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.     

The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane

In Gram-negative bacteria, lipopolysaccharide and phospholipid biosynthesis takes place at the inner membrane. How the completed lipid molecules are subsequently transported to the outer membrane remains unknown. Omp85 of Neisseria meningitidis is representative for a family of outer membrane proteins conserved among Gram-negative bacteria. We first demonstrated that the omp85 gene is co-transcribed with genes involved in lipid biosynthesis, suggesting an involvement in lipid assembly. A meningococcal strain was constructed in which Omp85 expression could be switched on or off through a tac promoter-controlled omp85 gene. We demonstrated that the presence of Omp85 is essential for viability. Depletion of Omp85 leads to accumulation of electron-dense amorphous material and vesicular structures in the periplasm. We demonstrated, by fractionation of inner and outer membranes, that lipopolysaccharide and phospholipids mostly disappeared from the outer membrane and instead accumulated in the inner membrane, upon depletion of Omp85. Omp85 depletion did not affect localization of integral outer membrane proteins PorA and Opa. These results provide compelling evidence for a role for Omp85 in lipid transport to the outer membrane.     

Human neutrophil response to recombinant neisserial Opa proteins

Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.     

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.     

Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins: intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth

Neisseria gonorrhoeae opacity-associated (Opa) proteins are a family of outer membrane proteins involved in gonococcal adherence to and invasion of human cells. We wanted to identify additional roles for Opa in the infectious process and used the yeast two-hybrid system to identify human epithelial cell proteins that interact with Opa proteins. Although this system has been used successfully to identify many types of interacting proteins, it has not been used to screen a human cell cDNA library for binding partners of a prokaryotic outer membrane protein. Therefore, we were also interested in exploring the versatility of the yeast two-hybrid system in identifying bacteria–host interactions. Using OpaP from strain F62SF as bait, we screened a HeLa cell cDNA library for Opa-interacting proteins (OIPs). We identified five different OIPs, designated OIP1–OIP5, two of which are homologous to human proteins — thyroid hormone receptor interacting protein (TRIP6) and pyruvate kinase isoenzyme M2 (PK). In the studies presented here, we investigated the interaction between Opa proteins and PK in more depth. Opa–PK interactions were confirmed by in vitro and in vivo assays independent of the yeast two-hybrid system. Escherichia coli expressing six different Opa proteins from gonococcal strain FA1090 all bound more PK than Opa-negative E. coli in in vitro binding assays. Using anti-PK antibody and fluorescence microscopy, we showed that human epithelial cell PK co-localizes with intracellular Opa+ gonococci and E. coli expressing Opa proteins. Using a mutant of N. gonorrhoeae unable to grow on pyruvate or lactate, it appears that intracellular pyruvate is essential for gonococcal growth and survival. These results suggest a novel mechanism in bacterial pathogenesis, i.e. the requirement for direct molecular interaction with a host metabolic enzyme (PK) for the acquisition of an essential intracellular carbon source and growth substrate (pyruvate). These results demonstrate that the yeast two-hybrid system is a valuable tool for identifying biologically relevant interactions between bacteria and host proteins, providing valuable leads for further investigations into novel mechanisms of bacterial pathogenesis.     

Critical determinants of the interactions of capsule-expressing Neisseria meningitidis with host cells: the role of receptor density in increased cellular targeting via the outer membrane Opa proteins

