Anne Collignon

GroEL (Hsp60) of Clostridium difficile is involved in cell adherence

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.

Immunization of hamsters against Clostridium difficile infection using the Cwp84 protease as an antigen

Clostridium difficile is a pathogen responsible for diarrhoea and colitis, particularly after antibiotic treatment. We evaluated the C. difficile protease Cwp84, found to be associated with the S-layer proteins, as a vaccine antigen to limit the C. difficile intestinal colonization and therefore the development of the infection in a clindamycin-treated hamster model. First, we evaluated the immune response and the animal protection against death induced by several immunization routes: rectal, intragastric and subcutaneous. Antibody production was variable according to the immunization routes. In addition, serum Cwp84 antibody titres did not always correlate with animal protection after challenge with a toxigenic C. difficile strain. The best survival rate was observed with the rectal route of immunization. Then, in a second assay, we selected this immunization route to perform a larger immunization assay including a Cwp84 immunized group and a control group. Clostridium difficile intestinal colonization and survival rate, as well as the immune response were examined. Clostridium difficile hamster challenge resulted in a 26% weaker and slower C. difficile intestinal colonization in the immunized group. Furthermore, hamster survival in the Cwp84 immunized group was 33% greater than that of the control group, with a significant statistical difference.

Development of an integrative vector for the expression of antisense RNA in Clostridium difficile

A method was developed to use the conjugative transposon Tn916 as a vector for introducing recombinant DNA into Clostridium difficile. This was used to introduce antisense RNA for the adhesin encoding gene cwp66 into C. difficile 79-685. RT-PCR demonstrated that cwp66 specific antisense RNA was produced. However, there was no statistically significant difference in the protein expression or in the adherence of recombinant C. difficile strains. This may be due to the amount of transcripts of the wild-type (sense) cwp66 outnumbering the antisense transcripts or secondary structures present within the cwp66 mRNA. Unlike in other strains of C. difficile, where Tn916 inserts into the genome at highly preferred sites, in C. difficile 79-685, it integrates into multiple sites opening up the possibility of using Tn916 as a mutagen in this strain.

Amoebal host range, host-free survival and disinfection susceptibility of environmental Chlamydiae as compared to Chlamydia trachomatis

The term Chlamydia-like organisms encompasses obligate intracellular bacterial species phylogenetically close to Chlamydiaceae. Most are associated with free-living amoebae, and several could be responsible for respiratory tract infections and abortion in human and animals. Despite increasing concern about their pathogenic role, the prevalence, biodiversity and ecology of Chlamydia-related bacteria still remain largely unknown. In this study, six members of the Chlamydiales were tested, including Parachlamydia acanthamoebae (two different strains), Protochlamydia naegleriophila, Waddlia chondrophila, Criblamydia sequanensis and Chlamydia trachomatis as a reference. Intracellular growth was tested in 11 different Acanthamoeba strains, demonstrating significant differences in host susceptibilities to infection depending on strains investigated. Survival of host-free bacteria in suspension or dried onto surfaces was also explored, demonstrating that Chlamydia-like organisms present better survival capacity than C. trachomatis. Longer survival times were observed for bacteria suspended in rich culture medium, with survivors being detected after 10 weeks incubation. We also tested susceptibility of host-free Chlamydia-like organisms to several disinfection treatments. Each chemical biocide tested reduced viability of host-free Chlamydia by more than 4 logs. Conversely, all Chlamydia-like organisms tested resisted exposure at 55 °C for 10 min, while C. trachomatis was completely inactivated.

The Effect of In Vitro Growth Conditions on the Resistance of Acanthamoeba Cysts

Despite increasing concerns of direct pathogenicity and/or their role as hosts for other microorganisms there are currently no standard methods for the inactivation of amoebae that belong to the genus Acanthamoeba. Methods used to grow amoebae and produce cysts for these tests may be important as they can dramatically modify cyst susceptibility. We compared resistance of cysts produced from trophozoites grown in peptone‐yeast extract‐glucose broth or by feeding on HEp‐2 cells and then encysted in Neffs medium. We observed that trophozoites grown using HEp‐2 cells as a nutrient source produce cysts that are significantly more resistant to SDS and to most biocides tested, including heat. Increased resistance is likely due to a higher proportion of mature cysts presenting thicker cell walls as demonstrated using transmission electron microscopy. This was confirmed by calcofluor white staining demonstrating higher cellulose content in cysts produced from trophozoites grown using HEp‐2 cells as a feeding source. These results demonstrate that not only methods used to produce cysts from trophozoites are critical, but that methods used to grow trophozoites before encystment should also be chosen carefully. This should be taken into account for the development of protocols to evaluate biocides and antimicrobials against amoebal cysts.

