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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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