Katherine Makepeace

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.

Meningococcal Opa and Opc proteins: their role in colonization and invasion of human epithelial and endothelial cells

Neisseria meningitidis (Nm) isolates from disease or during carriage express, on their outer membranes, one or more of a family of closely related proteins designated Opa proteins. In this study, we have examined the potential rotes of Nm Opa proteins in bacterial attachment and invasion of endothelial as well as epithelial cells and compared the influence of Opa proteins with that of Ope protein, which has been previously shown to increase bacterial interactions with eukaryotic cells. Several variants expressing different Opa proteins (A, B, D) or Opc were selected from a culture of capsule‐deficient non‐piliated bacteria of strain C751. Although the Opa proteins increased bacterial attachment and invasion of endothelial cells, Opc was the most effective protein in increasing bacterial interactions with these cells. In contrast, attachment to several human epithelial cells was facilitated at least as much by OpaB as Opc protein. OpaA was largely without effect whereas OpaD conferred intermediate attachment. OpaB also increased invasion of epithelial cells; more bacteria were internalized by Chang conjunctival cells compared with Hep‐2 larynx carcinoma or A549 lung carcinoma cells. Monoclonal antibody reacting with OpaB inhibited bacterial interactions with the host cells. Opa‐mediated interactions were also eliminated or significantly reduced in variants expressing capsule or those with sialylated lipopolysaccharide. These data are consistent with the notion that environmental factors controlling capsule and lipopolysaccharide phenotype may modulate bacterial interactions mediated by these OM proteins. In permissive microenvironments, some Opa proteins may be important in bacterial colonization and translocation in addition to Opc. The data also support the notion that Nm Opa may confer tissue tropism.

Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization.

A survey of Haemophilus influenzae strains indicated that around one‐third of capsular strains and over two‐thirds of non‐typeable strains included sialic acid in their lipopolysaccharides (LPS). Mutation of the CMP‐Neu5Ac synthetase gene (siaB) resulted in a sialylation‐deficient phenotype. Isogenic pairs, wild type and siaB mutant of two non‐typeable strains were used to demonstrate that sialic acid influences resistance to the killing effect of normal human serum but has little effect on attachment to, or invasion of, cultured human epithelial cells or neutrophils. We determine for the first time the site of attachment of sialic acid in the LPS of a non‐typeable strain and report that a small proportion of glycoforms include two sialic acid residues in a disaccharide unit.

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.

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.

The Class A Macrophage Scavenger Receptor Is a Major Pattern Recognition Receptor for Neisseria meningitidis Which Is Independent of Lipopolysaccharide and Not Required for Secretory Responses

ABSTRACT Macrophages (Mφ) play a key role in the pathogenesis of invasive meningococcal infections. The roles of two pattern recognition molecules, the Mφ scavenger receptor (SR-A) and Toll-like receptor 4 (TLR-4), have been investigated using bone marrow culture-derived Mφ (BMMφ). Surprisingly, a comparison of BMMφ from wild-type and SR-A knockout (SR-A−/−) mice showed that nonopsonic phagocytosis of meningococci was mediated almost exclusively via SR-A. Previous studies have demonstrated only a partial involvement of the receptor in the uptake of other bacteria, such as Escherichia coli. Interestingly, we also show that lipopolysaccharide (LPS) was not the ligand for the receptor on these organisms. Further study of the downstream events of SR-A-mediated ingestion of Neisseria meningitidis demonstrated that SR-A was not required for cytokine production. To determine the bacterial and host factors required to stimulate Mφ activation, we examined TLR-4-deficient Mφ from C3H/HeJ mice and LPS-deficient meningococci. TLR-4-deficient cells elaborated reduced amounts of tumor necrosis factor alpha, interleukin-12 (IL-12), and IL-10, even though ingestion via SR-A was unaffected in these cells. Similarly, although there was no change in SR-A-mediated ingestion of LPS-deficient meningococci, the mutant failed to stimulate a Mφ-dependent cytokine response. Thus, we show that Mφ SR-A mediates opsonin-independent uptake of N. meningitidis independently of lipid A and that this activity is uncoupled from the Mφ secretion of proinflammatory cytokines, which provides a basis for further investigation of the role of this receptor in meningococcal disease in humans.

Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae.

We have identified a gene for the addition of N‐acetylneuraminic acid (Neu5Ac) in an α‐2,3‐linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase‐variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N‐acetyl‐lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase‐variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non‐typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl‐α‐(2–3)‐lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an α‐2,3‐sialyltransferase activity, is the first reported phase‐variable sialyltransferase gene.