Ultrastructural Variability of the Exosporium Layer of Clostridium difficile Spores

Abstract

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 ( , 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.

Ultrastructure Variability of the Exosporium Layer of Clostridium difficile Spores from Sporulating Cultures and Biofilms

“…The composition of the exosporium layer of C. difficile spores has been recently identified (19), revealing a complex composition of 184 proteins, among which several proteins known to be involved in pathogenesis and evasion of immunity in other pathogens were identified (e.g., elongation factor Tu and alpha-enolase) (19). Notably, our recent results have shown that during spore formation, C. difficile is capable of producing spores with two distinctive morphotypes of the exosporium layer, either thin or thick (13). However, whether these two different morphotypes of the exosporium layer emerge directly from the mother cell and whether these two morphotypes are formed during biofilm development remain unclear.…”

“…In this context, recent studies have revealed that the ultrastructure of C. difficile spores is similar to that of other Gram-positive bacteria (13,14). The outermost layer is an electron-dense layer that, in most epidemic strains, is covered by hair-like projections and has been shown to be ultrastructurally stable (13).…”

“…The outermost layer is an electron-dense layer that, in most epidemic strains, is covered by hair-like projections and has been shown to be ultrastructurally stable (13). Underlying the exosporium layer is the spore coat, which has laminations (i.e., lamellae) similar to those present in other bacterial spores, although it differs in protein composition from the spore coat of other bacterial species (13,14). The …”

“…Although not demonstrated experimentally, it has been suggested that development of spores might be relevant for biofilm formation, since it has been reported that a Spo0A mutant (Spo0A is a transcription factor that controls spore formation) is defective in biofilm formation (12). Recently, the presence of two exosporium proteins (i.e., CdeC and the N-terminal domain of BclA1) was detected by indirect immunofluorescence analysis of spores from C. difficile biofilms (13). However, whether the ultrastructure of biofilmformed spores is similar to that of sporulating cultures remains unclear.…”

“…The outermost layer is an electron-dense layer that, in most epidemic strains, is covered by hair-like projections and has been shown to be ultrastructurally stable (13). Underlying the exosporium layer is the spore coat, which has laminations (i.e., lamellae) similar to those present in other bacterial spores, although it differs in protein composition from the spore coat of other bacterial species (13,14). The C. difficile spore coat, as similarly reported for Bacillus subtilis (15), exhibits enzymatic digestion resistance to proteases (i.e., proteinase K and trypsin) (16), which might be related to the apparent resistance of C. difficile spores to macrophages (17).…”