Biomedical subjects

Marie-Claude Barc

Publications and source records attributed to Marie-Claude Barc.

5 recordsLinked to original sources

PubMed2005

Immunological properties of surface proteins of Clostridium difficile.

Sera from patients with Clostridium difficile-associated disease (CDAD) and sera from a control group were analysed by an ELISA to detect antibodies directed against four surface proteins and toxins A and B of C. difficile. The surface proteins were the flagellar cap protein FliD, the flagellin FliC, the adhesin Cwp66 divided into two domains, Cwp66-Nterminal and Cwp66-Cterminal, and the fibronectin-binding protein Fbp68. For each antigen, antibody levels in the CDAD patient group and in the control group were compared. In the CDAD patient group, the mean of the antibody levels decreased from Cwp66-Cterminal to Fbp68, FliD, toxins B and A, Cwp66-Nterminal and finally FliC, suggesting different immunogenic properties among these adhesins. For Cwp66-Nterminal, FliC, FliD and Fbp68, the antibody level observed in the control group was higher than in the CDAD group with a statistically significant difference whereas the antibody level for toxins A and B was not statistically different. In conclusion, this study suggests that during the clinical course of disease, C. difficile adhesins are able to induce an immune response which could play a role in the defence mechanism of the host.

Adhesins, Bacterial↗

PubMed2003

Identification and characterization of a fibronectin-binding protein from Clostridium difficile.

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.

Adhesins, Bacterial↗

PubMed2002

Inhibition of in vitro cell adherence of Clostridium difficile by Saccharomyces boulardii.

The influence on the adherence of Clostridium difficile to Vero cells of the yeast Saccharomyces boulardii, the yeast fractions (cytoplasm and cell wall) and the culture supernatant was investigated in vitro. C. difficile adherence was significantly inhibited when bacteria were pre-incubated with the whole yeast and the cell wall fraction; this adherence inhibition was dose-dependent. The cell wall fraction also acts upon the target cultured cells inasmuch as the level of adherence was significantly decreased when Vero cells were preincubated with it. The same experiments carried out in the presence of an inhibitor of serine proteases resulted in no inhibition of bacterial adherence. These results suggest that the yeast could inhibit adherence of C. difficile to cells thanks to its proteolytic activity but also through steric hindrance.

Animals↗

PubMed2002

Clostridium difficile genotyping based on slpA variable region in S-layer gene sequence: an alternative to serotyping.

Recent investigations of Clostridium difficile cell wall components have revealed the presence of an S-layer encoded by the slpA gene. The aim of this study was to determine whether slpA genotyping can be used as an alternative to serotyping. The variable regions of slpA were amplified by PCR from serogroup reference strains and various clinical isolates chosen randomly. Amplified products were analyzed after restriction enzyme digestion and DNA sequencing. The sequences of the variable region of the SlpA protein were found to be strictly identical within a given serogroup but divergent between serogroups. These preliminary results suggest that PCR-restriction fragment length polymorphism, in conjunction with DNA sequencing of the slpA variable region, could constitute an alternative typing method for determining C. difficile serotypes.

Amino Acid Sequence↗

PubMed2000

A Clostridium difficile gene encoding flagellin.

Six strains of Clostridium difficile examined by electron microscopy were found to carry flagella. The flagella of these strains were extracted and the N-terminal sequences of the flagellin proteins were determined. Four of the strains carried the N-terminal sequence MRVNTNVSAL exhibiting up to 90% identity to numerous flagellins. Using degenerate primers based on the N-terminal sequence and the conserved C-terminal sequence of several flagellins, the gene encoding the flagellum subunit (fliC) was isolated and sequenced from two virulent strains. The two gene sequences exhibited 91% inter-strain identity. The gene consists of 870 nt encoding a protein of 290 amino acids with an estimated molecular mass of 31 kDa, while the extracted flagellin has an apparent molecular mass of 39 kDa on SDS-PAGE. The FliC protein displays a high degree of identity in the N- and C-terminal amino acids whereas the central region is variable. A second ORF is present downstream of fliC displaying homology to glycosyltransferases. The fliC gene was expressed in fusion with glutathione S-transferase, purified and a polyclonal monospecific antiserum was obtained. Flagella of C. difficile do not play a role in adherence, since the antiserum raised against the purified protein did not inhibit adherence to cultured cells. PCR-RFLP analysis of amplified flagellin gene products and Southern analysis revealed inter-strain heterogeneity; this could be useful for epidemiological and phylogenetic studies of this organism.

Amino Acid Sequence↗