Protection against Clostridium difficile infection in a hamster model by oral vaccination using flagellin FliC-loaded pectin beads

Clostridium difficile flagellin FliC is a highly immunogenic pathogen-associated molecular pattern playing a key role in C. difficile pathogenesis and gut colonization. Here, we designed an oral vaccine against C. difficile with FliC encapsulated into pectin beads for colonic release. Bead stability and FliC retention was confirmed in vitro using simulated intestinal media (SIM), while bead degradation and FliC release was observed upon incubation in simulated colonic media (SCM). The importance of FliC encapsulation into pectin beads for protection against C. difficile was assessed in a vaccination assay using a lethal hamster model of C. difficile infection. Three groups of hamsters orally received either FliC-loaded beads or unloaded beads in gastro-resistant capsule to limit gastric degradation or free FliC. Two other groups were immunized with free FliC, one intra-rectally and the other intra-peritoneally. Hamsters were then challenged with a lethal dose of C. difficile VPI 10463. Fifty percent of hamsters orally immunized with FliC-loaded beads survived whereas all hamsters orally immunized with free FliC died within 7u202fdays post challenge. No significant protection was observed in the other groups. Only intra-peritoneally immunized hamsters presented anti-FliC IgG antibodies in sera after immunizations. These results suggest that an oral immunization with FliC-loaded beads probably induced a mucosal immune response, therefore providing a protective effect. This study confirms the importance of FliC encapsulation into pectin beads for a protective oral vaccine against C. difficile.

Facteurs de virulence de Clostridium difficile

Resume Clostridium difficile est lagent causal des colites pseudo-membraneuses et de 20 a 30% des diarrhees associees aux antibiotiques. C. difficile colonise le tractus digestif le plus souvent apres perturbation de la flore de barriere digestive par les antibiotiques. Le pouvoir pathogene de C. difficile repose principalement sur les toxines A et B. Cependant, la premiere etape du pouvoir pathogene est letape de colonisation digestive. C. difficile est capable dadherer a la muqueuse intestinale de lhomme et du hamster, ainsi qua differentes cellules en culture. Plusieurs adhesines ont ete identifiees chez C. difficile comme les proteines de la couche S (proteines P36 et P47), les proteines Cwp66 (Clostridial Wall Protein de 66 kDa), Fbp68 (Fibronectin binding protein de 68 kDa), GroEL (proteine de choc thermique de 58 kDa) et les proteine flagellaires FliC (flagelline) et FliD (coiffe flagellaire). Par ailleurs, toutes les souches de C. difficile produisent des enzymes hydrolytiques telles que hyaluronidase, chondroitine-4-sulphatase et une protease Cwp84 ( Clostridial Wall Protein de 84 kDA). Les toxines A et B sont des toxines de haut poids moleculaires (large clostridial toxins LCTs). La toxine A est une enterotoxine, letale et cytotoxique. La toxine B est une cytotoxine environ 1 000 fois plus puissante que la toxine A. Les toxines A et B possedent une activite toxique directe sur les enterocytes grâce a leur activite glucosyl-transferase, mais aussi une activite indirecte sur les cellules de la lamina propria , principalement la toxine A, via la production de mediateurs proinflammatoires. Les toxines A et B sont codees par les genes tcdA (8133 pb) et tcdB (7098 pb) localises sur le locus de pathogenicite (PaLoc) ou element toxigenique. Des techniques de PCR-RFLP ont montre la variabilite du locus PaLoc. Les polymorphismes se retrouvent dans les deux genes des toxines et permettent de distinguer des toxinotypes. Certaines souches de C. difficile synthetisent un autre type de toxine, la toxine CDT, toxine binaire a activite ADP-ribosylation actine-specifique dont le role dans la virulence nest pas encore elucide. La variabilite de la reponse de lhote associee a la variabilite des facteurs de colonisation, des toxines A et B, la presence dans certaines souches de la toxine binaire refletent bien la complexite du pouvoir pathogene de cette bacterie.