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.     

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.     

CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes.

Colonization of urogenital tissues by the human pathogen Neisseria gonorrhoeae is characteristically associated with purulent exudates of polymorphonuclear phagocytes (PMNs) containing apparently viable bacteria. Distinct variant forms of the phase-variable opacity-associated (Opa) outer membrane proteins mediate the non-opsonized binding and internalization of N. gonorrhoeae by human PMNs. Using overlay assays and an affinity isolation technique, we demonstrate the direct interaction between Opa52-expressing gonococci and members of the human carcinoembryonic antigen (CEA) family which express the CD66 epitope. Gonococci and recombinant Escherichia coli strains synthesizing Opa52 showed specific binding and internalization by transfected HeLa cell lines expressing the CD66 family members BGP (CD66a), NCA (CD66c), CGM1 (CD66d) and CEA (CD66e), but not that expressing CGM6 (CD66b). Bacterial strains expressing either no opacity protein or the epithelial cell invasion-associated Opa50 do not bind these CEA family members. Consistent with their different receptor specificities, Opa52-mediated interactions could be inhibited by polyclonal anti-CEA sera, while Opa50 binding was instead inhibited by heparin. Using confocal laser scanning microscopy, we observed a marked recruitment of CD66 antigen by Opa52-expressing gonococci on both the transfected cell lines and infected PMNs. These data indicate that members of the CEA family constitute the cellular receptors for the interaction with, and internalization of, N. gonorrhoeae.     

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.     

Neisseria meningitidis producing the Opc adhesin binds epithelial cell proteoglycan receptors

Neisseria meningitidis possesses a repertoire of surface adhesins that promote bacterial adherence to and entry into mammalian cells. Here, we have identified heparan sulphate proteoglycans as epithelial cell receptors for the meningococcal Opc invasin. Binding studies with radiolabelled heparin and heparin affinity chromatography demonstrated that Opc is a heparin binding protein. Subsequent binding experiments with purified ³⁵SO₄-labelled epithelial cell proteoglycan receptors and infection assays with epithelial cells that had been treated with heparitinase to remove glycosaminoglycans confirmed that Opc-expressing meningococci exploit host cell-surface proteoglycans to gain access to the epithelial cell interior. Unexpectedly, Opa28-producing meningococci lacking Opc also bound proteoglycans. These bacteria also bound CEA receptors in contrast to the Opc-expressing phenotype, suggesting that Opa28 may possess domains with specificity for different receptors. Opa/Opc-negative meningococci did not bind either proteoglycan or CEA receptors. Using a set of genetically defined mutants with different lipopolysaccharide (LPS) and capsular phenotype, we were able to demonstrate that surface sialic acids interfere with the Opc–proteoglycan receptor interaction. This effect may provide the molecular basis for the reported modulatory effect of capsule and LPS on meningococcal adherence to and entry into various cell types.     

The CGM1a (CEACAM3/CD66d)-mediated phagocytic pathway of Neisseria gonorrhoeae expressing opacity proteins is also the pathway to cell death

Phagocytosis of Opa+ Neisseria gonorrhoeae (gonococcus, GC) by neutrophils is in part dependent on the interaction of Opa proteins with CGM1a (CEACAM3/CD66d) antigens, a neutrophil-specific receptor. However, the signaling pathways leading to phagocytosis have not been characterized. Here we show that interaction of OpaI bacteria with neutrophils or CGM1a-transfected DT40 cells induces calcium flux, which correlates with phagocytosis of bacteria. We identified an immunoreceptor tyrosine-based activation motif (ITAM) in CGM1a, and showed that the ability of CGM1a to transduce signals and mediate phagocytosis was abolished by mutation of the ITAM tyrosines. We also demonstrated that CGM1a-ITAM-mediated bacterial phagocytosis is dependent on Syk and phospholipase C activity in DT40 cells. Unexpectedly, the activation of the CGM1a-ITAM phagocytic pathway by Opa+ GC results in induction of cell death.     

Conformational analysis of opacity proteins from Neisseria meningitidis.

