Abstract

BACKGROUND & AIMS: Collagen like proteins are present in the spore of several spore producing bacteria. In Clostridium difficile, most of the strains described exhibit collagen like proteins, called BclA1, BclA2 and BclA3, in the external layer of the spores: the exosporium. The exosporium of epidemic strains of C. difficile and other spore former bacteria exhibit exosporium-filaments which in B. cereus group correspond to collagen like proteins and are implicated in adherence to the host. In this work we evaluated the role of BclA3 in the spore properties of epidemic R20291 strain and its adherence to Caco-2 cells. METHODS: By homology recombination we constructed a bclA3 mutant strain in R20291 (CD3193) and evaluated the resulting phenotype of the spores. To asses the influence of this protein in the localization of other exosporium proteins, we used immunofluorescence and western blot with serum immunized against several exosporium proteins. In order to evaluate whether the hair-like filaments are made of BclA2 and/or BclA3, wild-type and bclA3 mutant spores we evaluated by immunogold transmission electron microscopy. Since the exosporium is an hydrophobic layer, the contribution of BclA3 to hydrophobicity was assessed. Finally, we evaluated if the absence of BclA3 affected the adherence of R20291 spores to monolayers of differentiated Caco-2 cells. RESULTS: The different protein profiles of spore-coat/exosporium extracts observed between wild-type and bclA3 spores by SDS-PAGE indicates that absence of BclA3 profoundly affects the composition of the outer layers. Western blot analysis shows that the absence of BclA3 leads to the formation of spores that lack CdeM, while affecting the molecular mass of species that immunoreacted with anti-BclA2 antibodies. Through immunofluorescence we observed that the distribution of the N-terminal domain (NTD) of BclA1, the C-terminal domain (CTD) of BclA2, CdeC and CdeM is profoundly affected in bclA3 mutant spores as compared to wild-type spores. Transmission electron micrographs demonstrate that spores of a bclA3 mutant present a defective exosporium layer, loosely attached from the spore coat. Immunogold transmission electron micrographs provide evidence that the hair-like filaments are composed, at least in part, by BclA2 and BclA3. Finally, spores of a bclA3 mutant strain adhered 30% less than wild-type spores to Caco-2 cells. CONCLUSIONS: BclA3 seems to have implications in the assembly of the outer layers of C. difficile spores, and therefore also in the ability of C. difficile spores to adhere to intestinal epithelial Caco-2 cells. In vivo studies are necessary to determine the implications of BclA3 in the pathogenesis of the epidemic R20291 strain.