Mumtaz Virji

Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic neisseriae

Opa protein‐expressing pathogenic neisseriae interact with CD66a‐transfected COS (African green monkey kidney) and CHO (Chinese hamster ovary) cells. CD66a (BGP) is a member of carcinoembryonic antigen (CEA, CD66) family. The interactions occur at the N‐terminal domain of CD66a, a region that is highly conserved between members of the CEA subgroup of the CD66 family. In this study, we have investigated the roles of CD66 expressed on human epithelial cells and polymorphonuclear phagocytes (PMNs) in adhesion mediated via Opa proteins. Using human colonic (HT29) and lung (A549) epithelial cell lines known to express CD66 molecules, we show that these receptors are used by meningococci. A monoclonal antibody, YTH71.3, against the N‐terminal domain of CD66, but not 3B10 directed against domains, A1/B1, inhibited meningococcal adhesion to host cells. When acapsulate bacteria expressing Opa proteins were used, large numbers of bacteria adhered to HT29 and A549 cells. In addition, both CD66a‐transfected CHO cells and human epithelial cells were invaded by Opa‐expressing meningococci, suggesting that epithelial cell invasion may occur via Opa–CD66 interactions. In previous studies we have shown that serogroup A strain C751 expresses three Opa proteins, all of which mediate non‐opsonic interactions with neutrophils. We have examined the mechanisms of these interactions using antibodies and soluble chimeric receptors. The results indicate that the nature of their interactions with purified CD66a molecules and with CD66 on neutrophils is alike and that these interactions occur at the N‐terminal domain of CD66. Thus, the Opa family of neisserial ligands may interact with several members of the CD66 family via their largely conserved N‐terminal domains.

The N-domain of the human CD66a adhesion molecule is a target for Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae

Using COS (African green monkey kidney) cells transfected with cDNAs encoding human cell surface molecules, we have identified human cellular receptors for meningococcal virulence‐associated Opa proteins, which are expressed by the majority of disease and carrier isolates. These receptors belong to the immunoglobulin superfamily of adhesion molecules and are expressed on epithelial, endothelial and phagocytic cells. Using soluble chimeric receptor molecules, we have demonstrated that meningococcal Opa proteins bind to the N‐terminal domain of biliary glycoproteins (classified as BGP or CD66a) that belong to the CEA (CD66) family. Moreover, the Opa proteins of the related pathogen Neisseriagonorrhoeae, responsible for urogenital infections, also interact with this receptor, making CD66a a common target for pathogenic neisseriae. Over 95% of Opa‐expressing clinical and mucosal isolates of meningococci and gonococci were shown to bind to the CD66 N‐domain, demonstrating the presence of a conserved receptor‐binding function in the majority of neisserial Opa proteins.

Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose

Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N‐linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O‐linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1‐4‐linked digalactose moiety covalently linked to a 2,4‐diacetamido‐2,4,6‐trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase (galE) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper‐adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.

The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells.

The influence of the two surface structures of Neisseria meningitidis, capsule and pili, in bacterial interactions with human endothelial cells was investigated. Increased association correlated with the presence of pili on bacteria while capsule type had no apparent effect. Strains expressing both Class I and Class II pili associated with endothelial cells in significantly larger numbers compared with the non‐piliated variants of the same strains (>10×). Variants of Neisseria gonorrhoeae strain P9 expressing antigenically distinct pili also associated with endothelial cells in larger numbers (>30×) compared with the non‐piliated variant. Electron microscopic studies confirmed these data and showed that gonococci were internalized more frequently compared with meningococci. One consequence of increased association was an increase in the cytopathic effect of bacteria on the target cells.

Recognition of sialylated meningococcal lipopolysaccharide by siglecs expressed on myeloid cells leads to enhanced bacterial uptake

Sialic acid‐binding immunoglobulin‐like lectins (siglecs) are expressed predominantly in the haemopoietic and immune systems and exhibit specificities for both the linkage and the nature of sialic acids in N‐glycans, O‐glycans and glycolipids. Several siglecs, including sialoadhesin (Sn, siglec‐1) and siglec‐5, bind to NeuAcα2,3Gal, a terminal capping structure that can also be displayed on the lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). In the present study, we examined the potential of siglecs expressed on cells of the immune system to function as receptors for sialylated Nm. We used sialylated and non‐sialylated LPS derivatives of two serogroups (A and B) of Nm in this study. Using recombinant chimeric soluble receptors, siglec‐transfected cell lines and macrophages from wild‐type and Sn‐deficient mice, we observed that sialylated but not non‐sialylated variants of either genetic background were specifically recognized by Sn and siglec‐5, whereas other siglecs examined were ineffective. In addition, macrophages expressing Sn, as well as transfectants expressing Sn or siglec‐5, bound and phagocytosed sialylated bacteria in a siglec‐ and sialic acid‐dependent manner. This study demonstrates that Nm LPS sialylation can lead to increased bacterial susceptibility to phagocytic uptake, a phenomenon in direct contrast to previously reported protective effects of LPS sialylation.

Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis.