Opacity-associated (Opa) proteins are outer membrane proteins which play a critical role in the adhesion of pathogenic Neisseria spp. to epithelial and endothelial cells and polymorphonuclear neutrophils. The adherence is mainly mediated by the CD66-epitope-containing members of the carcinoembryonic-antigen family of human cell-adhesion molecules (CEACAM). For the analysis of the specific interactions of individual Opa proteins with their receptors, pure protein is needed in its native conformation. In this study, we describe the isolation and structural analysis of opacity proteins OpaJ129 and OpaB128 derived from Neisseria meningitidis strain H44/76. When the Opa proteins were produced with the phoE signal sequence in Escherichia coli, they were localized at the cell surface and the recombinant bacteria were found to specifically interact with CEACAM1. For refolding and purification, the proteins were overproduced without their signal sequences in E. coli, resulting in its cytoplasmic accumulation in the form of inclusion bodies. After solubilization of the inclusion bodies in urea, the proteins could be folded efficiently in vitro, under alkaline conditions by dilution in ethanolamine and the detergent n-dodecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate (SB12). The structure of the refolded and purified proteins, determined by circular dichroism, indicated a high content of beta-sheet conformation, which is consistent with previously proposed topology models for Opa proteins. A clear difference was found between the binding of refolded vs. denatured OpaJ protein to the N-A1 domain of CEACAM1. Almost no binding was found with the denatured Opa protein, showing that the Opa-receptor interaction is conformation-dependent.     

Molecular analysis of neisserial Opa protein interactions with the CEA family of receptors: identification of determinants contributing to the differential specificities of binding

The carcinoembryonic antigen (CEA) gene family members, CEACAM1, CEACAM3, CEACAM5 and CEACAM6, are bound by the Opa outer membrane proteins of pathogenic Neisseria spp., whereas CEACAM8 is not. In this study, we demonstrate that the closely related CEACAM4 and CEACAM7, which are also members of the CEA family, are not Opa receptors. We exploited the high conservation between CEACAM6 and CEACAM8 to generate an extensive set of chimeric receptors in order to delineate the sequences necessary for Opa binding. Using a transfection-based infection system, we showed that binding of Opa₅₂ involves residues 27–42, which are predicted to form β-strand C and short loops adjacent to it, and residues lying between amino acids 60 and 108 in the amino-terminal domain. The replacement of residues 27–29 in CEACAM6 with the CEACAM1 or CEACAM5 sequences generated recombinant CEACAM6 receptors that are bound by CEACAM1/CEACAM5-specific Opa variants. Together, our data demonstrate that Opa proteins bind to residues exposed on the GFCC' face of the N-terminal domain of CEACAM receptors, and identify an amino acid triplet sequence that is responsible for the differential binding of Opa proteins to CEACAM1, CEACAM5 and CEACAM6.     

Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC)

Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI-anchored proteins CD55 (decay-accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA-related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM-expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr-I, including Dr, F1845 and AfaE-III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr-I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells. The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not. Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli-like extensions, which are elongated. Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli-like extensions.     

Opa proteins and CEACAMs: pathways of immune engagement for pathogenic Neisseria

Neisseria meningitidis and Neisseria gonorrhoeae are globally important pathogens, which in part owe their success to their ability to successfully evade human immune responses over long periods. The phase-variable opacity-associated (Opa) adhesin proteins are a major surface component of these organisms, and are responsible for bacterial adherence and entry into host cells and interactions with the immune system. Most immune interactions are mediated via binding to members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family. These Opa variants are able to bind to different receptors of the CEACAM family on epithelial cells, neutrophils, and T and B lymphocytes, influencing the innate and adaptive immune responses. Increased epithelial cell adhesion creates the potential for prolonged infection, invasion and dissemination. Furthermore, Opa proteins may inhibit T-lymphocyte activation and proliferation, B-cell antibody production, and innate inflammatory responses by infected epithelia, in addition to conferring increased resistance to antibody-dependent, complement-mediated killing. While vaccines containing Opa proteins could induce adhesion-blocking and bactericidal antibodies, the consequence of CEACAM binding by a candidate Opa-containing vaccine requires further investigation. This review summarizes current knowledge of the immunological consequences of the interaction between meningococcal and gonococcal Opa proteins and human CEACAMs, considering the implications for pathogenesis and vaccine development.     

