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 degrees 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 degrees 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.
Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, F-92296 Châtenay-Malabry cedex, France.
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Waligora
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C
Hennequin
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Mullany
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Bourlioux
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Collignon
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Karjalainen
D018829:Adhesins, Bacterial / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D016360:Clostridioides difficile / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D011817:Rabbits; D014709:Vero Cells
D018829:Adhesins, Bacterial