Clostridium difficile is an important nosocomial pathogen that has become a major cause of antibiotic-associated diarrhea. There is a general consensus that C. difficile spores play an important role in C. difficile pathogenesis, contributing to infection, persistence, and transmission. Evidence has demonstrated that C. difficile spores have an outermost layer, termed the exosporium, that plays some role in adherence to intestinal epithelial cells. Recently, the protein encoded by CD1067 was shown to be present in trypsin-exosporium extracts of C. difficile 630 spores. In this study, we renamed the CD1067 protein Clostridium difficile exosporium cysteine-rich protein (CdeC) and characterized its role in the structure and properties of C. difficile spores. CdeC is expressed under sporulation conditions and localizes to the C. difficile spore. Through the construction of an ΔcdeC isogenic knockout mutant derivative of C. difficile strain R20291, we demonstrated that (i) the distinctive nap layer is largely missing in ΔcdeC spores; (ii) CdeC is localized in the exosporium-like layer and is accessible to IgGs; (iii) ΔcdeC spores were more sensitive to lysozyme, ethanol, and heat treatment than wild-type spores; and (iv) despite the almost complete absence of the exosporium layer, ΔcdeC spores adhered at higher levels than wild-type spores to intestinal epithelium cell lines (i.e., HT-29 and Caco-2 cells). Collectively, these results indicate that CdeC is essential for exosporium morphogenesis and the correct assembly of the spore coat of C. difficile.

Clostridium difficile is a Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea that can culminate in fatal colitis. During the infection, C. difficile produces metabolically dormant spores, which persist in the host and can cause recurrence of the infection. The surface of C. difficile spores seems to be the key in spore-host interactions and persistence. The proteome of the outermost exosporium layer of C. difficile spores has been determined, identifying two cysteine-rich exosporium proteins, CdeC and CdeM. In this work, we explore the contribution of both cysteine-rich proteins in exosporium integrity, spore biology and pathogenesis. Using targeted mutagenesis coupled with transmission electron microscopy we demonstrate that both cysteine rich proteins, CdeC and CdeM, are morphogenetic factors of the exosporium layer of C. difficile spores. Notably, cdeC, but not cdeM spores, exhibited defective spore coat, and were more sensitive to ethanol, heat and phagocytic cells. In a healthy colonic mucosa (mouse ileal loop assay), cdeC and cdeM spore adherence was lower than that of wild-type spores; while in a mouse model of recurrence of the disease, cdeC mutant exhibited an increased infection and persistence during recurrence. In a competitive infection mouse model, cdeC mutant had increased fitness over wild-type. Through complementation analysis with FLAG fusion of known exosporium and coat proteins, we demonstrate that CdeC and CdeM are required for the recruitment of several exosporium proteins to the surface of C. difficile spores. CdeC appears to be conserved exclusively in related Peptostreptococcaeace family members, while CdeM is unique to C. difficile. Our results sheds light on how CdeC and CdeM affect the biology of C. difficile spores and the assembly of the exosporium layer and, demonstrate that CdeC affect C. difficile pathogenesis. We discovered a mechanism of assembly of the outer most layer of Clostridium difficile spores, the exosporium. While CdeC is conserved in several Peptostreptococcaeace family members, CdeM is unique to C. difficile. We show that two proteins that are rich in cysteine amino acid residues, CdeC and CdeM, are essential for the recruitment of additional spore coat and exosporium proteins. The absence of CdeC, had profound implications in the correct spore coat assembly which were related to decreased spore resistant properties that are relevant for in vivo infection such as lysozyme resistance, macrophage infection. Notably, the absence of either cysteine rich proteins leads to a decrease in spore adherence of C. difficile spores to healthy colonic mucosa; but only the absence of CdeC affected in vivo competitive fitness in a mouse model, recurrence of the disease in a mouse model of recurrent infection. Considering the importance of the outer layers of C. difficile spores in spore-host interactions, our findings have broad implications on the biology of C. difficile spores and to C. difficile pathogenesis.

Cfr is a radical S -adenosyl- l -methionine (SAM) enzyme that confers cross-resistance to antibiotics targeting the 23S rRNA through hypermethylation of nucleotide A2503. Three cfr -like genes implicated in antibiotic resistance have been described, two of which, cfr (B) and cfr (C), have been sporadically detected in Clostridium difficile . However, the methylase activity of Cfr(C) has not been confirmed.

Clostridium difficile is the main cause of nosocomial infections including antibiotic associated diarrhea, pseudomembranous colitis and toxic megacolon. During the course of Clostridium difficile infections (CDI), C. difficile undergoes sporulation and releases spores to the colonic environment. The elevated relapse rates of CDI suggest that C. difficile spores has a mechanism(s) to efficiently persist in the host colonic environment. In this work, we provide evidence that C. difficile spores are well suited to survive the host’s innate immune system. Electron microscopy results show that C. difficile spores are recognized by discrete patchy regions on the surface of macrophage Raw 264.7 cells, and phagocytosis was actin polymerization dependent. Fluorescence microscopy results show that >80% of Raw 264.7 cells had at least one C. difficile spore adhered, and that ∼60% of C. difficile spores were phagocytosed by Raw 264.7 cells. Strikingly, presence of complement decreased Raw 264.7 cells’ ability to phagocytose C. difficile spores. Due to the ability of C. difficile spores to remain dormant inside Raw 264.7 cells, they were able to survive up to 72 h of macrophage infection. Interestingly, transmission electron micrographs showed interactions between the surface proteins of C. difficile spores and the phagosome membrane of Raw 264.7 cells. In addition, infection of Raw 264.7 cells with C. difficile spores for 48 h produced significant Raw 264.7 cell death as demonstrated by trypan blue assay, and nuclei staining by ethidium homodimer-1. These results demonstrate that despite efficient recognition and phagocytosis of C. difficile spores by Raw 264.7 cells, spores remain dormant and are able to survive and produce cytotoxic effects on Raw 264.7 cells.

The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the transmission, infectious, and persistent morphotype, and the outermost exosporium layer is likely to play a major role in spore-host interactions during the first contact of C. difficile spores with the host and for spore persistence during recurrent episodes of infection. Although some studies on the biology of the exosporium have been conducted (J. Barra-Carrasco et al., J Bacteriol 195:3863–3875, 2013, http://dx.doi.org/10.1128/JB.00369-13 ; J. Phetcharaburanin et al., Mol Microbiol 92:1025–1038, 2014, http://dx.doi.org/10.1111/mmi.12611 ), there is a lack of information on the ultrastructural variability and stability of this layer. In this work, using transmission electron micrographs, we analyzed the variability of the spore's outermost layers in various strains and found distinctive variability in the ultrastructural morphotype of the exosporium within and between strains. Through transmission electron micrographs, we observed that although this layer was stable during spore purification, it was partially lost after 6 months of storage at room temperature. These observations were confirmed by indirect immunofluorescence microscopy, where a significant decrease in the levels of two exosporium markers, the N-terminal domain of BclA1 and CdeC, was observed. It is also noteworthy that the presence of the exosporium marker CdeC on spores obtained from C. difficile biofilms depended on the biofilm culture conditions and the strain used. Collectively, these results provide information on the heterogeneity and stability of the exosporium surface of C. difficile spores. These findings have direct implications and should be considered in the development of novel methods to diagnose and/or remove C. difficile spores by using exosporium proteins as targets.