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Development of MAbs to cell surface adhesins acting as colonisation factors in EHEC strains

Among other properties, EPEC/EHEC differ from other pathogenic and non-pathogenic Escherichia coli by their surface antigens. Recognition of those could help in the specific diagnosis and description of specific virulence factors, including putative colonisation factors. The general objectives of WP16 were to raise MAbs to specific surface structures involved in the development of lesions by EPEC/EHEC strains and to identify the genetic basis of any newly recognised specific antigen. The factors targeted include those that are well defined, such as intimin, but also as yet undefined surface antigens.

Major finding during the project:
- Genetic basis of the MAb 2F3 recognized antigen.
Prior to the project, a MAb has been raised to an undefined surface antigen of an O26 EHEC strain (Mab 2F3). Continued diagnostic work with an sELISA developed with this MAB has provided evidence that it is largely specific to EPEC/EHEC O26 strains (see result 21: development of immuno-capture and ELISA test diagnostic). A genomic DNA library from the O26 bovine EHEC strain 4276 was constructed using cosmid vectors (derivatives from cosmid pHC79) of different sizes and different cloning capacity. The constructs were packaged into l bacteriophages, which were subsequently able to infect an E. coli. More then 2000 clones were obtained. The entire library was screened by sELISA with the 2F3 MAb.

One positive clone harboured a genomic locus of 30 kb, containing the entire O-antigen gene cluster and the second half of the colanic acid gene cluster. Six independent mutants obtained by in vitro insertional mutagenesis assay (Tn5 transposition system) had the transposon inserted into the O26 antigen gene cluster. The O26 O-antigen gene cluster from the EHEC strain 4276 was also cloned into an E. coli DH5a strain using long range PCR assay. Several clones expressing the 2F3 antigen and the O26 O antigen were obtained. The O26 O antigen gene cluster from an EHEC strain is therefore sufficient to synthesise the epitope recognised by the2F3 Mab. Finally Western blot assay on LPS extracts with the 2F3 MAb gave a positive result. The sequencing of one O26 antigen gene cluster from a 2F3-negative E. coli is complete. Comparison to the published sequence of an O26 antigen gene cluster from a 2F3-positive E. coli failed to highlight any significant difference.

Derivation of other monoclonal antibodies (Mab):
- Derivation of monoclonal antibodies (Mab) to the intimin adhesin. Prior to and following the start of the project, MAbs were raised to the intimin adhesin of the EPEC/EHEC: to a conserved intimin region and to specific regions of the b-intimin and g2-intimin. They have been examined for immunohistological staining of gut tissue (see below) collected from calves experimentally infected with O26 EPEC/EHEC strains (see result 14: identification of colonisation factors of bovine EHEC strains).

- Derivation of monoclonal antibodies (Mab) to additional surface antigens. Outer membrane protein (OMP) and lipopolysaccharide (LPS) extracts from EPEC/EHEC strains of the O5, O111, O103, O118 and O157 serogroups were used to immunise mice. Hybridoma fusions were carried out on mice that demonstrated a better serum titre to the equivalent EPEC/EHEC whole cells than to corresponding non-EPEC/EHEC strains. Three MAbs with specificity to O111 LPS were obtained, one of which, was shown to be specific for the O111 EPEC/EHEC strains, was successfully applied in a sELISA to produce an EPEC/EHEC specific assay (see result 21: developement of immuno-capture and ELISA test diagnostic).

- Identification of O26 specific surface antigens. In a different approach, the strains from the O26 strain cosmid library were examined for expression of O26 specific surface antigens using polyclonal sera raised to whole cell and surface antigen fractions of O26. Five of the clones reacted to polyclonal sera (Pabs). Unfortunately antisera raised with all five did not show O26 specific activity, since cross-reaction to other E. coli strains, EPEC/EHEC and non-EPEC/EHEC was observed by immunoblot and ELISA.

Immunocytochemical work has been conducted with samples obtained from the experimental infections already carried out (see result 14: identification of colonisation factors of bovine EHEC strains). The b-intimin MAb 4H4 demonstrated clear, specific staining in the infected tissue. In addition, Pabs to EspA, EspB, EspD and Tir (obtained from CR2), all demonstrated specific staining in infected tissue. Attempts to apply the antibodies available to immunogold staining for electron microcopy examination have been successfully achieved with MAb 2F3 (O26 LPS) and 4H4 (b intimin), but staining with PAbs to EspA, Esp B, EspD and Tir showed little evidence of specificity.

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