Characterization of a cell surface protein of Clostridium difficile with adhesive properties

Our laboratory has previously shown that Clostridium difficile adherence to cultured cells is enhanced after heat shock at 60 degreesC and that it is mediated by a proteinaceous surface component. The present study was undert aken 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 sur face-associated protein of C. difficile 79-685, was isolated by immunoscree ning of a C. difficile gene library with polyclonal antibodies against C. d ifficile heated at 60 degreesC. The Cwp66 protein (66 kDa) contains two dom ains, each carrying three imperfect repeats and one presenting homologies t o the autolysin Cw1B of Bacillus subtilis, A survey of 36 strains of C. dif ficile representing 11 serogroups showed that the 3' portion of the cwp66 g ene is variable; this was confirmed by sequencing of cwp66 from another str ain, C-253, Two recombinant protein fragments corresponding to the two doma ins of Cwp66 were expressed in fusion with glutathione S-transferase in Esc herichia coli and purified by affinity chromatography using gluthatione-Sep harose 4B, Antibodies raised against the two domains recognized Cwp66 in ba cterial surface extracts. By immunoelectron microscopy, the C-terminal doma in was found to be cell surface exposed. When used as inhibitors in cell bi nding studies, the antibodies and protein fragments partially inhibited adh erence of C. difficile to cultured cells, confirming that Cwp66 is an adhes in, the first to be identified in clostridia.