In vitro and in vivo adhesive properties of flagella and recombinant flagellin FliC and flagellar cap FliD proteins of Clostridium difficile were analyzed. FliC, FliD, and crude flagella adhered in vitro to axenic mouse cecal mucus. Radiolabeled cultured cells bound to a high degree to FliD and weakly to flagella deposited on a membrane. The tissue association in the mouse cecum of a nonflagellated strain was 10-fold lower than that of a flagellated strain belonging to the same serogroup, confirming the role of flagella in adherence.Clostridium difficile is now well established as the main cause of nosocomial infections such as pseudomembranous colitis, antibiotic-associated diarrhea, and antibiotic-associated colitis (3,6,15,24), especially in elderly and immunocompromised patients (2, 6). Toxigenic C. difficile strains produce two virulence factors, toxins A and B (26). The proposed accessory virulence factors include (i) capsule, an antiphagocytic factor (10); (ii) fimbriae (7); (iii) hydrolytic enzymes, which are potentially involved in mucus degradation and penetration (5,30,34,35); and (iv) adhesins mediating adherence to mucosa (14,18,20,21,39,40).Flagella have been implicated in internalization of Campylobacter jejuni and Legionella pneumophila (12,17) and in cell adherence and colonization by C. jejuni (27), Helicobacter pylori (13), and Aeromonas caviae (31). Motility mediated by flagella is responsible for the invasiveness of Salmonella enterica serovar Typhi (25) and Borrelia burgdorferi (33) and the pathogenicity of Vibrio cholerae (32). The flagellin FliC is the major structural component of the flagellar filament, and assembly of a flagellum requires other proteins called hookassociated proteins (HAP1, HAP2, and HAP3). The fliD gene encodes structural component HAP2 of the flagellar cap at the distal end of the filament (4,19). In a previous study we characterized the fliC and fliD genes of C. difficile, which encode the 39-kDa flagellin protein (36, 37) and the 56-kDa flagellar cap protein (38), respectively. The aim of this work was to study the potential role of C. difficile flagella in adherence and colonization.In vitro adherence of recombinant FliC, FliD, and crude flagella to mucus. Inasmuch as during the colonization process C. difficile is likely to encounter a layer of mucus first in the intestine, the properties of adhesion of FliC, FliD, and crude flagella to cecal axenic mouse mucus were investigated. Thirteen-week-old C 3 H axenic mice, obtained from l'Institut National de Recherche Agronomique (Jouy-en-Josas, France) and from our breeding program, were maintained in sterile isolators (Isoconcept, Orléans, France) and received standard nutrients sterilized by irradiation. Mucus was obtained from excised ceca that were opened lengthwise after the contents were removed by gentle shaking twice in phosphate-buffered saline (PBS) (10 mM phosphate buffer, 150 mM NaCl; pH 7.2). The mucus was gently scraped off and suspended in 10 ml of PBS containing 0.02% (wt/vol) sodium azide by stirring ...

Our laboratory has previously shown that Clostridium difficile adherence to cultured cells is enhanced after heat shock at 60°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°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.

Gene hpaA, which codes for the receptor-binding subunit of the N-acetylneuraminyllactose-binding fibrillar hemagglutinin (NLBH) of Helicobacter pylori, was cloned and sequenced. The protein expressed by hpaA, designated HpaA, was identified as the adhesin subunit on the basis of its fetuin-binding activity and its reactivity with a polyclonal, monospecific rabbit serum prepared against NLBH purified from H. pylori. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blots (immunoblots) showed that the cloned adhesin has the same molecular weight (20,000) as that found on H. pylori. Also, HpaA contains a short sequence of amino acids (KRTIQK) which are all either identical or functionally similar to those which compose the sialic acid-binding motif of Escherichia coli SfaS, K99, and CFA/I. Affinity-purified antibody specific for a 12-residue synthetic peptide that included this sequence blocked the hemagglutinating activity of H. pylori and was shown by immuno-gold electron microscopy to react with almost transparent material on unstained H. pylori cells, which is consistent with previous observations concerning the location and morphology of the NLBH.

Previous results have demonstrated that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses ; this study focussed on whether the GroEL heat shock protein is implicated in this process. The 1940 bp groESL operon of C. difficile was isolated by PCR. The 1623 bp groEL gene is highly conserved between various C. difficile isolates as determined by RFLP-PCR and DNA sequencing, and the operon is present in one copy on the bacterial chromosome. The 58 kDa GroEL protein was expressed in Escherichia coli in fusion with glutathione S-transferase and the fusion protein was purified from IPTG-induced bacterial lysates by affinity chromatography on glutathione-Sepharose. A polyclonal, monospecific antiserum was obtained for GroEL which established by immunoelectron microscopy, indirect immunofluorescence and immunoblot analysis that GroEL is released extracellularly after heat shock and can be surface associated. Cell fractionation experiments suggest that GroEL is predominantly cytoplasmic and membrane bound. GroEL-specific antibodies as well as the purified protein partially inhibited C. difficile cell attachment and expression of the protein was induced by cell contact, suggesting a role for GroEL in cell adherence.