The CPR1953 and CPR1954 orphan histidine kinases profoundly affect sporulation initiation and Clostridium perfringens enterotoxin (CPE) production by C. perfringens type F strain SM101, whether cultured in vitro (modified Duncan–Strong sporulation medium (MDS)) or ex vivo (mouse small intestinal contents (MIC)). To help distinguish whether CPR1953 and CPR1954 act independently or in a stepwise manner to initiate sporulation and CPE production, cpr1953 and cpr1954 null mutants of SM101 were transformed with plasmids carrying the cpr1954 or cpr1953 genes, respectively, causing overexpression of cpr1954 in the absence of cpr1953 expression and vice versa. RT-PCR confirmed that, compared to SM101, the cpr1953 mutant transformed with a plasmid encoding cpr1954 expressed cpr1954 at higher levels while the cpr1954 mutant transformed with a plasmid encoding cpr1953 expressed higher levels of cpr1953. Both overexpressing strains showed near wild-type levels of sporulation, CPE toxin production, and Spo0A production in MDS or MIC. These findings suggest that CPR1953 and CPR1954 do not function together in a step-wise manner, e.g., as a novel phosphorelay. Instead, it appears that, at natural expression levels, the independent kinase activities of both CPR1953 and CPR1954 are necessary for obtaining sufficient Spo0A production and phosphorylation to initiate sporulation and CPE production.

C. perfringens type F isolates are a leading cause of food poisoning and antibiotic-associated diarrhea. Type F isolate virulence requires production of C. perfringens enterotoxin [CPE], which acts by forming large pore complexes in host cell plasma membranes. During GI disease, CPE is produced in the intestines when type F strains undergo sporulation. The toxin is then released into the intestinal lumen when the mother cell lyses at the completion of sporulation. Once present in the lumen, CPE encounters proteases. This study examined the in vitro, ex vivo, and in vivo processing of CPE by intestinal proteases and the effects of this processing on CPE activity. Results using purified trypsin or mouse intestinal contents detected the rapid cleavage of CPE to a major band of ~32 kDa and studies with Caco-2 cells showed that this processed CPE still forms large complexes and retains cytotoxic activity. When mouse small intestinal loops were challenged with CPE, the toxin caused intestinal histologic damage, despite rapid proteolytic processing of most CPE to 32 kDa within 15 min. Intestinal large CPE complexes became more stable with longer treatment times. These results indicate that CPE processing involving trypsin occurs in the intestines and the processed toxin retains enterotoxicity.