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

ABSTRACT: 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.

SUBMITTER: Waligora AJ

PROVIDER: S-EPMC98141 | biostudies-literature | 2001 Apr

REPOSITORIES: biostudies-literature

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

Waligora A J AJ Hennequin C C Mullany P P Bourlioux P P Collignon A A Karjalainen T T

Infection and immunity 20010401 4

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 polyclon ...[more]

PMID: 11254569

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Project description:Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Autolysin is a lytic enzyme that hydrolyzes peptidoglycans of the bacterial cell wall, with a catalytic domain and cell wall-binding domains, proven to be involved in bacterial cell wall remodeling and cell division. Although autolysins in C. difficile have been reported, the autolysins have failed to yield impressive results when used as exogenous lytic agents. In this study, we expressed and characterized the binding domains (Cwp19-BD and Acd-BD) and catalytic domains (Cwp19-CD, Acd-CD, and Cwl-CD) of C. difficile autolysins, and the domains with the best binding specificity and lytic activity were selected towards C. difficile to design a novel lytic protein Cwl-CWB2. Cwl-CWB2 showed good biosafety with significantly low hemolysis and without cytotoxicity. The results of fluorescence analysis and lytic assay demonstrated that Cwl-CWB2 has higher binding specificity and stronger lytic activity with a minimum inhibitory concentration at 13.39 ± 5.80 μg/mL against living C. difficile cells, which is significantly stronger than commercial lysozyme (3333.33 ± 1443.37 μg/mL) and other reported C. difficile autolysins. Besides, Cwl-CWB2 exhibited good stability as about 75% of the lytic activity was still retained when incubated at 37 °C for 96 h, which is considered to be a potential antimicrobial agent to combat C. difficile. KEY POINTS: • Several binding domains and catalytic domains, deriving from several Clostridium difficile autolysins, were expressed, purified, and functionally characterized. • A novel C. difficile lytic protein Cwl-CWB2 was designed from C. difficile autolysins. • The binding specificity and lytic activity of Cwl-CWB2 against C. difficile showed advantages compared with other reported C. difficile autolysins. • Cwl-CWB2 exhibited significantly low hemolysis and cytotoxicity against normal-derived colon mucosa 460 cell.

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

Publications

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

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