Structure and assembly of aClostridioides difficilespore 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.

Characterization of Exosporium Layer Variability of Clostridioides difficile Spores in the Epidemically Relevant Strain R20291

“…In the other hand, another characteristic that bifurcates the differentiation pathway in spore formation is the polar appendage, that can be present as a robust structure, a short appendage or be absent (Antunes et al, 2018). Additionally, short or absence of appendage are related to poor germination in response to taurocholate (Antunes et al, 2018).…”

“…In the other hand, another characteristic that bifurcates the differentiation pathway in spore formation is the polar appendage, that can be present as a robust structure, a short appendage or be absent (Antunes et al, 2018). Additionally, short or absence of appendage are related to poor germination in response to taurocholate (Antunes et al, 2018). Phase contrast microscopy shows polar appendage in two different morphologies: appendage type 1, an exosporium prolongation in a polar region ( Figure 2C), and appendage type 2, diffuse, round and loose prolongation of the exosporium.…”

“…To date, two morphogenetic cysteine-rich proteins, CdeC and CdeM, have been identified as essential for the correct assembly of the exosporium layer (Barra-Carrasco et al, 2013;Antunes et al, 2018;Calderón-Romero et al, 2018). Both are required for the location of several spore-coat and exosporium proteins to the outer spore outer layer (Calderón-Romero et al, 2018) and the absence of CdeM seems to be involved in the conformation of electron dense material in the spore surface (Antunes et al, 2018). How these proteins contribute to the variability observed in the outermost layer of C. difficile spores remains unclear.…”

Characterization of exosporium layer variability ofClostridioides difficilespores in the epidemically relevant strain R20291

“…In our experimental 316 conditions, we observed no difference in the adherence of spores from the upper and lower 317 fractions to monolayers of epithelial Caco-2 cells. By contrast, Antunes et al (2018), demonstrated 318 that 630 spores enriched in appendage by density gradient germinated faster than fractions of 319 spores with no appendage [14]. The exact role of these phenotypes in C. difficile pathogenesis is 320 unclear and require further research.…”

“…): i) a homogenous distribution surrounding the spore; 298 and ii) a defined pattern at one or both poles of the spore Antunes et al (2018). reported similar 299 variability in the fluorescence pattern of SNAP-translational fusion with the exosporium protein, 300 CdeC [14].…”