The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile

Abstract

Toxin synthesis in Clostridium difficile increases as cells enter into stationary phase. We first compared the expression profiles of strain 630E during exponential growth and at the onset of stationary phase and showed that genes involved in sporulation, cellular division, and motility, as well as carbon and amino acid metabolism, were differentially expressed under these conditions. We inactivated the sigH gene, which encodes an alternative sigma factor involved in the transition to post-exponential phase in Bacillus subtilis. Then, we compared the expression profiles of strain 630E and the sigH mutant after 10 h of growth. About 60% of the genes that were differentially expressed between exponential and stationary phases, including genes involved in motility, sporulation, and metabolism, were regulated by SigH, which thus appears to be a key regulator of the transition phase in C. difficile. SigH positively controls several genes required for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The spo0A and CD2492 genes, encoding the master regulator of sporulation and one of its associated kinases, and the spoIIA operon were transcribed from a SigH-dependent promoter. The expression of tcdA and tcdB, encoding the toxins, and of tcdR, encoding the sigma factor required for toxin production, increased in a sigH mutant. Finally, SigH regulates the expression of genes encoding surface-associated proteins, such as the Cwp66 adhesin, the S-layer precursor, and the flagellum components. Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting a SigH consensus in their promoter regions are good candidates for direct SigH targets.

The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

“…dnaG-sigA1 operon did not show a dependence on H (48). However, sigA2, which was presumed to be a second copy of the sigA gene on the C. difficile genome (6), showed a significant reduction in expression (by 180-fold) in the sigH mutant, thus suggesting that its expression was strongly dependent on H .…”

“…4). Interestingly, the genes encoding the toxins tcdA and tcdB, as well as the sigma factor (encoded by tcdR) that transcribes these genes, showed increased expression in the sigH mutant, indicating that they are negatively regulated by H , probably through an intermediate transcriptional regulator (48). By using the B. subtilis H consensus binding motif (49) as a template, the promoter regions of the genes with reduced expression in the sigH mutant were examined to identify genes that were putatively directly regulated by H .…”

“…There has been only one reported sigH inactivation mutant that was successfully generated in Clostridium, that of the enteropathogen C. difficile (48). Global gene expression profiling of the sigH mutant of C. difficile relative to the wild-type (WT) strain revealed that 286 genes showed reduced expression, indicating that H plays a role in their expression (48).…”

Esporas de Clostridium difficile y su relevancia en la persistencia y transmisión de la infección

“…Muchos factores ambientales influencian la producción de toxina, como por ejemplo, la disponibilidad de nutrientes, el potencial redox y la temperatura del entorno. Al respecto, se han encontrado tres reguladores involucrados en la producción de toxina por parte de C. difficile: Ccpa, mediando la represión dependiente de glucosa; CodY y Spo0A; estos dos últimos controlando eventos previos y posteriores a la fase exponencial de crecimiento 104 . En C. perfringens, σ K es producido en células en la fase pre-divisional y se encuentra involucrado en la producción de enterotoxina 105 .…”

“…Dos estudios independientes reportaron que mientras la mutación de Spo0A reduce la producción de TcdA, la mutación de SigH (factor sigma que regula positivamente algunos genes requeridos para la esporulación) estimularía la producción de TcdA 104,107 . Sin embargo, el efecto de estas dos proteínas en la expresión de toxinas por parte de C. difficile es indirecta y sus mecanismos todavía no han sido dilucidados.…”