Porin PorB Research Articles

Serological correlates of protection against meningococci in a cohort of university students, before and during an outbreak of serogroup C infection.

The association between individual meningococcal antigens and the development of protective immunity to both serogroup C and B meningococci was studied before and during an outbreak of serogroup C infection among university students. Persons who became infected showed, in serum taken either before infection or on admission to the hospital, low levels of bactericidal activity against the outbreak strain; patients who survived infection developed bactericidal activity that correlated with production of antibodies to serogroup C capsular polysaccharide but not to either lipopolysaccharide or major outer-membrane proteins. Uninfected classmates also showed a strong correlation between bactericidal activity and the presence of anti-capsular antibodies. In contrast, bactericidal activity against serogroup B did not correlate with the presence of antibodies to capsular polysaccharide but did correlate with antibodies reacting with the porin proteins PorA and PorB. These studies support the introduction of conjugate MenC vaccines, validate strategies for prevention of serogroup B infection that are based on vaccines containing PorA, and suggest that PorB may also be an important component of such vaccines.

Neisserial PorB is translocated to the mitochondria of HeLa cells infected with Neisseria meningitidis and protects cells from apoptosis.

We have previously shown that purified meningococcal porin PorB associates with mitochondria and prevents apoptosis of B cells, Jurkat cells and HeLa cells (Massari et al., 2000, Proc Natl Acad Sci USA 97: 9070-9075). This work examines if intact meningococci have a similar effect as purified porins. It was first determined that intact live meningococci do not induce apoptosis of HeLa cells and do not perturb mitochondrial physiology. This latter consideration is important as Neisserial porins affect the susceptibility of cells to apoptosis by preventing mitochondrial depolarization and cytochrome c release, events involved in the apoptosis cascade. Purified PorB or PorB from live bacteria were found to translocate into and interact with mitochondria. It was then determined whether treatment of HeLa cells with meningococci could prevent staurosporine-mediated apoptosis due to an effect of PorB on the mitochondrial parameters. Incubation of HeLa cells with live meningococci prevented staurosporine-induced apoptosis, as ascertained by measurements of mitochondrial potential, translocation of mitochondrial cytochrome c to the cytosol, caspases activation, and nuclear DNA degradation. These data are consistent with our previous findings that purified PorB associates with mitochondria and prevents apoptosis, and demonstrates that the mechanism by which whole meningococci protects cells from apoptosis is a result of direct interaction of neisserial porin with mitochondria.

Sequential immunization with vesicles prepared from heterologous Neisseria meningitidis strains elicits broadly protective serum antibodies to group B strains.

The capsular polysaccharide of Neisseria meningitidis group B is an autoantigen, whereas noncapsular antigens are highly variable. These factors present formidable challenges for development of a broadly protective and safe group B vaccine. Mice and guinea pigs were sequentially immunized with three doses of micovesicles or outer membrane vesicles prepared from three meningococcal strains that were each antigenically heterologous with respect to the two major porin proteins, PorA and PorB, and the group capsular polysaccharide. The resulting antisera conferred passive protection against meningococcal group B bacteremia in infant rats and elicited complement-mediated bactericidal activity against genetically diverse group B strains that were either homologous or heterologous with respect to PorA of the strains used to prepare the vaccine. By using knockout strains, a portion of the bactericidal antibody was directed against the highly conserved protein, neisserial surface protein A (NspA). Further, an anti-NspA monoclonal antibody elicited by the sequential immunization was highly bactericidal against strains that were previously shown to be resistant to bacteriolysis by anti-NspA antibodies produced by immunization with recombinant NspA. Sequential immunization with heterologous vesicle preparations offers a novel approach to eliciting broadly protective immunity against N. meningitidis strains. An NspA-based vaccine prepared from protein expressed by Neisseria also may be more effective than the corresponding recombinant protein made in Escherichia coli.

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.

Neisseria meningitidisporin PorB interacts with mitochondria and protects cells from apoptosis

Neisserial porins are strong immune adjuvants and B cell activators. The effect of neisserial porin PorB on activation-induced cell death was investigated, as a potential additional mechanism of the porin's immunopotentiating ability. Neisserial porins interact with target cells to localize intracellularly in the mitochondrial compartment without negatively affecting cellular survival. Pretreatment with Neisseria meningitidis PorB porin decreased or abrogated the mitochondrial damage induced by apoptotic stimuli. In addition, end stage determinants of apoptosis, including DNA breakdown, were diminished by PorB. Immunoprecipitation experiments revealed that PorB interacts with the mitochondrial porin VDAC (voltage-dependent anion channel). The mechanism of the antiapoptotic effect of neisserial porins could be explained by the protein-protein interaction of PorB with VDAC, similar to the interaction of VDAC with antiapoptotic Bcl-2 proteins, resulting in an enhancement of cell survival and continued activation of B cells.

