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Infection and Immunity, April 2001, p. 2144-2153, Vol. 69, No. 4
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.4.2144-2153.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Characterization of a Cell Surface Protein of Clostridium difficile with Adhesive Properties

Anne-Judith Waligora,1 Claire Hennequin,1 Peter Mullany,2 Pierre Bourlioux,1 Anne Collignon,1 and Tuomo Karjalainen1,*

Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, F-92296 Châtenay-Malabry cedex, France,1 and Eastman Dental Institute, University of London, London WC1X8LD, United Kingdom2

Received 14 September 2000/Returned for modification 22 November 2000/Accepted 9 January 2001

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.

* Corresponding author. Mailing address: Département de Microbiologie, Faculté de Pharmacie, Université de Paris-Sud, F-92296 Châtenay-Malabry cedex, France. Phone: (33)-1-46 83 55 49. Fax: (33)-1-46 83 58 83. E-mail: tuomo.karjalainen{at}cep.u-psud.fr.

Infection and Immunity, April 2001, p. 2144-2153, Vol. 69, No. 4
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.4.2144-2153.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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