The intestine is an extremely complex living system that participates in the protection of the host through a strong defense against aggressions from the external environment. This defensive task is based on 3 constituents that are in permanent contact and dialog with each other: the microflora, mucosal barrier, and local immune system. We review herein current knowledge about these important functions. The gut microflora play a major role against exogenous bacteria through colonization resistance, but the mechanism of action is not yet established, although it is linked to the bacteria colonizing the gut. This colonization involves bacteria-bacteria dialog, bacteria-mucins interactions, and bacteria-colonocytes cross-talk associated with environmental factors. The intestinal mucosa is a cellular barrier and the main site of interaction with foreign substances and exogenous microorganisms. It is a complex physicochemical structure consisting of a mucous layer linked to cellular and stromal components that participate in the defense of the host through mucosal blood flow, mucosal secretions, epithelial cell functionals, surface hydrophobicity, and defensin production. The intestine is the primary immune organ of the body represented by the gut-associated lymphoid tissue through innate and acquired immunity. This immune system can tolerate dietary antigens and the gut-colonizing bacteria and recognizes and rejects enteropathogenic microorganisms that may challenge the body's defenses. In cooperation with these endogenous barriers, some in-transit bacteria, such as probiotics, can act as partners of the defense system of the intestine.
Our laboratory has previously shown that Clostridium difficile adherence to cultured cells is enhanced after heat shock at 60°C and that it is mediated by a proteinaceous surface component. The present study was undertaken to identify the surface molecules of this bacterium that could play a role in its adherence to the intestine. The cwp66 gene, encoding a cell surface-associated protein of C. difficile 79-685, was isolated by immunoscreening of a C. difficile gene library with polyclonal antibodies against C. difficile heated at 60°C. The Cwp66 protein (66 kDa) contains two domains, each carrying three imperfect repeats and one presenting homologies to the autolysin CwlB of Bacillus subtilis. A survey of 36 strains of C. difficile representing 11 serogroups showed that the 3 portion of the cwp66 gene is variable; this was confirmed by sequencing of cwp66 from another strain, C-253. Two recombinant protein fragments corresponding to the two domains of Cwp66 were expressed in fusion with glutathione S-transferase in Escherichia coli and purified by affinity chromatography using gluthatione-Sepharose 4B. Antibodies raised against the two domains recognized Cwp66 in bacterial surface extracts. By immunoelectron microscopy, the C-terminal domain was found to be cell surface exposed. When used as inhibitors in cell binding studies, the antibodies and protein fragments partially inhibited adherence of C. difficile to cultured cells, confirming that Cwp66 is an adhesin, the first to be identified in clostridia.
Previous results have demonstrated that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses ; this study focussed on whether the GroEL heat shock protein is implicated in this process. The 1940 bp groESL operon of C. difficile was isolated by PCR. The 1623 bp groEL gene is highly conserved between various C. difficile isolates as determined by RFLP-PCR and DNA sequencing, and the operon is present in one copy on the bacterial chromosome. The 58 kDa GroEL protein was expressed in Escherichia coli in fusion with glutathione S-transferase and the fusion protein was purified from IPTG-induced bacterial lysates by affinity chromatography on glutathione-Sepharose. A polyclonal, monospecific antiserum was obtained for GroEL which established by immunoelectron microscopy, indirect immunofluorescence and immunoblot analysis that GroEL is released extracellularly after heat shock and can be surface associated. Cell fractionation experiments suggest that GroEL is predominantly cytoplasmic and membrane bound. GroEL-specific antibodies as well as the purified protein partially inhibited C. difficile cell attachment and expression of the protein was induced by cell contact, suggesting a role for GroEL in cell adherence.
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