Neisseria meningitidis has two independent modes of recognizing its human receptor CEACAM1

Background

Several human-restricted Gram-negative bacteria exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) for host colonization. For example, Neisseria meningitidis engages these human receptors via outer membrane proteins of the colony opacity-associated (Opa) protein family triggering internalization into non-phagocytic cells.

Principal Findings

We report that a non-opaque strain of N. meningitidis selectively interacts with CEACAM1, but not other CEACAM family members. Using functional assays of bacterial adhesion and internalisation, microscopic analysis, and a panel of CEACAM1 deletion mutants we demonstrate that the engagement of CEACAM1 by non-opaque meningococci occurs in a manner distinct from Opa protein-mediated association. In particular, the amino-terminal domain of CEACAM1 is necessary, but not sufficient for Opa protein-independent binding, which requires multiple extracellular domains of the human receptor in a cellular context. Knock-down of CEACAM1 interferes with binding to lung epithelial cells, whereas chemical or pharmacological disruption of host protein glycosylation does not abrogate CEACAM1 recognition by non-opaque meningococci. The previously characterized meningococcal invasins NadA or Opc do not operate in a CEACAM1-dependent manner.

Conclusions

The results demonstrate a mechanistically distinct, Opa protein-independent interaction between N. meningitidis and human CEACAM1. Our functional investigations suggest the presence of a second CEACAM1-binding invasin on the meningococcal surface that associates with the protein backbone and not the carbohydrate structures of CEACAM1. The redundancy in meningococcal CEACAM1-binding factors further highlights the important role of CEACAM recognition in the biology of this human-adapted pathogen.     

Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells

The pathogenic Neisseriae Neisseria meningitidis and Neisseria gonorrhoeae, initiate colonization by attaching to host cells using type IV pili. Subsequent adhesive interactions are mediated through the binding of other bacterial adhesins, in particular the Opa family of outer membrane proteins. Here, we have shown that pilus-mediated adhesion to host cells by either meningococci or gonococci triggers the rapid, localized formation of dramatic cortical plaques in host epithelial cells. Cortical plaques are enriched in both components of the cortical cytoskeleton and a subset of integral membrane proteins. These include: CD44v3, a heparan sulphate proteoglycan that may serve as an Opa receptor; EGFR, a receptor tyrosine kinase; CD44 and ICAM-1, adhesion molecules known to mediate inflammatory responses; f-actin; and ezrin, a component that tethers membrane components to the actin cytoskeleton. Genetic analyses reveal that cortical plaque formation is highly adhesin specific. Both pilE and pilC null mutants fail to induce cortical plaques, indicating that neisserial type IV pili are required for cortical plaque induction. Mutations in pilT, a gene required for pilus-mediated twitching motility, confer a partial defect in cortical plaque formation. In contrast to type IV pili, many other neisserial surface structures are not involved in cortical plaque induction, including Opa, Opc, glycolipid GgO4-binding adhesins, polysialic acid capsule or a particular lipooligosaccharide variant. Furthermore, it is shown that type IV pili allow gonococci to overcome the inhibitory effect of heparin, a soluble receptor analogue, on gonococcal invasion of Chang and A431 epithelial cells. These and other observations strongly suggest that type IV pili play an active role in initiating neisserial infection of the mucosal surface in vivo. The functions of type IV pili and other neisserial adhesins are discussed in the specific context of the mucosal microenvironment, and a multistep model for neisserial colonization of mucosal epithelia is proposed.     

Variable opacity (Opa) outer membrane proteins account for the cell tropisms displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells.

Opacity proteins (Opa) of Neisseria gonorrhoeae, a family of variant outer membrane proteins implicated in pathogenesis, are subject to phase variation. In strain MS11, 11 different opa gene alleles have been identified, the expression of which can be turned on and off independently. Using a reverse genetic approach, we demonstrate that a single Opa protein variant of strain MS11, Opa50, enables gonococci to invade epithelial cells. The remaining variant Opa proteins show no, or very little, specificity for epithelial cells but instead confer interaction with human polymorphonuclear neutrophils (PMNs). Thus, depending on the opa allele expressed, gonococci are capable of invading epithelial cells or of interacting with human leukocytes. The respective properties of Opa proteins are maintained independent of the gonococcal strain; thus, the specificity for epithelial cells or leukocytes is intrinsic to Opa proteins. Significant homology exists in the surface exposed variable regions of two invasion supporting Opa proteins from independent strains. Efficient epithelial cell invasion is favoured by high level Opa production, however, a 10-fold reduction still allows significant invasion by gonococci. In contrast, recombinant Escherichia coli expressing Opa proteins adhered or invaded poorly under similar experimental conditions, thus indicating that additional factors besides Opa are required in the Opa-mediated interaction with human cells.     

