Clostridium perfringens sporulation and gliding motility

Graduation date: 2007Clostridium perfringens type A isolates, an anaerobic enterotoxigenic spore forming bacterium, are the third leading cause of food-borne disease in the United States. Factors that contribute to the virulence of C. perfringens include the ability of the bacterium to form heat resistant spores and to produce an enterotoxin (CPE). Although much progress has been made in the past 30 years in understanding the mechanism of sporulation in another spore forming bacteria Bacillus subtilis, genomic comparisons have revealed fundamental differences in the initiation of sporulation between Bacillus and Clostridium species. In addition, understanding of the sporulation mechanism in other Clostridium species has been hampered by the lack of molecular genetic tools and the restriction on handling pathogenic Clostridia such as C. botulinum and C. tetani. Completion of C. perfringens genome allowed for opportunities to analyze the mechanism of sporulation and its association with toxin production.\ud \ud \ud \ud In the current study, the role of the transcriptional regulator Spo0A in C. perfringens sporulation was analyzed. Sequencing analyses of four C. perfringens CPE positive isolates identified functional spo0A homologs. Inactivation of spo0A in C. perfringens strain SM101 blocked spore formation as well as CPE production. Sporulation and CPE production was restored when the spo0A mutant was complemented with wild-type spo0A, demonstrating that Spo0A is required for initiation of sporulation and CPE production. Due to the lack of genetic tools available to study sporulation in other Clostridium species, we evaluated the role of Spo0A from other Clostridium species in restoring spore formation in our C. perfringens spo0A mutant. Our results demonstrated that Spo0A from C. acetobutylicum and C. tetani, but not from C. botulinum and C. difficile, were able to restore both spore formation and CPE production in the spo0A mutant. Analysis of the transcriptional level of spo0A from C. botulinum and C. difficile suggested that the lack of complementation from these two Clostridial Spo0As could be due to inherent transcript stability or requirement for additional co-factors. \ud A second focus of the study was to identify environmental or metabolic signals required for the initiation of sporulation in C. perfringens. Analysis of the commonly used sporulation medium identified inorganic phosphate as a signal required for spore formation. The optimum level of inorganic phosphate necessary for maximal sporulation was demonstrated to be 35 milimolar. Microscopic and Northern blot analyses demonstrated that the sensing of inorganic phosphate in initiating sporulation occurs at stage 0 of the spore formation stage. \ud The third focus of this study was to characterize the regulatory mechanism of gliding motility, another stationary phase behavior in C. perfringens. Long believed to be a non-motile organism, recent study identified Type IV pilus formation in C. perfringens and demonstrated gliding motility on solid agar surface. In the present study, we found that gliding motility in C. perfringens is repressed by the presence of rapidly metabolized carbohydrates. Inactivation of the catabolite control protein (CcpA) in C. perfringens strain 13 blocks the ability of the bacteria to repress gliding motility in the presence of glucose, suggesting that CcpA is required for catabolite repression of C. perfringens motility. Study of the transcriptional profile of Type IV pilus (TFP) biosynthesis genes pilT and pilD demonstrated that catabolite repression of C. perfringens gliding motility is due partially to the down regulation of TFP biosynthesis genes.\ud Collectively, the present study contributes to the understanding of the mechanism of spore formation and gliding motility in the enterotoxigenic C. perfringens. Molecular techniques utilized in this study offer valuable tools in future sporulation and motility research in other Clostridium species