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ABSTRACTIn vitro and in vivo adhesive properties of flagella and recombinant flagellin FliC and flagellar cap FliD proteins ofClostridium difficilewere analyzed. FliC, FliD, and crude flagella adhered in vitro to axenic mouse cecal mucus. Radiolabeled cultured cells bound to a high degree to FliD and weakly to flagella deposited on a membrane. The tissue association in the mouse cecum of a nonflagellated strain was 10-fold lower than that of a flagellated strain belonging to the same serogroup, confirming the role of flagella in adherence.

ABSTRACTOur laboratory has previously shown that Clostridium difficileadherence 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 cwp66gene, encoding a cell surface-associated protein of C. difficile79-685, was isolated by immunoscreening of a C. difficilegene library with polyclonal antibodies against C. difficileheated 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 ofC. difficilerepresenting 11 serogroups showed that the 3′ portion of the cwp66gene is variable; this was confirmed by sequencing of cwp66from another strain, C-253. Two recombinant protein fragments corresponding to the two domains of Cwp66 were expressed in fusion with glutathione S-transferase inEscherichia coliand 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 ofC. difficileto cultured cells, confirming that Cwp66 is an adhesin, the first to be identified in clostridia.