and includes an upstream IS1469 sequence and flanking IS1470 sequences [11,12]
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