Nitrate salts suppress sporulation and production of enterotoxin in Clostridium perfringens strain NCTC8239

Clostridium perfringens type A is a common source of food‐borne illness in humans. Ingested vegetative cells sporulate in the small intestinal tract and in the process produce C. perfringens enterotoxin (CPE). Although sporulation plays a critical role in the pathogenesis of food‐borne illness, the molecules triggering/inhibiting sporulation are still largely unknown. It has previously been reported by our group that sporulation is induced in C. perfringens strain NCTC8239 co‐cultured with Caco‐2 cells in Dulbecco's Modified Eagle Medium (DMEM). In contrast, an equivalent amount of spores was not observed when bacteria were co‐cultured in Roswell Park Memorial Institute‐1640 medium (RPMI). In the present study it was found that, when these two media are mixed, RPMI inhibits sporulation and CPE production induced in DMEM. When a component of RPMI was added to DMEM, it was found that calcium nitrate (Ca[NO₃]₂) significantly inhibits sporulation and CPE production. The number of spores increased when Ca(NO₃)₂‐deficient RPMI was used. The other nitrate salts significantly suppressed sporulation, whereas the calcium salts used did not. qPCR revealed that nitrate salts increased expression of bacterial nitrate/nitrite reductase. Furthermore, it was found that nitrite and nitric oxide suppress sporulation. In the sporulation stages, Ca(NO₃)₂ down‐regulated the genes controlled by Spo0A, a master regulator of sporulation, but not spo0A itself. Collectively, these results indicate that nitrate salts suppress sporulation and CPE production by down‐regulating Spo0A‐regulated genes in C. perfringens strain NCTC8239. Nitrate reduction may be associated with inhibition of sporulation.