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Infection and Immunity, October 2002, p. 5770-5778, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5770-5778.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Binding of Clostridium difficile Surface Layer Proteins to Gastrointestinal Tissues

Emanuela Calabi,1 Franco Calabi,2 Alan D. Phillips,3 and Neil F. Fairweather1*

Department of Biological Sciences, Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology and Medicine, London SW7 2AY,1 Developmental Biology Unit, Institute of Child Health, London WC1 1EH,2 Centre for Paediatric Gastroenterology, Royal Free Hospital, London NW3 2QG, United Kingdom3

Received 21 February 2002/ Returned for modification 9 May 2002/ Accepted 3 July 2002

Clostridium difficile is the etiological agent of antibiotic-associated diarrhea, a potentially serious condition frequently affecting elderly hospitalized patients. While tissue damage is primarily induced by two toxins, the mechanism of gut colonization, and particularly the role of bacterial adherence to the mucosa, remains to be clarified. Previous studies have shown binding of C. difficile whole cells to cultured cell lines and suggested the existence of multiple adhesins, only one of which has been molecularly characterized. In this paper, we have investigated tissue binding of C. difficile surface layer proteins (SLPs), which are the predominant outer surface components and are encoded by the slpA gene. The adherence of C. difficile to HEp-2 cells was studied by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis, which showed that antibodies to the high-molecular-weight (MW) SLP inhibited adherence. Immunohistochemical analysis of human gastrointestinal tissue sections revealed strong binding both to the surface epithelium lining the digestive cavities and to the subjacent lamina propria, while glands were negative. A similar pattern was observed in the mouse. By using purified recombinant SLPs, we show that binding is largely mediated by the high-MW SLP. By Western blotting analysis, we have identified two potential ligands of the C. difficile SLPs, one of which may be specific to the gut. By using purified extracellular matrix components immobilized on nitrocellulose, we also show SLP binding to collagen I, thrombospondin, and vitronectin, but not to collagen IV, fibronectin, or laminin. These results raise the possibility that the SLPs play a role both in the initial colonization of the gut by C. difficile and in the subsequent inflammatory reaction.

* Corresponding author. Mailing address: Department of Biological Sciences, Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology and Medicine, London SW7 2AY, United Kingdom. Phone: 44 (0)20 7594 5247. Fax: 44 (0)20 7594 3609. E-mail: n.fairweather{at}ic.ac.uk.

Editor: B. B. Finlay

Infection and Immunity, October 2002, p. 5770-5778, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5770-5778.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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