The pilus and porin of Neisseria gonorrhoeae cooperatively induce Ca(2+) transients in infected epithelial cells

Purified pili and porin from Neisseria quickly mobilize calcium (Ca(2+)) stores in monocytes and epithelial cells, ultimately influencing host cell viability as well as bacterial intracellular survival. Here, we examined the Ca(2+) transients induced in human epithelial cells during infection by live, piliated N. gonorrhoeae. Porin induced an influx of Ca(2+) from the extracellular medium less than 60 s post infection. The porin-induced transient is followed by a pilus-induced release of Ca(2+) from intracellular stores. The timing of these events is similar to that observed using purified proteins. Interestingly, the porin-induced Ca(2+) flux is required for the pilus-induced transient, indicating that the pilus-induced Ca(2+) release is, itself, Ca(2+) dependent. Several lines of evidence indicate that porin is present on pili. Moreover, pilus retraction strongly influences the porin- and pilus-induced Ca(2+) fluxes. These and other results strongly suggest that the pilus and porin cooperate to modulate calcium signalling in epithelial cells, and propose a model to explain how N. gonorrhoeae triggers Ca(2+) transients in the initial stages of pilus-mediated attachment.     

Neisserial porin-induced dendritic cell activation is MyD88 and TLR2 dependent

Neisserial porins have been shown to act as B cell mitogens and immune adjuvants. PorA and PorB are the major outer membrane porin proteins of the human pathogen Neisseria meningitidis. We have shown that the mechanism of the immunopotentiating capability of porin involves up-regulation of the T cell costimulatory ligand, CD86. Due to neisserial porin's ability to activate B cells and potentiate immune responses, we hypothesized that porin also employs the potent immune stimulatory function of dendritic cells (DC). We examined the ability of purified N. meningitidis PorB to induce maturation of murine splenic and bone marrow-derived DC. PorB treatment induced DC maturation, as demonstrated by increased expression of CD86 and class I and II MHC molecules. In addition, PorB not only enhanced the allostimulatory activity of DC, but also augmented the ability of DC to stimulate T cells in an Ag-specific manner. PorB-matured DC secreted the inflammatory cytokine IL-6, which may have implications for the adjuvant property of porin. Induction of IL-6 by PorB is also significant because IL-6 is one of a number of cytokines produced during infection with N. meningitidis and may be involved in the inflammatory process observed during infection and disease. We previously demonstrated the requirement of MyD88 and TLR2 for PorB-induced B cell activation. In the present study, MyD88 and TLR2 were also essential for PorB-induced DC activation. This work is significant for elucidating the mechanism(s) of neisserial porin's immune stimulatory activity.     

Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria gonorrhoeae to mitochondria of infected cells

Infection of cell cultures with Neisseria gonorrhoeae results in apoptosis that is mediated by the PorB porin. During the infection process porin translocates from the outer bacterial membrane into host cell membranes where its channel activity is regulated by nucleotide binding and voltage-dependent gating, features that are shared by the mitochondrial voltage-dependent anion channel (VDAC). Here we show that porin is selectively and efficiently transported to mitochondria of infected cells. Prevention of porin translocation also blocked the induction of apoptosis. Mitochondria of cells treated with porin both in vitro and in vivo were depleted of cytochrome c and underwent permeability transition. Overexpression of Bcl-2 blocked porin-induced apoptosis. The release of cytochrome c occurred independently of active caspases but was completely prevented by Bcl-2. Our data suggest that the Neisseria porin can, like its eukaryotic homologue, function at the mitochondrial checkpoint to mediate apoptosis.     

Sequence analysis and relationships between meningococcal class 3 serotype proteins and other porins from pathogenic and non-pathogenic Neisseria species

The presence of highly conserved regions within previously determined porin gene sequences from Neisseria meningitidis and Neisseria gonorrhoeae permitted the construction of oligonucleotide primers for PCR amplification of other neisserial porin genes. Although two separate porin genes (porA and porB) are present in N. meningitidis only a single fragment, corresponding to porB, could be amplified from this species. The amplified porB genes from four different meningococcal serotypes, which express the class 3 outer membrane protein, were sequenced. Amplified fragments corresponding to porin genes from N. lactamica and N. sicca were also sequenced. In common with the known neisserial porins, models of the organisation of the predicted proteins indicated trans-membrane structures with eight surface exposed loops. In the meningococcal class 3 proteins the main regions of sequence variation, which must be responsible for serotype specificity, were located on loops 5 and 7. A phylogenetic analysis of the family of porins from the Neisseria confirmed the close relationship of the meningococcal class 3 protein with the gonococcal PIA protein, while the gonococcal PIB protein was shown to be closely related to the N. lactamica porin. The close relationship seen between porins of the pathogenic and non-pathogenic Neisseriae identified no obvious virulence-associated regions in the proteins, but did suggest that the current nomenclature for neisserial porin genes may need reviewing.     