Lipopolysaccharide (LPS) is a major determinant of Neisseria meningitidis virulence. A key feature of meningococcal LPS is the phase‐variable expression of terminal structures which are proposed to have disparate roles in pathogenesis. In order to identify the biosynthetic genes for terminal LPS structures and the control mechanisms for their phase‐variable expression, the lic2A gene, which is involved in LPS biosynthesis in Haemophilus influenzae, was used as a hybridization probe to identify a homologous gene in N. meningitidis strain MC58. The homologous region of DNA was cloned and nucleotide sequence analysis revealed three open reading frames (ORFs), two of which were homologous to the H. influenzae lic2A gene. All three ORFs were mutagenized by the insertion of antibiotic‐resistance cassettes and the LPS from these mutant strains was analysed to determine if the genes had a role in LPS biosynthesis. Immunological and tricine—SDS—PAGE analysis of LPS from the mutant strains indicated that all three genes were probably transferases in the biosynthesis of the terminal lacto‐N‐neotetraose structure of meningococcal LPS. The first ORF of the locus contains a homopolymeric tract of 14 guanosine residues within the 5′‐end of the coding sequence. As the lacto‐N‐neotetraose structure in meningococcal LPS is subject to phase‐variable expression, colonies that no longer expressed the terminal structure, as determined by monoclonal antibody binding, were isolated. Analysis of an ‘off’ phase variant revealed a change in the number of guanosine residues resulting in a frameshift mutation, indicating that a slipped‐strand mispairing mechanism, operating in the first ORF, controls the phase‐variable expression of lacto‐N‐neotetraose.

OPC- AND PILUS-DEPENDENT INTERACTIONS OF MENINGOCOCCI WITH HUMAN ENDOTHELIAL CELLS : MOLECULAR MECHANISMS AND MODULATION BY SURFACE POLYSACCHARIDES

The interplay between four surface‐expressed virulence factors of Neisseria meningitidis (pili, Opc, capsule and lipopolysaccharide (LPS)) in host cell adhesion and invasion was examined using derivatives of a serogroup B strain, MC58, created by mutation (capsule, Opc) and selection of variants. To examine the role of Opc and of additional expression of pili, bacteria lacking the expression of Opa proteins were used. The effects of different LPS structures were examined in variants expressing either sialylated (L3 immunotype) or truncated non‐sialylated (L8 immuno‐type) LPS. Studies showed that (i) pili were essential for meningococcal interactions with host cells in both capsulate and acapsulate bacteria with the sialylated L3 LPS immunotype, (ii) the Opc‐mediated invasion of host cells by piliated and non‐piliated bacteria was observed only in acapsulate organisms with L8 LPS immunotype, and (iii) expression of pili in Opc‐expressing bacteria resulted in increased invasion. Investigations on the mechanisms of cellular invasion indicated that the Opc‐mediated invasion was dependent on the presence of serum in the incubation medium and was mediated by serum proteins with arginine‐glycine‐aspartic acid (RGD) sequence. Cellular invasion in piliated Opc+ phenotype also required bridging molecules containing the RGD recognition sequence and appeared to involve the integrin αvβ3 as a target receptor on endothelial cells. These studies extend the previous observations on variants of a serogroup A strain (C751) and show that Opc mediates cellular invasion in distinct meningococcal strains and provide confirmation of its mechanism of action. This is the first investigation that evaluates, using derivatives of a single strain, the interplay between four meningococcal surface virulence factors in host cell invasion.

Pilus‐facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin

Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (<150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non‐piliated and failed to adhere to endothelial cells (<1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent Mr and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper‐adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent Mr values on SDS‐PAGE. Hyper‐adherent pilins shared unique changes in sequence including substitution of Asn‐113 for Asp‐113 and changes from Asn‐Asp‐Thr‐Asp to Thr‐Asp‐Ala‐Lys at residues 127‐130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N‐glycosylation motif Asn‐X‐Ser/Thr and could potentially be glycosylated post‐translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS‐PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn‐60‐Asn‐61‐Thr‐62 in an adherent variant was replaced with Asp‐60‐Asn‐61‐Ser‐62 in a non‐adherent variant. Fully adherent backswitchers obtained from the non‐adherent variant always regained Asn‐60 but retained Ser‐62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N‐linked glycosylation sites on the pilin subunit.

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

The 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 define 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, efficient 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 identified within the Opa adhesiotopes on the N‐domain. These studies define 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.

Expression of the Opc protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis

Whereas capsulate strains of Neisseria meningitidis are dependent on pili for adhesion to human endothelial and epithelial cells, strains which lacked assembled pili and were partially capsule‐deficient adhered to and invaded human endothelial and epithelial cells if they expressed the Opc protein. Bacteria expressing low or undetectable levels of Opc protein failed to adhere to or invade eukaryotic cells. In addition, the presence of OpaAc751 protein on the surface of bacteria did not increase bacterial interactions with host cells. Association of Opc‐expressing bacteria was inhibited by antibodies against Opc. Invasion was dependent on the host‐cell cytoskeletal activity and was inhibited by cytochalasin D. In some cells, infected at the apical surface, bacteria emerging from basal surface were detected by electron microscopy. Opc is found in diverse meningococci and may represent a common virulence factor, which facilitates adherence and invasion by these bacteria.