PubMed Health⌕ Search

Biomedical subjects

M Virji

Publications and source records attributed to M Virji.

At least 37 records · Page 2Linked to original sources
PubMed1996

Posttranslational modifications of meningococcal pili. Identification of a common trisaccharide substitution on variant pilins of strain C311.

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. Recent studies have shown that a novel O-linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 50 to 73 of the PilE molecule. The structure was shown to be Gal beta 1-4 Gal alpha 1-3 diacetamidotrideoxyhexose which is directly attached to pilin. Pilins derived from galactose epimerase (galE) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. These studies confirm our previous observations that meningococcal pili are glycosylated and provide the first structural evidence for the presence of covalently linked carbohydrate on pili. We have identified a completely novel protein/carbohydrate linkage on a multimeric protein that is an essential virulence determinant in N. meningitidis.

Amino Acid Sequence↗
PubMed1996

Discovery of a novel protein modification: alpha-glycerophosphate is a substituent of meningococcal pilin.

Pili, which are filamentous protein structures on the surface of the meningitis-causing organism Neisseria meningitidis, are known to be post-translationally modified with substituents that affect their mobility in SDS/PAGE and which might play a crucial role in adherence and bloodstream invasion. Tryptic digests of pili were analysed by fast atom bombardment and electrospray MS to identify putative modifications. Serine-93 was found to carry a novel modification of alpha-glycerophosphate. This is the first time that alpha-glycerophosphate has been observed as a substituent of a prokaryotic or eukaryotic protein.

Amino Acid Sequence↗
PubMed1996

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 Neisseria gonorrhoeae, 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.

Animals↗
PubMed1996

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.

Animals↗
PubMed1995

Cloning and molecular analysis of the Isi1 (rfaF) gene of Neisseria meningitidis which encodes a heptosyl-2-transferase involved in LPS biosynthesis: evaluation of surface exposed carbohydrates in LPS mediated toxicity for human endothelial cells.

Neisseria meningitidis, but not Haemophilus influenzae, damage cultured human endothelial cells. We have undertaken a study to generate genetically and structurally defined lipopolysaccharide (LPS) mutant strains of meningococci for functional studies to assess the role of surface exposed oligosaccharides in imparting specificity of toxic damage to human endothelial cells. The Isi1 gene, which had been shown to be involved in LPS biosynthesis of Neisseria gonorrhoeae, was amplified by PCR and cloned. Nucleotide sequence analysis confirmed the identity of the clone and revealed homology with Isi1 of N. gonorrhoeae and the rfaF gene of Salmonella typhimurium which encodes a heptosyl-2-transferase involved in LPS biosynthesis. The identity of the cloned Isi1 gene, as a functional rfaF homologue, was confirmed by the complementation of a S. typhimurium rfaF mutant using a P22 phage sensitivity test. An Isi1 mutant meningococcal strain was constructed, and structural analysis of the mutant LPS molecule revealed a single heptose in the core structure, consistent with a heptosyl-2-transferase deficient mutant. In order to investigate the relative cytotoxicities of meningococci expressing native and altered LPS, wild type, Isi1, and galE strains were compared in cytotoxicity assays using human umbilical vein endothelial cells (Huvecs) in culture. Analysis using Huvecs derived from several individuals (cords) showed that the three phenotypes were almost equally cytotoxic. Removal of the terminal portion (galE mutant) or the majority (Isi mutant) of the oligosaccharide did not effect LPS-mediated cytopathic damage to Huvecs in a culture suggesting that the oligosaccharide portion did not play a major role in cytotoxicity.

Amino Acid Sequence↗
PubMed1995

Investigations into the molecular basis of meningococcal toxicity for human endothelial and epithelial cells: the synergistic effect of LPS and pili.

Using human umbilical vein endothelial cells as an in vitro model of toxicity, it was found that Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria lactamica and Neisseria sicca caused damage to these cells, in contrast to the lack of cytotoxicity exhibited by Haemophilus influenzae type b. N. meningitidis was also found to be toxic for human epithelial cells. The major toxic factor of N. meningitidis was found to be a heat-stable component of outer membrane vesicles, and could be inhibited by polymyxin B, suggesting that lipopolysaccharide plays a major role in toxicity. However, the toxicity mediated by lipopolysaccharide was modulated significantly by pilus-dependent adherence. Intra-strain variants expressing altered pilins which exhibited different levels of adherence to epithelial and endothelial cells were used to study the role of pilus. The degree of toxicity observed correlated with their relative level of adherence to cultured cells. In contrast, Opc-dependent increased adherence did not result in increased toxicity for endothelial cells, suggesting that pili have a synergistic effect, contributing to the overall damage.

Bacterial Adhesion↗
PubMed1995

The effect of variations in the expression of pili on the interaction of Neisseria meningitidis with human nasopharyngeal epithelium.

