Document Detail


Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus.
MedLine Citation:
PMID:  7927694     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Our laboratory has previously shown that Clostridium difficile adherence to Caco-2 cells is greatly enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component. The experiments described here show that C. difficile could adhere to several types of tissue culture cells (Vero, HeLa, and KB) after heat shock. The type of culture medium (liquid or solid, with or without blood) had little effect on adhesion. To clone the adhesin gene, polyclonal antibodies against C. difficile heated at 60 degrees C were used to screen a genomic library of C. difficile constructed in lambda ZapII. Ten positive clones were identified in the library, one of which (pCL6) agglutinated several types of erythrocytes in the presence of mannose. In Western blots (immunoblots), this clone expressed in Escherichia coli a 40- and a 27-kDa protein; a 27-kDa protein has been previously identified in the surface extracts of heat-shocked C. difficile as a possible adhesin. The clone adhered to Vero, Caco-2, KB, and HeLa cells; the adherence was blocked by anti-C. difficile antibodies, by a surface extract of C. difficile, and by mucus isolated from axenic mice. Furthermore, the clone could attach ex vivo to intestinal mucus isolated from axenic mice. Preliminary studies on the receptor moieties implicated in C. difficile adhesion revealed that glucose and galactose could partially block adhesion to tissue culture cells, as did di- or trisaccharides containing these sugars, suggesting that the adhesin is a lectin. In addition, N-acetylgalactosamine, a component of mucus, and gelatin partially impeded cell attachment.
Authors:
T Karjalainen; M C Barc; A Collignon; S Trollé; H Boureau; J Cotte-Laffitte; P Bourlioux
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Infection and immunity     Volume:  62     ISSN:  0019-9567     ISO Abbreviation:  Infect. Immun.     Publication Date:  1994 Oct 
Date Detail:
Created Date:  1994-11-04     Completed Date:  1994-11-04     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  0246127     Medline TA:  Infect Immun     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  4347-55     Citation Subset:  IM    
Affiliation:
Département de Microbiologie et Immunologie, Faculté de Pharmacie, Université de Paris-Sud, Châtenay-Malabry, France.
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MeSH Terms
Descriptor/Qualifier:
Animals
Bacterial Adhesion*
Cell Line
Cloning, Molecular
Clostridium difficile / genetics*,  pathogenicity
Dogs
Gene Library
Genes, Bacterial*
Hemagglutination
Humans
Mice
Mucus / microbiology*
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