Structure and assembly of a Clostridioides difficile spore polar appendage

Abstract Clostridioides difficile , a strict anaerobic spore-former, is the main cause of nosocomial disease associated to antibiotic therapy in adults and a growing concern in the community. Spores are the main infectious, persistence and transmission vehicle. Spore germination occurs in the intestine and the resulting vegetative cells will produce the toxins responsible for the disease symptoms, and spores. During sporulation, a wild type population bifurcates into two main spore morphotypes, with or without a thick exosporium. We show that this bifurcation extends to the formation of spores with a robust polar appendage or spores with a short appendage or that lack this structure. The cysteine-rich CdeM protein localizes to the appendage and around the entire surface of the spore, and is a major structural component of the exosporium, which we show is continuous with the appendage. In a CdeM mutant, when present, the polar appendage is short and disorganized. We show that wild type and cdeM spores with a short or no appendage germinate poorly in response to taurocholate, compared to those with an appendage. cdeM spores of the two types, however, germinate faster than their wild type counterparts. Thus, while the absence of CdeM may increase the permeability of spores to taurocholate, proper assembly of the appendage is also important for germination. Consistent with an overall enhancement of germination, a cdeM mutant shows increased virulence in a hamster model of disease. For a wild type population, spores with a short or no appendage germinate slower than the appendage-bearing spores. Differences in transmission, persistence and disease severity may result, in part, from their proportion in a spore population.