Class-3 porin protein of Neisseria meningitidis: cloning and structure of the gene.

The gene coding for the class-3 protein of Neisseria meningitidis was cloned and sequenced. The deduced amino acid (aa) sequence was highly homologous (50-78%) to those of other neisserial porin proteins. Alignment of the aa sequence of five neisserial porin proteins pinpointed several regions of identity or near identity. These are assumed to be membrane-spanning beta-strands. A comparison of the homologies between these neisserial porins showed that the class-3 protein is most closely related to the Neisseria gonorrhoeae P1A protein.     

Mass tag-assisted identification of naturally processed HLA class II-presented meningococcal peptides recognized by CD4+ T lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.     

Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨ_m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨ_m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨ_m loss and apoptosis, demonstrating that dissipation of ΔΨ_m is a requirement for cell death caused by neisserial infection.     

Improved purification of native meningococcal porin PorB and studies on its structure/function

The outer membrane protein PorB of Neisseria meningitidis is a pore-forming protein which has various effects on eukaryotic cells. It has been shown to (1) up-regulate the surface expression of the co-stimulatory molecule CD86 and of MHC class II (which are TLR2/MyD88 dependent and related to the porin's immune-potentiating ability), (2) be involved in prevention of apoptosis by modulating the mitochondrial membrane potential, and (3) form pores in eukaryotic cells. As an outer membrane protein, its native trimeric form isolation is complicated by its insoluble nature, requiring the presence of detergent throughout the whole procedure, and by its tight association with other outer membrane components, such as neisserial LOS or lipoproteins. In this study, an improved chromatographic purification method to obtain an homogeneous product free of endotoxin and lipoprotein is described, without loss of any of the above-mentioned properties of the porin. Furthermore, we have investigated the requirement of the native trimeric structure for the porin's activity. Inactivation of functional PorB trimers into non-functional monomers was achieved by incubation on ice. Thus, routine long- and medium-term storage at low temperature may be a cause of porin inactivation.     

The role of porins in neisserial pathogenesis and immunity

Neisseria meningitidis and Neisseria gonorrhoeae are Gram-negative pathogenic bacteria responsible for bacterial meningitis and septicemia, and the sexually transmitted disease gonorrhea, respectively. Porins are the most represented outer membrane proteins in the pathogenic Neisseria species, functioning as pores for the exchange of ions, and are characterized by a trimeric beta-barrel structure. Neisserial porins have been shown to act as adjuvants in the immune response via activation of B cells and other antigen-presenting cells (APCs). Their effect on the immune response is mediated by upregulation of the costimulatory molecule B7-2 (CD86) on the surface of APCs, an effect that is Toll-like receptor 2- and MyD88-dependent. The effect of neisserial porins on the immune system also involves interaction with components of the complement cascade. Furthermore, neisserial porins co-localize with mitochondria of target cells, where they appear to modulate apoptosis.     

Identification and characterization of a cross-reactive and a unique B-cell epitope on the hsp60 homologue from Neisseria gonorrhoeae

Two monoclonal antibodies (G6 and 7B), generated against a 63-kDa stress protein (GSP63) from Neisseria gonorrhoeae strain VP1, were used to investigate the antigenic heterogeneity of GSP63 among the Neisseriaceae and its antigenic relationship with the Hsp60 heat-shock protein family. Immunoblotting experiments demonstrated antibody reactivity with all pathogenic Neisseria tested and with some of the commensal strains. One of the antibodies (7B) cross-reacted with the 65-kDa M. bovis BCG heat-shock protein and with 14 out of the 21 similarly sized proteins in other bacterial species. The other antibody (G6) specifically recognized neisserial GSP63 homologues. These results demonstrate that GSP63 is a conserved neisserial antigen bearing both a unique neisserial B-cell epitope and a more widely distributed Hsp60 epitope.     

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.     

