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)2down-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.
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan. shishimaru@vet.osakafu-u.ac.jp.
Mayo
M
Yasugi
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan.
Keisuke
K
Otsuka
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka 598-8531, Japan.
Masami
M
Miyake
D018938:Caco-2 Cells; D003016:Clostridium perfringens / Q000187:drug effects* / Q000502:physiology*; D003470:Culture Media / Q000737:chemistry; D004768:Enterotoxins / Q000096:biosynthesis*; D005517:Foodborne Diseases / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D006801:Humans; D009566:Nitrates / Q000494:pharmacology*; D009574:Nitro Compounds / Q000494:pharmacology; D013171:Spores, Bacterial / Q000187:drug effects*
D003470:Culture Media; D004768:Enterotoxins; D009566:Nitrates; D009574:Nitro Compounds
Clostridium perfringens; enterotoxin; nitrate; sporulation