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...

SummaryHaemophilus influenzae, a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We have recently shown that numerous strains of capsulate (typeable) and acapsulate (non-typeable) H. influenzae target the carcinoembryonic antigen (CEA) family of cell adhesion molecules (CEACAMs). Moreover, the ligands appeared to be antigenically variable and, when using viable typeable bacteria, their adhesive functions were inhibited by the presence of capsule. In this report, we show that the antigenically variable outer membrane protein, P5, expressed by typeable and non-typeable H. influenzae targets human CEA-CAM1. Variants and mutants lacking the expression of P5 of all strains tested were unable to target purified soluble receptors. A non-typeable strain that did not interact with CEACAM1 was made adherent to both the soluble receptors and CEACAM1-transfected Chinese hamster ovary cells by transformation with the P5 gene derived from the adherent typeable strain Rd. However, several H. influenzae mutants lacking P5 expression continued to bind the cell-bound CEACAM1 receptors. These observations suggest that (i) CEACAM1 alone can support P5 interactions and (ii) some strains contain additional ligands with the property to target CEACAM1 but require the receptor in the cellular context. The identification of a common ligand in diverse strains of H. influenzae and the presence of multiple ligands for the same receptor suggests that targeting of members of the CEACAM family of receptors may be of primary significance in colonization and pathogenesis of H. influenzae strains.

SummaryThe pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post-translationally modified by the addition of an O-linked trisaccharide, Gal (␤1-4) Gal (␣1-3) 2,4-diacetimido-2,4,6-trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with welldefined lipopolysaccharide (LPS) or pilin-linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin-linked trisaccharide structure.The pglA mutation, like the galE mutation reported previously, had no effect on pilus-mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (␤1-4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.

SummaryHaemophilus influenzae (Hi), a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We show that distinct strains belonging to typable (THi) and nontypable (NTHi) H. influenzae target human carcinoembryonic antigens (the membrane associated CEA family of cell adhesion molecules, are now termed CEACAMs). All strains of H. influenzae biogroup aegyptius (Hi-aeg) and more than 70% of THi and NTHi strains tested specifically recognize CEACAMIFc soluble constructs. Furthermore, transfection of Chinese hamster ovary cells with human CEACAM1 cDNA alone was sufficient for promoting Hi interactions with the transfected cells. The majority of the Hi-aeg strains tested interacted with soluble constructs containing only the N-terminal domain. In contrast, several THi and NTHi strains reacted with soluble constructs only when additional extracellular A and B domains of the receptor were present. The use of monoclonal antibodies confirmed that THi and NTHi strains also interact primarily at the N-domain. We used site-directed mutants of CEACAM1 that contained substitutions at surface exposed amino acids and a molecular model of the N-domain to identify the residues involved in interactions with Hi ligands. The studies show that a common region exposed at the CFG face of the molecule is targeted by diverse Hi strains. However, mutation at distinct sites within this area affected the interactions of distinct strains signifying the potential for tissue tropism via this receptor. Analyses of the molecular basis of interaction with human cell lines and purified CEA show that Hi strains, especially those belonging to Hi-aeg, interact with multiple CEACAMs. Because Neisseria meningitidis (Nm) strains are also known to bind at the CFG face of the receptor, we used Nm and Hi strains in co-infection experiments and demonstrate competition between these mucosal pathogens in colonization of target cells via CEACAMs.

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