Inactivation of the gene (cpe ) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe‐positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops

Abstract

SummaryPrevious epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses con®rmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced signi®cant ileal loop¯uid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wildtype cpe gene, which con®rms that the observed loss of virulence for the cpe knock-out mutants results from the speci®c inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a ®nding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.

Clostridium perfringensin poultry: an emerging threat for animal and public health

“…C. perfringens type A strains produce the chromosomal-encoded alpha-toxin, while C. perfringens type C strains produce alpha toxin together with beta toxin (Petit et al, 1999). Some strains of C. perfringens type A produce an enterotoxin at the moment of sporulation, causing disease in humans (Ridell et al, 1998;Sarker et al, 1999). The mechanism of action of the enterotoxin will be discussed further in the text.…”

The importance of calcium influx, calpain and calmodulin for the activation of CaCo-2 cell death pathways by Clostridium perfringens enterotoxin

“…CPE is both necessary and sufficient for obtaining the gastrointestinal (GI) symptoms of C. perfringens type A food poisoning (Sarker et al , 1999;, the third most frequently identified foodborne illness in the USA (Bean et al ., 1996). This enterotoxin also contributes (Sarker et al ., 1999) to the GI symptoms of up to 5-20% of all cases of non-foodborne human GI diseases (Carman, 1997) and may be involved in certain veterinary diarrhoeas (Songer, 1996).…”

Detection of Enterotoxigenic Clostridium perfringens Type A Isolates in American Retail Foods

“…This toxin is both necessary and sufficient for the enteric virulence of C. perfringens type A food poisoning isolates. Specific inactivation of the enterotoxin gene (cpe) by allelic exchange was shown to render a C. perfringens food poisoning isolate avirulent in rabbit ileal loops, and intestinal virulence could be restored by complementing that mutant with a cloned copy of the wild-type cpe gene (16). Furthermore, ingestion of purified CPE by human volunteers was determined to be sufficient for reproducing the cramping and diarrheic symptoms of the natural food poisoning (18).…”