Neisseria meningitidis capsule is an important virulence determinant required for survival in the blood but is reportedly involved in inhibiting cellular interactions mediated by meningococcal outer membrane adhesins. However, evidence from our previous studies suggested that target receptor density on host cells may determine whether or not capsulate bacteria can adhere via outer membrane proteins such as Opa. To confirm this and evaluate the impact of capsulation on bacterial interactions, we used Opa(+) and Opa(-) derivatives of capsulate and acapsulate meningococcal isolates and transfected cell lines expressing CEACAM1, a receptor targeted by Opa proteins. To assess the extent and rate of cell association, subpopulations of stably transfected Chinese hamster ovary cells with different receptor levels were derived. A quantitative correlation of CEACAM1 levels and Opa-dependent binding of both capsulate and acapsulate bacteria was demonstrated, which was accelerated at high receptor densities. However, it appears that invasion by Opa(+) capsulate bacteria only occurs when a threshold level of CEACAM density has been reached. Target cells expressing high levels of CEACAM1 (MFI c. 400) bound threefold more, but internalized 20-fold more Opa(+) capsulate bacteria than those with intermediate expression (MFI c. 100). No overall selection of acapsulate phenotype was observed in the internalized population. These observations confirm that capsule may not be an adequate barrier for cellular interactions and demonstrate the role of a host factor that may determine capsulate bacterial invasion potential. Upregulation of CEACAMs, which can occur in response to inflammatory cytokines, could lead to translocation of a small number of fully capsulate bacteria across mucosal epithelium into the bloodstream sufficient to cause a rapid onset of disseminated disease. Thus the data also suggest a novel rationale for the epidemiological observations that individuals with prior infectious/inflammatory conditions carry a high risk of invasive meningococcal disease.     

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.     

Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.

Neisseria gonorrhoeae is a facultative intracellular bacterium capable of penetrating into certain human epithelial cell types. In order to identify gonococcal factors essential for invading Chang human conjunctiva cells, a gentamicin selection assay for the quantification of viable intracellular bacteria was used in conjunction with microscopy. The results demonstrate a correlation between the invasive behaviour of gonococci and the expression of Opa proteins, a family of variable outer membrane proteins present in all pathogenic Neisseria species. However, only particular Opa proteins supported invasion into Chang cells as indicated by the use of two unrelated gonococcal strains. Invasion was sensitive to cytochalasin D, and strong adherence mediated by the Opa proteins appeared to be essential for the internalization of gonococci. In contrast pili, which also conferred binding to Chang conjunctiva cells, did not support cellular invasion but rather were inhibitory.     

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.     

Recognition of saccharides by the OpcA, OpaD, and OpaB outer membrane proteins from Neisseria meningitidis

The adhesion of the pathogen Neisseria meningitidis to host cell surface proteoglycan, mediated by the integral outer membrane proteins OpcA and Opa, plays an important part in the processes of colonization and invasion by the bacterium. The precise specificities of the OpcA and Opa proteins are, however, unknown. Here we use a fluorescence-based binding assay to show that both proteins bind to mono- and disaccharides with high affinity. Binding of saccharides caused a quench in the intrinsic fluorescence emission of both proteins, and mutation of selected Tyr residues within the external loop regions caused a substantial decrease in fluorescence. We suggest that the intrinsic fluorescence arises from resonance energy transfer from Tyr to Trp residues in the beta-barrel portion of the structure. OpcA bound sialic acid with a Kd of 0.31 microM and was shown to be specific for pyranose saccharides. The binding specificities of two different Opa proteins were compared; unlike OpcA, neither protein bound to monosaccharides, but both bound to maltose, lactose, and sialic acid-containing oligosaccharides, with Kd values in the micromolar range. OpaB had a 10-fold higher affinity for sialic acid-containing ligands than OpaD as a result of the mutation Y165V, which was shown to restore this specificity to OpaD. Finally, the OpcA- and Opa-dependent adhesion of meningococci to epithelial cells was shown to be partially inhibited by exogenously added sialic acid and maltose. The results show that OpcA and the Opa proteins can be thought of as outer membrane lectins and that simple saccharides can modulate their recognition of complex proteoglycan receptors.     

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.     

Moraxella catarrhalis stimulates the release of proinflammatory cytokines and prostaglandin E from human respiratory epithelial cells and monocyte-derived macrophages