The Neisseria type 2 IgA1 protease cleaves LAMP1 and promotes survival of bacteria within epithelial cells

Infection of human epithelial cells by Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) increases the rate of degradation of LAMP1, a major integral membrane glycoprotein of late endosomes and lysosomes. Several lines of evidence indicate that the neisserial IgA1 protease is directly responsible for this LAMP1 degradation. LAMP1 contains an IgA1-like hinge region with potential cleavage sites for the neisserial type 1 and type 2 IgA1 proteases. Neisserial type 2 IgA1 protease cleaves purified LAMP1 in vitro . Unlike its wild-type isogenic parent, an iga ⁻ mutant of N. gonorrhoeae cannot affect LAMP1 turnover and its growth in epithelial cells is dramatically reduced. Thus, IgA1 protease cleavage of LAMP1 promotes intracellular survival of pathogenic Neisseria spp.     

Host glycoprotein Gp96 and scavenger receptor SREC interact with PorB of disseminating Neisseria gonorrhoeae in an epithelial invasion pathway

Neisseria gonorrhoeae expresses numerous surface proteins that mediate bacterial adherence and invasion during infection. Gonococci expressing serotype A of the major outer membrane porin PorB (PorB(IA)) are frequently isolated from patients with severe disseminating infections. PorB(IA) triggers efficient adherence and invasion under low phosphate conditions mimicking systemic bloodstream infections. Here, we identify the human heat shock glycoprotein Gp96 and the scavenger receptor SREC as PorB(IA)-specific receptors. Gonococci expressing PorB(IA), but not those expressing PorB serotype B instead, bind to purified native or recombinant Gp96. Depletion of Gp96 from host cells prevented adherence but significantly triggered gonococcal invasion. Furthermore, such invasion was blocked by chemical inhibitors of scavenger receptors, and we identified SREC as the scavenger receptor involved in PorB(IA)-dependent invasion. Thus, we establish Gp96 as an anti-invasion factor and SRECs as receptors mediating host cell entry of highly invasive disseminating gonococci.     

Calcium signalling during cell interactions with bacterial pathogens

The ability of a pathogenic microorganism to cause a disease is conditioned by its ability to colonise a given niche and implicates the expression of specific determinants, i.e. virulence factors, that allow the pathogen to adhere to or to invade epithelial cells. Diseases may be induced by bacteria that replicate extracellularly and alter the epithelial mucosa by producing toxins. Ca2+ signalling has been implicated in various steps of bacterial infection. Bacterial toxins can induce an increase in free cytosolic Ca2+ in host cells, itself required for the toxin-mediated effects. Such toxins, by diffusing in the extracellular media, can act at a distance from the site of infection and have a global effect on the integrity of the epithelium by promoting the expression of pro-inflammatory cytokines. Independent on toxins, bacteria can induce Ca2+ responses that play a role in cytoskeletal rearrangements required for cell binding or internalisation of the microorganism. In some instances, invasion of the epithelium may be followed by bacterial access to deeper tissue, dissemination to other organs, and sometimes persistence in host cells in a parasitic-like mode. Such strategies underline the pathogen abilities to control innate defence cells such as professional phagocytes, and may implicate the diversion of Ca(2+)-dependent cellular processes that normally result in killing of the ingested bacteria. Finally, bacterial pathogens can also induce the cell release of ATP, a Ca2+ agonist, that may expand bacterial cell signalling by a paracrine or autocrine route, leading to enhanced colonisation or enhanced host cell responses to the invading microorganism.     

Pathophysiological changes of gram-negative bacterial infection can be reproduced by a synthetic peptide mimicking loop L7 sequence of Haemophilus influenzae porin

Several in vivo models have been used to dissect the molecular mechanisms that contribute to activate the coagulation and fibrinolytic systems by bacteria and bacterial products but many aspects remain poorly understood. In this study we examined the in vivo effect of the synthetic peptide corresponding to loop L7 from Haemophilus influenzae type b (Hib) porin to evaluate its role on the coagulative/fibrinolytic cascade and the circulating markers of endothelial injury. Plasma was obtained from rats injected intravenously with loop L7, Hib porin or a scrambled peptide and tested for fragment 1+2 (F1+2), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type I (PAI-1) antigen, von Willebrand factor (vWF) and soluble E-selectin (sE-selectin). The coagulative/fibrinolytic cascade was impaired as shown by PAI-1 level increased. Concomitantly, E-selectin, a marker of endothelial injury, was also significantly elevated. In addition either loop L7 or Hib porin injection induced hyperglycaemia and inflammatory cytokine production. The data were correlated with hemodynamic functions. The results indicate that loop L7 plays an essential role in the pathophysiologic events observed during gram-negative infection. These findings may have implications for the development of alternative therapies to counteract excessive inflammatory responses during septic shock.     

