Global Analysis of the Sporulation Pathway of Clostridium difficile

Abstract

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 σ ^F, σ ^E, σ ^G, and σ ^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 σ ^F, σ ^E, σ ^G, and σ ^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 σ ^F-dependent, 169 were σ ^E-dependent, 34 were σ ^G-dependent, and 31 were σ ^K-dependent. In contrast with B. subtilis, C. difficile σ ^E was dispensable for σ ^G activation, σ ^G was dispensable for σ ^K activation, and σ ^F was required for post-translationally activating σ ^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.

Author Summary

C. difficile is the leading cause of healthcare-associated infectious diarrhea in the United States in large part because of its ability to form spores. Since spores are resistant to most disinfectants and antibiotics, C. difficile infections frequently recur and are easily spread. Despite the importance of spores to C. difficile transmission, little is known about how spores are made. We set out to address this question by generating C. difficile mutants lacking regulatory factors required for sporulation and identifying genes that are regulated by these factors during spore formation using whole-genome RNA-Sequencing. We determined that the regulatory pathway controlling sporulation in C. difficile differs from related Clostridium species and the non-pathogenic model spore-former Bacillus subtilis and identified 314 genes that are induced during C. difficile spore development. Collectively, our study provides a framework for identifying C. difficile gene products that are essential for spore formation. Further characterization of these gene products may lead to the identification of diagnostic biomarkers and the development of new therapeutics.