Role of the Agr-Like Quorum-Sensing System in Regulating Toxin Production by Clostridium perfringens Type B Strains CN1793 and CN1795

Abstract

Clostridium perfringens type B causes enteritis and enterotoxemia in domestic animals. By definition, these bacteria must produce alpha toxin (CPA), beta toxin (CPB) and epsilon toxin (ETX) although most type B strains also produce perfringolysin O (PFO) and beta2 toxin (CPB2). A recently identified Agr-like quorum-sensing (QS) system in C. perfringens controls all toxin production by surveyed type A, C, and D strains, but whether this QS is involved in regulating toxin production by type B strains has not been explored. Therefore, the current study introduced agrB null mutations into type B strains CN1795 and CN1793. Both type B agrB null mutants exhibited reduced levels of CPB, PFO, and CPA in their culture supernatants, and this effect was reversible by complementation. The reduced presence of CPB in culture supernatant involved decreased cpb transcription. In contrast, the agrB null mutants of both type B strains retained wild-type production levels of ETX and CPB2. In a Caco-2 cell model of enteritis, culture supernatants of the type B agrB null mutants were less cytotoxic than supernatants of their wild-type parents. However, in an MDCK cell in vitro model for enterotoxemic effects, supernatants from the agrB null mutants or wildtype parents were equally cytotoxic after trypsin activation. Coupling these and previous results, it is now evident that straindependent variations exist in Agr-like QS system regulation of C. perfringens toxin production. The cell culture results further support a role for trypsin in determining which toxins contribute to disease involving type B strains.

Structure-Function Analysis of Peptide Signaling in the Clostridium perfringens Agr-Like Quorum Sensing System

“…Since the cysteine residue in the CLWFT sequence is conserved among the AgrD sequences of both CN1795 and CN3685 (15,16), this C residue could be essential for signaling activity. To test this hypothesis, synthetic peptides were prepared where the C residue Wild-type CN3685, the CN3685agrBko null mutant, wild-type CN1795, and the CN1795agrBko null mutant were used to compare natural CPB production levels by overnight TGY cultures.…”

“…Guinea pig heart blood 14 etx containing 15 g ml Ϫ1 chloramphenicol (Cm) were used for screening the CN1795 cpb-knockout mutant (CN1795cpbko) constructed in the current study. CN3685cpbko (an isogenic cpb null mutant of CN3685, also known as BMC100 [18]), CN3685agrBko (an agrB null mutant of CN3685, also known as BMJV10 [16]), and CN1795agrBko (an agrB null mutant of CN1795 [15]) had been previously constructed and characterized, both genotypically and phenotypically (Table 1).…”

“…ETX) in type B strains CN1793 and CN1795 (15), the production of CPA, CPB, and PFO in type C strain CN3685 (16), and the production of CPE during sporulation by CPE-positive type A strain F5603 (17). In addition, the Agr-like system was found to be essential for type C strain CN3685 to cause necrotic enteritis in a rabbit small intestinal loop model or lethal enterotoxemia in a mouse oral challenge model (16).…”

Toxin Plasmids of Clostridium perfringens

“…It also controls the production of several plasmid-encoded C. perfringens toxins, including CPB2 and CPE expression in type A strain F5603 (157), CPB production in type C strain CN3685 (158) and type B strains CN1793 and CN1795 (159), and ETX production in type D strain CN3718 (154). However, this Agr-like regulatory system is not required for wild-type levels of production of all C. perfringens toxins, since inactivating this system in type B strains CN1793 and CN1795 had no effect on their ETX or CPB2 production (159). The Agr-like regulatory system plays a role in the virulence of some C. perfringens strains.…”

Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

“…CPB, produced by C. perfringens type B and type C strains, is very sensitive to endogenous trypsin degradation in the intestines of natural host animals (18,19). In contrast, a major toxin of both type B and D strains, i.e., ETX, is produced as an inactive prototoxin that must be proteolytically activated by intestinal proteases (such as trypsin, chymotrypsin, and carboxypeptidases) or, perhaps, proteases produced by C. perfringens (20)(21)(22)(23)(24)(25). Until now, no information has been available regarding whether TpeL, which can be encoded by strains causing diseases originating in the intestines (26,27), is trypsin sensitive.…”