Spore Assembly in Clostridioides difficile

Clostridioides difficile is the leading cause of nosocomial infections in the US because antibiotic-induced gut dysbiosis is a major risk factor for C. difficile infections. Since the antibiotics used as the first-line therapy for treating C. difficile infections perpetuate this dysbiosis and increase the susceptibility of patients to recurrent infections, highly selective therapies that spare the native gut microbiota are urgently needed to treat and prevent C. difficile infections. We recently discovered that C. difficile’s division mechanism is a promising target for developing such therapies because C. difficile divides using a mechanism that is fundamentally distinct from previously studied organisms. Cell division in bacteria is mediated by a highly conserved multiprotein complex known as the divisome. The genes encoding essential divisome proteins are so conserved that they have been traced to the last common bacterial ancestor. However, we found that C. difficile is missing or does not require many of these divisome proteins for vegetative cell division. For example, functional orthologs of the septal peptidoglycan synthesizing enzymes, FtsW and FtsI, which were thought to be essential for cell division in all walled bacteria, are not encoded in C. difficile. In the absence of FtsW and FtsI, we showed that C. difficile uses a Class A penicillin-binding protein, PBP1, to drive septal peptidoglycan synthesis, revealing a novel function for this class of enzymes in bacteria. C. difficile is also missing the highly conserved FtsEX complex, which coordinates cell separation with cell division in most bacteria. Consistent with this absence, we recently found using time-lapse microscopy that cell division and cell separation are not as tightly coordinated in C. difficile relative to many bacteria. Since our preliminary data indicate that C. difficile uses a distinct machinery to mediate cell division and cell separation, this proposal seeks to identify the proteins that comprise C. difficile’s unique divisome and gain mechanistic insight into how they are assembled into a functional divisome. We will use genetic and biochemical approaches to identify proteins required for C. difficile cell division and cell separation, and we will determine how these proteins are assembled into a functional divisome. We will also define the protein-protein interactions that allow divisome assembly and identify functional domains within key divisome components in C. difficile. By providing novel mechanistic insight into C. difficile’s unique mode of cell division, these analyses will facilitate the development of narrow-spectrum antibiotics against C. difficile.