Arrested maturation of Neisseria-containing phagosomes in the absence of the lysosome-associated membrane proteins, LAMP-1 and LAMP-2

Mature, microbicidal phagosomes are rich in the lysosome-associated membrane proteins, LAMP-1 and LAMP-2, two highly glycosylated proteins presumed to form a protective barrier lining the phagosomal membrane. Pathogenic Neisseria secrete a protease that selectively cleaves LAMP-1, suggesting a critical role for LAMP proteins in the microbicidal competence of phagosomes. To determine the requirement for LAMP proteins in bacterial phagocytosis, we employed embryonic fibroblasts isolated from knockout mice lacking lamp-1, lamp-2 or both genes, as well as small interfering RNA (siRNA)-mediated knockdown of LAMP expression in a human epithelial cell line. Like wild-type cells, those lacking either LAMP-1 or LAMP-2 alone formed phagosomes that gradually acquired microbicidal activity and curtailed bacterial growth. In contrast, LAMP-1 and LAMP-2 double-deficient fibroblasts failed to kill engulfed Neisseria gonorrhoeae. In these cells, maturation was arrested prior to the acquisition of Rab7. As a result, the Rab7-interacting lysosomal protein (RILP, a Rab7 effector) was not recruited to the phagosomes, which were consequently unable to undergo dynein/dynactin-mediated centripetal displacement along microtubules and remained in a predominantly peripheral location. The inability of such phagosomes to migrate towards lysosomes likely contributed to their incomplete maturation and inability to eliminate bacteria. These findings suggest that neisserial degradation of LAMP-1 is not sufficient to affect its survival within the phagosome, and establish LAMP proteins as critical components in the process whereby phagosomes acquire microbicidal capabilities.     

Binding of the complement inhibitor C4bp to serogroup B Neisseria meningitidis

Neisseria meningitidis (meningococcus) is an important cause of meningitis and sepsis. Currently, there is no effective vaccine against serogroup B meningococcal infection. Host defense against neisseriae requires the complement system (C) as indicated by the fact that individuals deficient in properdin or late C components (C6-9) have an increased susceptibility to recurrent neisserial infections. Because the classical pathway (CP) is required to initiate efficient complement activation on neisseriae, meningococci should be able to evade it to cause disease. To test this hypothesis, we studied the interactions of meningococci with the major CP inhibitor C4b-binding protein (C4bp). We tested C4bp binding to wild-type group B meningococcus strain (H44/76) and to 11 isogenic mutants thereof that differed in capsule expression, lipo-oligosaccharide sialylation, and/or expression of either porin (Por) A or PorB3. All strains expressing PorA bound radiolabeled C4bp, whereas the strains lacking PorA bound significantly less C4bp. Increased binding was observed under hypotonic conditions. Deleting PorB3 did not influence C4bp binding, but the presence of polysialic acid capsule reduced C4bp binding by 50%. Bound C4bp remained functionally active in that it promoted the inactivation of C4b by factor I. PorA-expressing strains were also more resistant to C lysis than PorA-negative strains in a serum bactericidal assay. Binding of C4bp thus helps Neisseria meningitidis to escape CP complement activation.     

Pore formation and mitogenicity in blood cells by the class 2 protein of Neisseria meningitidis.

The class 2 outer membrane protein (MIEP) of Neisseria meningitidis has recently been shown to be mitogenic for lymphocytes (Liu, M.A., Friedman, A., Tai, J., Martinez, D., Deck, R. R., Hawe, L. A., Shieh, J. T.-C., Jenkins, T. D., Donnelly, J. J., and Oliff, A. I. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4633-4637. In this study, a possible connection between MIEP's mitogenicity and its possible action as a porin was investigated. MIEP, purified from the bacterial outer membrane protein complex under denaturing conditions, caused a modest but specific release of ions (86Rb) from both erythrocytes and lymphocytes, ultimately resulting in cell lysis. The dose-response of MIEP on erythrocyte lysis was qualitatively similar to a known porin (protein I from Neisseria gonorrhoeae) but was much less efficient. Induction or preservation of native structure in MIEP increased pore formation, resulting in levels comparable to that of the protein I porin. These observations suggest that native MIEP, free of the other outer membrane proteins of Neisseria meningitidis, can efficiently form pores in cells, but that denatured MIEP is variably and marginally effective. However, pore formation by MIEP was not related to its mitogenicity in lymphocytes, based on: (i) native MIEP was not mitogenic; (ii) denatured MIEP was highly mitogenic; and (iii) denatured MIEP was mitogenic at concentrations below the threshold level for pore formation. Therefore, mitogenicity is dependent upon MIEP being in a denatured, monomeric state and is masked by native conformation.