Cloning, nucleotide sequencing, and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli

Abstract

A complete copy of the gene (cpe) encoding Clostidium perfingens enterotoxin (CPE), an important virulence factor involved in C. perfringens food poisoning and other gastrointestinal illnesses, has been cloned, sequenced, and expressed in Escherichia coli. The cpe gene was shown to encode a 319-amino-acid polypeptide with a deduced molecular weight of 35,317. There was no consensus sequence for a typical signal peptide present in the 5' region of cpe. Cell lysates from recombinant cpe-positive E. coli were shown by quantitative immunoblot analysis to contain moderate amounts of CPE, and this recombinant CPE was equal to native CPE in cytotoxicity for mammalian Vero cells. CPE expression in recombinant E. coli appeared to be largely driven from a clostridial promoter. Immunoblot analysis also demonstrated very low levels of CPE in vegetative cell lysates of enterotoxin-positive C. perfringens. However, when the same C. perfringens strain was induced to sporulate, much stronger CPE expression was detected in these sporulating cells than in either vegetative C. perfringens cells or recombinant E. coli. Collectively, these results strongly suggest that sporulation is not essential for cpe expression, but sporulation does facilitate high-level cpe expression. Clostridium perfringens ranks among the most important of the anaerobic bacterial pathogens for humans and domestic animals, causing myonecrosis (gas gangrene), anaerobic cellulitis, septicemia, uterine infections, and gastrointestinal illnesses (28). The virulence of this endospore-forming, gram-positive bacterium results from its prolific ability to produce protein toxins (28). Among the 13 types of toxins known to be produced by this organism is C. perfringens enterotoxin (CPE), which is a single polypeptide of 35 kDa with a unique amino acid sequence (9, 23, 24, 28). CPE causes the symptoms associated with C. perfringens type A food poisoning, which is among the most common human food-borne illnesses (23, 24, 28), and it also appears to be involved in other important human and veterinary gastrointestinal illnesses (2, 5, 24). Recent studies (11, 15, 26, 27, 43) have shown CPE action to involve sequentially (i) specific binding of enterotoxin to a proteinaceous receptor on mammalian membranes, (ii) insertion of CPE into plasma membranes, (iii) formation of a complex between enterotoxin and two mammalian membrane proteins of 70 and 50 kDa, (iv) production of membrane permeability alterations for small molecules such as ions, and (v) induction of secondary effects which culminate in cell death. CPE binding to mammalian receptors is mediated by a 30-amino-acid region at the extreme C terminus of enterotoxin (12, 14). Cytotoxicity also requires sequences in the N-terminal half of the enterotoxin, suggesting the existence of separate binding and cytotoxicity domains on CPE (13). Upon entering the human host, it is common for many bacterial pathogens to undergo global changes in gene expression (30). These changes often include de novo synthesis of virulence factors...

Clostridium perfringensType E Animal Enteritis Isolates with Highly Conserved, Silent Enterotoxin Gene Sequences

“…Consistent with previous reports demonstrating that CPE expression by type A isolates is strongly associated with sporulation (7,8,16), CPE-specific Western blotting detected no CPE expression during vegetative growth of the cpe-positive type A isolates F4406 and NCTC 10239 (data not shown) but showed that both of these cpe-positive type A strains (but not the cpe-negative type A strain ATCC 3624) produced moderate to high levels of CPE ( Fig. 2) when grown in Duncan-Strong sporulation medium supplemented with 1.5% bile and 0.005% theophylline (DS-B).…”

“…Combining these sequencing results with the CPE expression and Southern blot results presented above, it appears likely that our type E animal enteritis isolates and NCIB 10748 carry a single cpe sequence which is silent not only because it lacks promoters, a ribosome binding site, and an initiation codon but also because it contains numerous nonsense and frameshift mutations. This finding is remarkable given recent results (4,6,8) demonstrating that not a single base pair variation is present in the cpe ORF of eight different type A isolates, and it confirms that the mutations present in the silent NCIB 10748 cpe sequence are not simply an artifactual consequence of long-term laboratory cultivation.…”

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

“…Furthermore, densitometric analyses of these Western blots revealed that, under the growth conditions used in this experiment, CPE expression was ,®vefold higher in F4969 compared with SM101 (Table 1). Collectively, these results are consistent with previous studies (Czeczulin et al, 1993;1996;Kokai-Kun et al, 1994;Collie Q 1999 perfringens wild-type and cpe mutant strains. DNA prepared from wild-type (SM101 and F4969) and cpe mutant (MRS101 and MRS4969) C. perfringens strains was digested with HpaI, subjected to electrophoresis, blotted and probed separately with either cpe-speci®c or catP -speci®c DIG-labelled probes.…”

Clostridium perfringens type E enteritis in calves: two cases and a brief review of the literature

“…Silent cpe sequences, found near the iota toxin genes on episomal DNA, were highly conserved among type E isolates, but contained nine nonsense and two frameshift mutations and lacked the initiation codon, promoters, and ribosome binding site. This is remarkable, given that sequencing of cpe from eight different isolates revealed 100% sequence homology [19,[23][24][25]. These strains were apparently not clonal; location of cpe sequences, with iap and ibp, on episomal DNA and lack of isolate-specific mutations suggests recent wide distribution among C. perfringens isolates.…”