Mutagenesis of the Neisseria gonorrhoeae porin reduces invasion in epithelial cells and enhances phagocyte responsiveness.

Porin (PorB), the major outer membrane protein of Neisseria gonorrhoeae, has been implicated in pathogenesis previously. However, the fact that porin deletion mutants are not viable has complicated investigations. Here, we describe a method of manipulating the porin gene site-specifically. N. gonorrhoeae MS11, which harbours the porB1B (P.1B) porin allele, was used to generate mutants carrying deletions in the surface loops 1 and 5. An 11-amino-acid deletion in loop 1 impaired Opa50-dependent invasion into human Chang epithelial cells, whereas loop 5 deletion exhibited no apparent phenotype. In a second approach, the complete gonococcal porB1B was replaced by the porBNia gene of Neisseria lactamica. Such mutants were unable to induce efficient uptake by epithelial cells but induced an enhanced respiratory response in HL60 phagocytic cells. The increased respiratory burst was accompanied by an enhanced phagocytic uptake of the mutant compared with the wild-type strain. Our data extend previous evidence for multiple central functions of PorB in the infection process.

Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases.

The porin (PorB) of Neisseria gonorrhoeae is an intriguing bacterial factor owing to its ability to translocate from the outer bacterial membrane into host cell membranes where it modulates the infection process. Here we report on the induction of programmed cell death after prolonged infection of epithelial cells with pathogenic Neisseria species. The underlying mechanism we propose includes translocation of the porin, a transient increase in cytosolic Ca2+ and subsequent activation of the Ca2+ dependent protease calpain as well as proteases of the caspase family. Blocking the porin channel by ATP eliminates the Ca2+ signal and also abolishes its pro-apoptotic function. The neisserial porins share structural and functional homologies with the mitochondrial voltage-dependent anion channels (VDAC). The neisserial porin may be an analogue or precursor of the ancient permeability transition pore, the putative central regulator of apoptosis.

Neisseria gonorrhoeae Porin Modulates Phagosome Maturation

The porin (PorB) of Neisseria gonorrhoeae has been implicated in the pathogenesis of this species. Porin is believed to translocate from the bacterial outer membrane into target cell membranes affecting various cell functions. Here we investigated the effect of porin on phagosome maturation. Phagocytosis of latex beads by human macrophages was allowed in the presence or absence of purified porin. Isolation of latex bead-containing phagosomes and subsequent two-dimensional gel electrophoresis revealed substantial differences in the phagosomal protein composition. Immunoblotting detected higher amounts of annexin II and the early endocytic markers Rab5 and transferrin receptor and decreased levels of the late endocytic markers Rab7 and cathepsin D in phagosomes obtained in the presence of porin compared with those obtained in its absence. Furthermore, association of Rab4 with the latex bead-containing phagosomes was revealed by flow cytometry. The amount of this small GTPase was markedly higher in the phagosomes isolated in the presence of porin. The data thus indicate that neisserial porin is itself able to arrest phagosome maturation within macrophages.

Modulation of Neisseria Porin (PorB) by Cytosolic ATP/GTP of Target Cells: Parallels between Pathogen Accommodation and Mitochondrial Endosymbiosis

PorB of the pathogenic Neisseria species belongs to the large family of pore-forming proteins (porins) produced by gram-negative bacteria. PorB is exceptional in that it is capable of translocating vectorially into membranes of infected target cells and functions in the infection process. Here we report on an unexpected similarity between Neisserial PorB and mitochondrial porins. Both porin classes interact with purine nucleoside triphosphates, which down-regulate pore size and cause a shift in voltage dependence and ion selectivity. Patch-clamp analyses indicate that PorB channel activity is tightly regulated in intact epithelial cells. In light of recent findings on the pivotal role of PorB in virulence and the prevention of phagosome lysosome fusion, these data provide important mechanistic clues on the intracellular pathogen accommodation reminiscent of mitochondrial endosymbiosis.