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A 68 kDa fibronectin-binding protein (Fbp68) from Clostridium difficile displaying significant homology to several established or putative Fbps from other bacteria was identified. The one-copy gene is highly conserved in C. difficile isolates. Fbp68 was expressed in Escherichia coli in fusion with glutathione S-transferase; the fusion protein and the native Fbp68 were purified. Immunoblot analysis and cell fractionation experiments revealed that Fbp68 is present on the surface of the bacteria. Far-immuno dot-blotting demonstrated that Fbp68 was capable of fixing fibronectin. Indirect immunofluorescence and ELISA were employed to demonstrate that C. difficile could bind both soluble and immobilized fibronectin. With competitive adherence inhibition assays it was shown that antibodies raised against Fbp68 partially inhibited attachment of C. difficile to fibronectin and Vero cells. Furthermore, Vero cells could fix purified membrane-immobilized Fbp68. Thus Fbp68 appears to be one of the several adhesins identified to date in C. difficile.

Recent investigations of the Clostridium difficile genome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins. Two of these genes have been proven to encode proteins involved in cell adherence: slpA encodes the precursor of the two proteins of the S-layer, P36 and P47, whereas cwp66 encodes the Cwp66 adhesin. To gain further insight into the function of this cluster, we further focused on slpA, cwp66, and cwp84, the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases. It displayed nonspecific proteolytic activity when expressed as a recombinant protein in Escherichia coli. Polymorphism of cwp66 and cwp84 genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots. The slpA gene is strongly transcribed during the entire growth phase as a bicistronic transcript; cwp66 is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream. The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence. No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains. The cwp84 gene is strongly transcribed as a monocistronic message. This feature, together with the highly conserved sequence pattern of cwp84, suggests a significant role in the physiopathology of C. difficile for the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster.

GroEL (Hsp60) of Clostridium difficile has been found to be involved in cell adherence in a study on whether the GroEL heat shock protein is implicated in the adherence of C. difficile to tissue culture cells, augmented by stresses. The 1623 bp groEL gene is highly conserved between some C. difficile isolates. The C. difficile groELS operon was isolated from two strains. Characterization and expression of the hsp60 gene was also carried out. PCR amplification of the groEL gene waS used with RFLP analysis.

Research demonstrates that flagellin gene sequences from two virulent strains of Clostridium difficile exhibit 91% inter-strain identity. Data further show that the gene product is a 31 kilodalton protein and the flagellin has a molecular mass of 39 kilodalton.

Research was conducted to examine the effect of environmental stresses on Clostridium difficile adherence in vitro in the Vero cell adherence model by the use of either various cultures or adherence assay conditions. The reference strain used in all of the adherence assays was the toxigenic C difficile isolate 79-685. Results indicate that Vero cells have the ability to bind to one, two or three bacterial surface proteins depending on the bacterial culture conditions.

The phase I variants of Xenorhabdus nematophilus F1 contains fimbriae which are composed of 16-kDa pilin subunits, but the phase II variants have no fimbriae and do not synthesize pilin. Also the phase I variants exhibit a agglutinating activity with the erythrocytes of sheep, rabbit and humans, and hemocytes of the insect Galleria mellonella. The purified fimbriae are responsible for the agglutination.

The cloning and sequencing of the Helicobacter pylori adhesin gene hpaA and characterization of the hpaA product were reported. The protein expressed by hpaA, designated HpaA, is the adhesin subunit responsible for the binding specificity of the N-acetylneuraminyllactose-binding fibrillar hemagglutinin. It was shown that HpaA has the same molecular weight (20 kDa) as that found in H. pylori and has the amino acid sequence KRTIQK. Affinity-purified antibody specific for a synthetic peptide containing the sequence blocked hemagglutination by H. pylori, showing that this sequence represents the receptor-binding site and that HpaA codes for the adhesin.

In this review we describe the most common mechanisms through which bacterial antagonisms occur in the GI-tract. These include depletion and:or competition for substrates; competition for receptor sites (competitive exclusion); generation of a restrictive physiological environment (role of pH, short-chain fatty acids, oxidation-reduction potential); and production of antimicrobial substances (bacteriocin- and non bacteriocin-related). [ABSTRACT FROM AUTHOR]

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was conducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27kDa... [ABSTRACT FROM AUTHOR]