Mapping the binding domains on meningococcal Opa proteins for CEACAM1 and CEA receptors

The opacity (Opa) proteins of pathogenic Neisseria spp. are adhesins, which play an important role in adhesion and invasion of host cells. Most members of this highly variable family of outer membrane proteins can bind to the human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). Several studies have identified the Opa-binding region on the CEACAM receptors; however, not much is known about the binding sites on the Opa proteins for the corresponding CEACAM-receptors. The high degree of sequence variation in the surface-exposed loops of Opa proteins raises the question how the binding sites for the CEACAM receptors are conserved. Neisseria meningitidis strain H44/76 possesses four different Opa proteins, of which OpaA and OpaJ bind to CEACAM1, while OpaB and OpaD bind to CEACAM1 and CEA. A sequence motif involved in binding to CEACAM1 was identified by alanine scanning mutagenesis of those amino acid residues conserved within the hypervariable (HV) regions of all four Opa proteins. Hybrid Opa variants with different combinations of HV-1 and HV-2 derived from OpaB and OpaJ showed a reduced binding to CEACAM1 and CEA, indicating that particular combinations of HV-1 and HV-2 are required for the Opa binding capacity. Homologue scanning mutagenesis was used to generate more refined hybrids containing novel combinations of OpaB and OpaJ sequences within HV-1 and HV-2. They could be used to identify residues determining the specificity for CEA binding. The combined results obtained with mutants and hybrids strongly suggest the existence of a conserved binding site for CEACAM receptors by the interaction of HV-1 and HV-2 regions.     

Retinoic acid treated HL60 cells express CEACAM1 (CD66a) and phagocytose Neisseria gonorrhoeae

Neisseria gonorrhoeae (gonococci, GC) are phagocytosed by neutrophils through the interaction between opacity proteins (Opa) and the CEA (CD66) family of antigens. In order to study this interaction, we used the human myeloid leukemia HL60 cell line, which differentiates into granulocyte-like cells upon treatment with dimethylsulfoxide (DMSO) or retinoic acid (RA). We found that RA-, but not DMSO- or untreated-HL60 cells, can phagocytose OpaI-expressing gonococci as well as Escherichia coli. The interaction of OpaI E. coli with RA-treated HL60 cells was inhibited by antibodies against CEACAM1. Phagocytosis of OpaI E. coli was found to be a result of the expression of CEACAM1 in RA-treated HL60 cells. Our results indicate that the level of expression of CEACAM1 in HL60 cells can be regulated by treatment with RA in a differentiation-dependent manner, and that this is important for phagocytosis of OpaI-expressing gonococci or E. coli.     

Profiling of bacterial adhesin--host receptor recognition by soluble immunoglobulin superfamily domains

Several gram-negative human pathogens recognize members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Pathogenic Neisseriae employ distinct isoforms of the colony opacity-associated proteins (Opa(CEA) proteins) to bind to the amino-terminal domains of CEACAMs. Here we present a novel approach to rapidly determine the CEACAM-binding properties of single bacteria. Expression of the isolated amino-terminal domains of various CEACAMs in eukaryotic cells yields soluble probes that selectively recognize Opa(CEA)-expressing bacteria in a pull-down assay format. Furthermore, by expressing soluble CEACAMs as fusions to green-fluorescent protein (CEACAM-N-GFP), CEACAM-binding bacteria can be decorated with a fluorescent label and analysed by flow cytometry allowing the specific detection of receptor binding events on the level of single bacteria. Besides its potential for rapid and quantitative analysis of pathogen-receptor interactions, this novel approach allows the detection of receptor recognition in heterogeneous bacterial populations and might represent a valuable tool for profiling the host binding capabilities of various microorganisms.     