The outer membrane proteins of Moraxella catarrhalis, a bacterial pathogen which causes disease in both children and adults, play an important role in its phenotypic properties. However, their proinflammatory potential with regard to respiratory epithelium and macrophages is unclear. To this end, we examined the cytokine- and mediator-inducing capacity of a heat-killed wild-type M. catarrhalis strain and a nonautoagglutinating mutant as well as their outer membrane proteins and secretory/excretory products using the A549 respiratory epithelial cell line. The outer membrane proteins and secretory/excretory products from both isolates as well as the heat-killed bacteria all induced interleukin (IL)-6, IL-8 and prostaglandin E2, but not IL-1beta, from the A549 cell line in a dose- and time-dependent manner. Heat-killed bacteria and secretory/excretory products stimulated the release of IL-1beta, IL-6, IL-8 and prostaglandin E2 from human monocyte-derived macrophages. Both heat-killed isolates also stimulated nuclear translocation and transactivation of nuclear factor-kappaB. The heat-killed wild-type autoagglutinating isolate induced significantly greater amounts of IL-6 and IL-8 from A549 cells than the nonautoagglutinating mutant compared with the monocyte-derived macrophages but no significant differences in the amounts induced by the two strains were observed. These differences were also evident when the respiratory cell line was stimulated with outer membrane proteins as well as in the degree of nuclear factor-kappaB transactivation. There was little difference in the stimulatory activity of the secretory/excretory products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses revealed some differences in the outer membrane proteins and secretory excretory products between the two isolates. Combined, these data show that M. catarrhalis secretory excretory products and outer membrane proteins are associated with the induction of inflammatory responses in both respiratory epithelium and macrophages.     

Branhamella catarrhalis: epidemiology, surface antigenic structure, and immune response.

Over the past decade, Branhamella catarrhalis has emerged as an important human pathogen. The bacterium is a common cause of otitis media in children and of lower respiratory tract infections in adults with chronic obstructive pulmonary disease. B. catarrhalis is exclusively a human pathogen. It colonizes the respiratory tract of a small proportion of adults and a larger proportion of children. Studies involving restriction enzyme analysis of genomic DNA show that colonization is a dynamic process, with the human host eliminating and acquiring new strains frequently. The surface of B. catarrhalis contains outer membrane proteins, lipooligosaccharide, and pili. The genes which encode several outer membrane proteins have been cloned, and some of these proteins are being studied as potential vaccine antigens. Analysis of the immune response has been limited by the lack of an adequate animal model of B. catarrhalis infection. New information regarding outer membrane structure should guide studies of the human immune response to B. catarrhalis. Immunoassays which specifically detect antibodies to determinants exposed on the bacterial surface will elucidate the most relevant immune response. The recognition of B. catarrhalis as an important human pathogen has stimulated research on the epidemiology and surface structures of the bacterium. Future studies to understand the mechanisms of infection and to elucidate the human immune response to infection hold promise of developing new methods to treat and prevent infections caused by B. catarrhalis.     

Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis.

Outer membrane fractions were prepared from 11 bacteria in the family Enterobacteriaceae: Escherichia coli serotypes O1K-, O4K2, O26K60, O75K-, and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumonia, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providencia stuartii. All strains studied were found to contain one non-peptidoglycan-bound, heat-modifiable outer membrane protein, and one or two peptidoglycan-associated major outer membrane proteins in the 27,000- to 40,000-dalton range. Crossed immunoelectrophoresis using sodium dodecyl sulfate-polyacarylamide gel electrophoresis for separation of the antigens in the first dimension of the procedure was shown to provide a useful model system for studying the antigenic relationships of the major outer membrane proteins in Enterobacteriaceae species. Peptidoglycan-bound major outer membrane proteins of all bacteria studied reacted with antiserum against the purified peptidogylcan-bound matrix protein I of E. coli O26K60 in this system. Non-peptidoglycan-associated proteins of all strains cross-reacted with protein II of E. coli O26K60 in both their unmodified and their heat-modified forms. These results indicate that the genes coding for the major outer membrane proteins in the family Enterobacteriaceae have been well enough conserved during the course of evolution to allow significant antigenic cross-reactivity between the corresponding proteins in different enterobacterial species.     

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.     

