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Protein composition of the outermost exosporium-like layer of Clostridium difficile 630 spores.

ABSTRACT: UNLABELLED:Clostridium difficile spores are considered the morphotype of infection, transmission and persistence of C. difficile infections. There is a lack of information on the composition of the outermost exosporium layer of C. difficile spores. Using recently developed exosporium removal methods combined with MS/MS, we have established a gel-free approach to analyze the proteome of the exosporium of C. difficile spores of strain 630. A total of 184 proteins were found in the exosporium layer of C. difficile spores. We identified 7 characterized spore coat and/or exosporium proteins; 6 proteins likely to be involved in spore resistance; 6 proteins possibly involved in pathogenicity; 13 uncharacterized proteins; and 146 cytosolic proteins that might have been encased into the exosporium during assembly, similarly as reported for Bacillus anthracis and Bacillus cereus spores. We demonstrate through Flag-fusions that CotA and CotB are mainly located in the spore coat, while the exosporium collagen-like glycoproteins (i.e. BclA1, BclA2 and BclA3), the exosporium morphogenetic proteins CdeC and CdeM, and the uncharacterized exosporium proteins CdeA and CdeB are mainly located in the exosporium layer of C. difficile 630 spores. This study offers novel candidates of C. difficile exosporium proteins as suitable targets for detection, removal and spore-based therapies. BIOLOGICAL SIGNIFICANCE:This study offers a novel strategy to identify proteins of the exosporium layer of C. difficile spores and complements previous proteomic studies on the entire C. difficile spores and spore coat since it defines the proteome of the outermost layer of C. difficile spores, the exosporium. This study suggests that C. difficile spores have several proteins involved in protection against environmental stress as well as putative virulence factors that might play a role during infection. Spore exosporium structural proteins were also identified providing the ground basis for further functional studies of these proteins. Overall this work provides new protein target for the diagnosis and/or therapeutics that may contribute to combat C. difficile infections.

SUBMITTER: Diaz-Gonzalez F

PROVIDER: S-EPMC6764588 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Protein composition of the outermost exosporium-like layer of Clostridium difficile 630 spores.

Díaz-González Fernando F Milano Mauro M Olguin-Araneda Valeria V Pizarro-Cerda Jaime J Castro-Córdova Pablo P Tzeng Shin-Chen SC Maier Claudia S CS Sarker Mahfuzur R MR Paredes-Sabja Daniel D

Journal of proteomics 20150404

Clostridium difficile spores are considered the morphotype of infection, transmission and persistence of C. difficile infections. There is a lack of information on the composition of the outermost exosporium layer of C. difficile spores. Using recently developed exosporium removal methods combined with MS/MS, we have established a gel-free approach to analyze the proteome of the exosporium of C. difficile spores of strain 630. A total of 184 proteins were found in the exosporium layer of C. diff ...[more]

PMID: 25849250

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Project description:Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CDI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.

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Protein composition of the outermost exosporium-like layer of Clostridium difficile 630 spores.

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Protein composition of the outermost exosporium-like layer of Clostridium difficile 630 spores.

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