Critical determinants of host receptor targeting by Neisseria meningitidisand Neisseria gonorrhoeae: identification of Opa adhesiotopes on the N-domain of CD66 molecules

The human pathogens Neisseria meningitidis and Neisseria gonorrhoeae expres s a family of variable outer membrane opacity-associated (Opa) proteins tha t recognize multiple human cell surface receptors. Most Opa proteins target the highly conserved N-terminal domain of the CD66 family of adhesion mole cules, although a few also interact with heparan sulphate proteoglycans. In this study, we observed that at least two Opa proteins of a N. meningitidi s strain C751 have the dual capacity to interact with both receptors. In ad dition, all three Opa proteins of C751 bind equally well to HeLa cells tran sfected with cDNA encoding the carcinoembryonic antigen [CEA (CD66e)] subgr oup of the CD66 family, but show distinct tropism for CGM1-(CD66d) and NCA (CD66c)-expressing cells. Because the C751 Opa proteins make up distinct st ructures via the surface-exposed hypervariable domains (HV-1 and HV-2), the se combinations appear to be involved in tropism for the distinct CD66 subg roups. To define the determinants of receptor recognition, we used mutant p roteins of biliary glycoprotein [BGP (CD66a)] carrying substitutions at sev eral predicted exposed sites in the N-domain and compared their interaction s 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 co nstructed show that the binding of all Opa proteins tested occurs at the no n-glycosylated (CFG) face of the molecule and, in general, appears to requi re Tyr-34 and Ile-91, Further, efficient interaction of distinct Opa protei ns 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 CF G face, making continuous binding domains (adhesiotopes). The epitope of th e monoclonal antibody YTH71.3 that inhibits Opa/CD66 interactions was also identified within the Opa adhesiotopes on the N-domain, These studies defin e the molecular basis that directs the Opa specificity for the CD66 family and the rationale for tropism of the Opa proteins for the CD66 subgroups.