Mannose-binding lectin binds to two major outer membrane proteins, opacity protein and porin, of Neisseria meningitidis

Human mannose-binding lectin (MBL) provides a first line of defense against microorganisms by complement activation and/or opsonization in the absence of specific Ab. This serum collectin has been shown to activate complement when bound to repeating sugar moieties on several microorganisms, including encapsulated serogroup B and C meningococci, which leads to increased bacterial killing. In the present study, we sought to identify the meningococcal cell surface components to which MBL bound and to characterize such binding. Outer membrane complex containing both lipooligosaccharide (LOS) and proteins and LOS from Neisseria meningitidis were examined for MBL binding by dot blot and ELISA. MBL bound outer membrane complex but not LOS. The binding to bacteria by whole-cell ELISA did not require calcium and was not inhibited by N-acetyl-glucosamine or mannose. With the use of SDS-PAGE, immunoblot analysis, and mAbs specific for meningococcal opacity (Opa) proteins and porin proteins, we determined that MBL bound to Opa and porin protein B (porB). The N-terminal amino acid sequences of the two MBL binding proteins confirmed Opa and PorB. Purified PorB inhibited the binding of MBL to meningococci. Escherichia coli with surface-expressed gonococcal Opa bound significantly more MBL than did the control strain. The binding of human factor H to purified PorB was markedly inhibited by MBL in a dose-dependent manner. Meningococci incubated with human serum bound MBL as detected by ELISA. We conclude that MBL binds to meningococci by a novel target recognition of two nonglycosylated outer membrane proteins, Opa and PorB.     

Differential Opa specificities for CD66 receptors influence tissue interactions and cellular response to Neisseria gonorrhoeae

The ability of all 11 variable opacity (Opa) proteins encoded by Neisseria gonorrhoeae MS11 to interact directly with the five CD66 antigens was determined. Transfected HeLa cell lines expressing individual CD66 antigens were infected with recombinant N. gonorrhoeae and Escherichia coli strains expressing defined Opas. Based upon the ability of these bacteria to bind and invade and to isolate specifically CD66 antigens from detergent-soluble extracts of the corresponding cell lines, distinct specificity groups of Opa interaction with CD66 were seen. Defining these specificity groups allowed us to assign a specific function for CD66a in the Opa-mediated interaction of gonococci with two different target cell types, which are both known to co-express multiple CD66 antigens. The competence of individual Opas to interact with CD66a was strictly correlated with their ability to induce an oxidative response by polymorphonuclear neutrophils. The same Opa specificity was observed for the level of gonococcal binding to primary endothelial cells after stimulation with TNFα, which was shown to increase the expression of CD66a rather than CD66e. As CD66e alone is expressed on other target tissues of gonococcal pathogenicity, Opa variation probably contributes to the cell tropism displayed by gonococci.     

Pathogenic neisseriae—Interplay between pro-and eukaryotic worlds

The pathogenicNeisseria species constitute a multi-faceted infection model of a highly adapted pathogen-host relationship. Several bacterial and host-cell factors involved in the cellular cross-talk have been recently unraveled. UsingNeisseria gonorrhoeae as a prototype, several structurally variable surface proteins, including pili and Opa proteins, have been revealed as adhesins recognizing distinct host-cell receptors. The Opa proteins, in particular, are important in facilitating interaction with heparan sulfate proteoglycan receptors and members of the CD66 and integrin receptor families. These interactions not only enable the pathogens' anchoring, and penetration into, the human mucosa but also stimulate cellular signaling cascades involving the phosphatidylcholinedependent phospholipase C, acidic sphingomyelinase and protein kinase C in epithelial cells, and Src-related kinases, Rac1, p21-activated kinase and Jun N-terminal kinase in phagocytic cells. Activation of these pathways is essential for the entry and intracellular accommodation of the pathogens but also leads to an early induction of cytokine release, thus priming the immune response. It is believed that detailed knowledge of cellular signaling cascades activated by infection will aid us in applying known and novel interfering drugs, in addition to classical antibiotic therapy, to the therapeutic and prophylactic treatment of persistent or otherwise difficult-to-treat bacterial infections. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.