Global Analysis of the Sporulation Pathway of Clostridium difficile

The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Despite the importance of spores to the pathogenesis of C. difficile, little is known about their composition or formation. Based on studies in Bacillus subtilis and other Clostridium spp., the sigma factors sigma(F), sigma(E), sigma(G), and sigma(K) are predicted to control the transcription of genes required for sporulation, although their specific functions vary depending on the organism. In order to determine the roles of sigma(F), sigma(E), sigma(G), and sigma(K) in regulating C. difficile sporulation, we generated loss-of-function mutations in genes encoding these sporulation sigma factors and performed RNA-Sequencing to identify specific sigma factor-dependent genes. This analysis identified 224 genes whose expression was collectively activated by sporulation sigma factors: 183 were sigma(F)-dependent, 169 were sigma(E)-dependent, 34 were (sigma)G-dependent, and 31 were sigma(K)-dependent. In contrast with B. subtilis, C. difficile sigma(E) was dispensable for sigma(G) activation, sigma(G) was dispensable for sigma(K) activation, and sigma(F) was required for post-translationally activating sigma(G). Collectively, these results provide the first genome-wide transcriptional analysis of genes induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes.