Functional Characterization of Clostridium difficile Spore Coat Proteins

Abstract

dSpores of Clostridium difficile play a key role in the dissemination of this important human pathogen, and until recently little has been known of their functional characteristics. Genes encoding six spore coat proteins (cotA, cotB, cotCB, cotD, cotE, and sodA) were disrupted by ClosTron insertional mutagenesis. Mutation of one gene, cotA, presented a major structural defect in spore assembly, with a clear misassembly of the outermost layers of the spore coat. The CotA protein is most probably subject to posttranslational modification and could play a key role in stabilizing the spore coat. Surprisingly, mutation of the other spore coat genes did not affect the integrity of the spore, although for the cotD, cotE, and sodA mutants, enzyme activity was reduced or abolished. This could imply that these enzymatic proteins are located in the exosporium or alternatively that they are structurally redundant. Of the spore coat proteins predicted to carry enzymatic activity, three were confirmed to be enzymes using both in vivo and in vitro methods, the latter using recombinant expressed proteins. These were a manganese catalase, encoded by cotD, a superoxide dismutase (SOD), encoded by sodA, and a bifunctional enzyme with peroxiredoxin and chitinase activity, encoded by cotE. These enzymes being exposed on the spore surface would play a role in coat polymerization and detoxification of H 2 O 2 . Two additional proteins, CotF (a tyrosine-rich protein and potential substrate for SodA) and CotG (a putative manganese catalase) were shown to be located at the spore surface.

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…In only one publication is there electron microscopic evidence of a possible traditional exosporium layer with a defined basal layer and interspace region (185). In this report, as in others, micrographs indicate an external crust-like layer (such as that shown in Fig.…”

“…In this report, as in others, micrographs indicate an external crust-like layer (such as that shown in Fig. 8) or fragments of material adherent to the spore coat in strains of C. difficile (13,174,185,186). Joshi et al also indicated a pronounced variation in spore hydrophobicity among strains, with the more hydrophobic strains possessing a more organized exosporium (173).…”

“…The CotA protein was shown to be important for proper assembly of the outermost spore layer, and the CotA, CotE (a bifunctional peroxiredoxin reductase and chitinase), CotF, and SodA (superoxide dismutase) proteins were found to be associated with the spore surface (185). The C. difficile bclA1, bclA2, bclA3, cdeC, cotA, cotCB, cotD, and cotE genes were determined to be dependent on K for expression; this is the same sigma factor involved in exosporium gene expression in the B. cereus family of organisms (191).…”

The Clostridium difficile Exosporium Cysteine (CdeC)-Rich Protein Is Required for Exosporium Morphogenesis and Coat Assembly

“…To date, the only protein that has been shown to belong exclusively to the exosporium is the collagen-like BclA1 protein (Pizarro-Guajardo et al, submitted). Other proteins identified to be located at the surface of proteinase K-treated spores (i.e., spores that lack the exosporium layer) are five proteins (i.e., CotA, CotB, CotCB, CotD, and CotE) (37), where only CotA is essential for the assembly of the coat and exosporium of C. difficile spores (38). In this context, this study has made several important contributions to the understanding of the formation of the outermost layers of C. difficile spores.…”

Esporas de Clostridium difficile y su relevancia en la persistencia y transmisión de la infección

“…Algunas especies como C. difficile, poseen una capa más externa llamada exosporium, que presenta proyecciones de filamentos que podrían jugar roles esenciales en la interacción espora-hospedero 39,43,66 y/o en la resistencia de las esporas. Los primeros estudios en factores de resistencia de esporas de C. difficile han identificado dos factores con un rol en la resistencia; estos son: CdeC, el que está involucrado en la resistencia de la espora a etanol, calor y lisozima 65 y CotA, involucrada en la pérdida de la resistencia al calor en un sub-grupo de la población total 67 . Ambos factores alteran el proceso de ensamblaje de las capas externas de las esporas de C. difficile.…”