Biogenesis of beta-barrel membrane proteins of mitochondria

beta-Barrel membrane proteins have several important functions in outer membranes of Gram-negative bacteria and in the organelles of endosymbiotic origin, mitochondria and chloroplasts. The biogenesis of beta-barrel membrane proteins was, until recently, an unresolved process. A breakthrough was achieved when a specific pathway for the insertion of beta-barrel outer-membrane proteins was identified in both mitochondria and Gram-negative bacteria. The key component of this pathway is Tob55 (also known as Sam50) in mitochondria and Omp85 in bacteria, both beta-barrel membrane proteins themselves. Tob55 is part of the hetero-oligomeric TOB (topogenesis of mitochondrial outer-membrane beta-barrel proteins) or SAM (sorting and assembly of mitochondria) complex, which is present in the mitochondrial outer membrane. Tob55 belongs to an evolutionarily conserved protein family, the members of which are present in almost all eukaryotes and in Gram-negative bacteria and chloroplasts. Thus, is it emphasized that the insertion pathway of mitochondrial beta-barrel membrane proteins was conserved during evolution of mitochondria from endosymbiotic bacterial ancestors.     

Bacterial lipoproteins; biogenesis,sorting and quality control

Bacterial lipoproteins are a subset of membrane proteins localized on either leaflet of the lipid bilayer. These proteins are anchored to membranes through their N-terminal lipid moiety attached to a conserved Cys. Since the protein moiety of most lipoproteins is hydrophilic, they are expected to play various roles in a hydrophilic environment outside the cytoplasmic membrane. Gram-negative bacteria such as Escherichia coli possess an outer membrane, to which most lipoproteins are sorted. The Lol pathway plays a central role in the sorting of lipoproteins to the outer membrane after lipoprotein precursors are processed to mature forms in the cytoplasmic membrane. Most lipoproteins are anchored to the inner leaflet of the outer membrane with their protein moiety in the periplasm. However, recent studies indicated that some lipoproteins further undergo topology change in the outer membrane, and play critical roles in the biogenesis and quality control of the outer membrane.This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Acid sphingomyelinase is involved in CEACAM receptor-mediated phagocytosis of Neisseria gonorrhoeae

The interaction with human phagocytes is a hallmark of symptomatic Neisseria gonorrhoeae infections. Gonococcal outer membrane proteins of the Opa family induce the opsonin-independent uptake of the bacteria that relies on CEACAM receptors and an active signaling machinery of the phagocyte. Here, we show that CEACAM receptor-mediated phagocytosis of Opa(52)-expressing N. gonorrhoeae into human cells results in a rapid activation of the acid sphingomyelinase. Inhibition of this enzyme by imipramine or SR33557 abolishes opsonin-independent internalization without affecting bacterial adherence. Reconstitution of ceramide, the product of acid sphingomyelinase activity, in imipramine- or SR33557-treated cells restores internalization of the bacteria. Furthermore, we demonstrate that CEACAM receptor-initiated stimulation of other signalling molecules, in particular Src-like tyrosine kinases and Jun N-terminal kinases, requires acid sphingomyelinase. These studies provide evidence for a crucial role of the acid sphingomyelinase for CEACAM receptor-initiated signalling events and internalization of Opa(52)-expressing N. gonorrhoeae into human neutrophils.     

The Moraxella catarrhalis outer membrane protein CD contains two distinct domains specifying adherence to human lung cells

Most Moraxella catarrhalis isolates express a highly-conserved outer membrane protein of 453 residues designated OMPCD, which has been previously shown to mediate binding to A549 human lung cells. Here, it is reported that two distinct domains of the M. catarrhalis strain O35E OMPCD protein specify adherence. Truncated proteins were expressed in Escherichia coli to demonstrate that OMPCD residues 1-240 as well as 241-400 are important for attachment to A549 cells, and database searches indicated that amino acids 285-299 resemble an adhesive motif found in eukaryotic proteins termed thrombospondin-type 3 repeat (TT3R). Cellular enzyme-linked immunosorbent assay using His-tagged proteins demonstrated that residues 236-300 of OMPCD, containing the TT3R motif, specify adhesive properties. Furthermore, these assays revealed that a purified protein encompassing residues 16-236 binds to A549 cells. The two cell-binding domains of OMPCD were further defined to amino acids 16-150 and 261-300 by utilizing a surface-display system, which was constructed from the M. catarrhalis autotransporter protein McaP, to express foreign peptides on the surface of recombinant bacteria.