Bacterial adherence is essential to colonization of the nasopharynx by Neisseria meningitidis, and pili may facilitate adherence. Scanning electron microscopy and immunogold labeling were used to study the interaction of 2 piliated and 1 nonpiliated variant of N. meningitidis with nasopharyngeal mucosa in an adenoid organ culture system with an air-mucosal interface. Meningococcal infection caused epithelial damage, loss of ciliated epithelium, and ciliary disorganization, which progressed with time and were greater with the piliated strains. Pili increased adherence of meningococci to the mucosa, and there was tropism of piliated strains for nonciliated cells containing microvilli, damaged epithelium, and sites of cell separation. Bacterial adherence was associated with a change in the appearance of the microvilli.

Adenoids↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

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 immunotype) 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 alpha v beta 3 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.

Antigens, Bacterial↗
PubMed1995

Functional implications of the expression of PilC proteins in meningococci.

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil- backswitcher isolated from the hyper-adherent variant was PilC+ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.

Bacterial Adhesion↗
PubMed1994

Interactions of Neisseria meningitidis with human monocytes.

The roles of capsule, pili and Class 5 outer-membrane proteins (Opa and Opc) of Neisseria meningitidis (Nm) in bacterial interactions with human monocytes were investigated using several meningococcal isolates of different serogroups. The presence of either Class I or Class II pili in capsulate strains of several serogroups had no significant effect on adherence to and internalisation by monocytes. Using clonal variants derived from a non-piliated serogroup A strain, C751, it was observed that capsulate bacteria (cap+) failed to interact with human monocytes in significant numbers whether or not they expressed outer-membrane proteins. These bacteria were also resistant to phagocytic killing. For capsule-deficient bacteria, expression of the Opc protein or OpaBC751 correlated with high levels of association, while the expression of OpaDC751 or OpaAC751 resulted in comparatively lower levels. Bacteria expressing undetectable levels of Opc or Opa proteins (Opc-, Opa-) failed to interact with monocytes. In phagocytic killing assays, Opc-expressing bacteria (Opc+) as well as Opa-expressing bacteria (Opa+) were killed more readily than Opc-, Opa- bacteria (30% decrease in viability of Opc+ bacteria; 18%, 10% and 8% decrease in viability of OpaB+, OpaD+ and OpaA+ bacteria). A study of intracellular survival showed a gradual decrease in viability of both capsulate and capsule-deficient bacteria. However, proportionately greater numbers of capsule-deficient bacteria were internalized and consequently larger numbers survived over a 4-h period. Prolonged bacterial survival within phagocytic cells may have implications in dissemination of bacteria by carriage within these cells.

Animals↗
PubMed1994

Distinct mechanisms of interactions of Opc-expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions.

Interactions of Opc-expressing Neisseria meningitidis with polarized and non-polarized human umbilical vein endothelial cells (Huvecs) were investigated. Metabolic inhibitors and cytochalasin D treatment showed that host cellular and cytoskeletal functions were important for Opc-expressing bacterial association with Huvecs at the apical surface. In addition, this interaction required the presence of serum in the incubation medium whilst association with non-polarized cells did not require serum. Pre-exposure of Opc-expressing bacteria to serum was sufficient to increase the number of bacterial interactions at the apical surface; B306, a monoclonal antibody (mAb) against Opc, inhibited these interactions, suggesting that Opc binds to serum factor(s) and this in turn increases adherence to Huvecs. The receptors involved in this 'sandwich' adherence belong to the integrin family since the interaction was inhibited by peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD) and the tetrapeptide RGDS (but not the peptide RGES) was inhibitory. Non-polarized cells appeared to expose receptors/sites that bound to Opc-expressing bacteria directly, did not require serum factors and were not inhibited by RGD-containing peptides. Serum-dependent interactions of Opc-expressing bacteria to apical surface was inhibited significantly by several mAbs against alpha v beta 3 integrins. Some mAbs against alpha 5 and beta 1 caused partial inhibition; antibodies that did not block the function of beta 1 integrins or the mAbs against alpha 2 integrins were not inhibitory to bacterial interactions with Huvecs. Purified vitronectin supported adherence of Opc-expressing bacteria to Huvecs but not of Opc- bacteria. These interactions were inhibited by mAb B306 against Opc, by RGDS peptides as well as by blocking antibodies directed against alpha v beta 3 but not antibodies against other integrins. These data suggest that a sequence of molecular events resulting in trimolecular complexes at the endothelial surface may drive neisserial invasion of Huvecs. The expression of Opc appears to enable bacteria to utilize the normal signal-transduction mechanism of host cells via ligands in sera that adhere to endothelial cell integrins.

Antibodies, Monoclonal↗