Critical determinants of host receptor targeting by Neisseria meningitidis and Neisseria gonorrhoeae : identification of Opa adhesiotopes on the N‐domain of CD66 molecules

Abstract

SummaryThe human pathogens Neisseria meningitidis and Neisseria gonorrhoeae express a family of variable outer membrane opacity-associated (Opa) proteins that recognize multiple human cell surface receptors. Most Opa proteins target the highly conserved N-terminal domain of the CD66 family of adhesion molecules, although a few also interact with heparan sulphate proteoglycans. In this study, we observed that at least two Opa proteins of a N. meningitidis strain C751 have the dual capacity to interact with both receptors. In addition, all three Opa proteins of C751 bind equally well to HeLa cells transfected with cDNA encoding the carcinoembryonic antigen [CEA (CD66e)] subgroup of the CD66 family, but show distinct tropism for CGM1-(CD66d) and NCA (CD66c)-expressing cells. Because the C751 Opa proteins make up distinct structures via the surface-exposed hypervariable domains (HV-1 and HV-2), these combinations appear to be involved in tropism for the distinct CD66 subgroups. To de®ne the determinants of receptor recognition, we used mutant proteins of biliary glycoprotein [BGP (CD66a)] carrying substitutions at several predicted exposed sites in the N-domain and compared their interactions with several Opa proteins of both N. meningitidis and N. gonorrhoeae. The observations applied to the molecular model of the BGP N-domain that we constructed show that the binding of all Opa proteins tested occurs at the non-glycosylated (CFG) face of the molecule and, in general, appears to require Tyr-34 and Ile-91. Further, ef®cient interaction of distinct Opa proteins depends on different non-adjacent amino acids. In the three-dimensional model, these residues lie in close proximity to Tyr-34 and Ile-91 at the CFG face, making continuous binding domains (adhesiotopes). The epitope of the monoclonal antibody YTH71.3 that inhibits Opa/CD66 interactions was also identi®ed within the Opa adhesiotopes on the N-domain. These studies de®ne the molecular basis that directs the Opa speci®city for the CD66 family and the rationale for tropism of the Opa proteins for the CD66 subgroups. NomenclatureOpacity-associated (Opa) proteins of distinct strains have been called OpaA, B, X, etc. In this study, because Opa proteins of the strain C751 are primarily studied, these have been referred to without a suf®x, whereas others have been referred to with the suf®x specifying the strain, e.g. OpaA FA1090 . The Opa protein of the strain MC58 studied has been termed OpaX in our previous investigations (McNeil and Virji, 1997) and this nomenclature is maintained. It should be noted that Neisseria gonorrhoeae (Ng) Opa proteins were initially called`P.II' and those of Neisseria meningitidis (Nm)`Class 5 proteins . The unifying term Opa replaces these nomenclatures and is derived from the fact that most Opa expression results in opaque colony phenotype (Hitchcock, 1989).The nomenclature for the members of the carcinoembryonic antigen family is clari®ed in Fig. 3. The term CD66 is reserved for the CEA family. The subgroups within the family are refer...

Two Variable Regions in Carcinoembryonic Antigen-related Cell Adhesion Molecule1 N-terminal Domains Located in or Next to Monoclonal Antibody and Adhesion Epitopes Show Evidence of Recombination in Rat but Not in Human

“…3), two regions of sequence variability (V-regions) were detected. Moreover, these V-regions were located adjacent to or within the regions corresponding to binding sites identified previously for opa proteins (55,56) and MHV (57-59) and adhesive domains mediating intercellular adhesion (25,28,33) (Fig. 3).…”

Selection for a CEACAM Receptor-Specific Binding Phenotype during Neisseria gonorrhoeae Infection of the Human Genital Tract

“…To validate the results of the Lec11-based infection assay, we used an established whole-cell blotting assay using soluble CEACAM1-Fc fusion protein (23) to reassess the CEACAM1-binding specificity of the commonly used MS11 strains versus the LPCIs. As expected, recombinant MS11 strains expressing Opa 57 N. gonorrhoeae showed binding to CEACAM1-Fc, while Opa Ϫ N. gonorrhoeae did not (Fig.…”

“…Alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG(HϩL), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG(HϩL) and AP-conjugated goat anti-human IgG(HϩL) antibodies were purchased from Jackson ImmunoResearch Laboratories (Mississauga, Ontario, Canada). The soluble CEACAM1-Fc fusion protein was prepared as described previously (23). The CEACAM-specific phycoerythrin (PE) conjugate (CD66-PE; clone B1.1) was obtained from Becton Dickinson.…”

CEACAM1 Dynamics during Neisseria gonorrhoeae Suppression of CD4+ T Lymphocyte Activation

“…23,24). Individual Opa variants are able to bind to the conserved N-terminal domains of CEACAM1, CEACAM3, CEACAM5, and/or CEACAM6 via specific proteinprotein interactions (25)(26)(27).…”