The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation.

The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis, only two of these morphogenetic proteins have homologs in the Clostridia: SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis. Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis, C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia, but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation.

Inducing and Quantifying Clostridium difficile Spore Formation.

The Gram-positive nosocomial pathogen Clostridium difficile induces sporulation during growth in the gastrointestinal tract. Sporulation is necessary for this obligate anaerobe to form metabolically dormant spores that can resist antibiotic treatment, survive exit from the mammalian host, and transmit C. difficile infections. In this chapter, we describe a method for inducing C. difficile sporulation in vitro. This method can be used to study sporulation and maximize spore purification yields for a number of C. difficile strain backgrounds. We also describe procedures for visualizing spore formation using phase-contrast microscopy and for quantifying the efficiency of sporulation using heat resistance as a measure of functional spore formation.

SpoIIID-mediated regulation of σK function during Clostridium difficile sporulation.

The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health-care-associated diarrhea worldwide. Although C. difficile spore formation is essential for disease transmission, the regulatory pathways that control this developmental process have only been partially characterized. In the well-studied spore-former Bacillus subtilis, the highly conserved σ(E) , SpoIIID and σ(K) regulatory proteins control gene expression in the mother cell to ensure proper spore formation. To define the precise requirement for SpoIIID and σ(K) during C. difficile sporulation, we analyzed spoIIID and sigK mutants using heterologous expression systems and RNA-Seq transcriptional profiling. These analyses revealed that expression of sigK from a SpoIIID-independent promoter largely bypasses the need for SpoIIID to produce heat-resistant spores. We also observed that σ(K) is active upon translation, suggesting that SpoIIID primarily functions to activate sigK. SpoIIID nevertheless plays auxiliary roles during sporulation, as it enhances levels of the exosporium morphogenetic protein CdeC in a σ(K) -dependent manner. Analyses of purified spores further revealed that SpoIIID and σ(K) control the adherence of the CotB coat protein to C. difficile spores, indicating that these proteins regulate multiple stages of spore formation. Collectively, these results highlight that diverse mechanisms control spore formation in the Firmicutes.