Gonococcal protein III. Purification and chemical characterization of the protein, and the DNA sequence of the structural gene

Gotschlich, Emil C.; Blake, M. S.; Lytton, E. J.; Seiff, Michael E.

doi:10.1007/bf00415502

Cited by 7 publications

(1 citation statement)

References 20 publications

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“…The extent of labeling was between 75 and 100%. Porin P.IB from N. gonorrheae strain MS11 was purified as a homogeneous preparation in a buffer containing 25 mM Tris-HCl, pH 8.0, 0.05% Zwittergent 3-14 (Calbiochem), and 200 mM NaCl according to Gotschlich et al (34). It was stored at concentrations not exceeding 1 mg/mL.…”

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Interaction of the Gonococcal Porin P.IB with G- and F-Actin

et al. 2000

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The invasion of epithelial cells by N. gonorrheae is accompanied by formation of a halo of actin filaments around the enveloped bacterium. The transfer of the bacterial major outer membrane protein, porin, to the host cell membrane during invasion makes it a candidate for a facilitator for the formation of this halo. Western analysis shows here that gonococcal porin P.IB associates with the actin cytoskeleton in infected cells. Using the pyrene-labeled Mg forms of yeast and muscle actins, we demonstrate that under low ionic strength conditions, P.IB causes formation of filamentous actin assemblies, although they, unlike F-actin, cannot be internally cross-linked with N,N'-4-phenylenedimaleimide (PDM). In F-buffer, low porin concentrations appear to accelerate actin polymerization. Higher P.IB concentrations lead to the formation of highly decorated fragmented F-actin-like filaments in which the actin can be cross-linked by PDM. Co-assembly of P.IB with a pyrene-labeled mutant actin, S(265)C, prevents formation of a pyrene excimer present with labeled S(265)C F-actin alone. Addition of low concentrations of porin to preformed F-actin results in sparsely decorated F-actin. Higher P.IB concentrations extensively decorate the filaments, thereby altering their morphology to a state like that observed when the components are copolymerized. With preformed labeled S(265)C F-actin, P.IB quenches the pyrene excimer. This decrease is prevented by the F-actin stabilizers phalloidin and to a lesser extent beryllium fluoride. P.IB's association with the actin cytoskeleton and its ability to interact with and remodel actin filaments support a direct role for porin in altering the host cell cytoskeleton during invasion.

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Interaction of the Gonococcal Porin P.IB with G- and F-Actin

et al. 2000

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Neisseria gonorrhoeae porin, P.IB, causes release of ATP from yeast actin

Wen

Blake

Rubenstein

2004

J Muscle Res Cell Motil

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Neisserial porins may play a role in the invasion of the host cell by the bacterium. The protein translocates to the host cell membrane and then to the cytosol during the invasive process, and we have shown it interacts with actin in vitro. Here, we have examined the nucleotide-dependence of the interaction of Neisseria porin, P.IB, with fluorescently labeled yeast G actin. Increasing free ATP between 0 to 0.5 mM retards complex formation between the two proteins. The ATP effect probably results from binding of the nucleotide to actin rather than to porin. Complex formation results in a biphasic release of bound nucleoside triphosphate from actin in the absence of free nucleotide at a rate slower than that of complex formation, but it does not induce hydrolysis of the actin-bound nucleotide. ATP prevents the porin-induced distortion of F-actin structure, and addition of ATP to the complex formed in the absence of free nucleotide induces actin polymerization indicating that P.IB stabilizes nucleotide-free G-actin. Our results suggest that P.IB causes an actin conformation change leading to the production of a polymerization-competent nucleotide-free protein.

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Immunobiology of purified recombinant outer membrane porin protein I of Neisseria gonorrhoeae

Elkins

Kb²,

et al. 1994

Molecular Microbiology

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Gonococcal porins (Por) from strains FA19 (Por-1, serogroup A), MS11 (Por-2, serogroup B) and FA6434 (Por-5, a hybrid porin containing epitopes from both serogroups), were expressed in Escherichia coli and purified under non-denaturing conditions. Porins were inserted into liposomes, and they were bound by monoclonal antibodies which bind native Por and intact gonococci, but not denatured Por. All three recombinant porins (rPor) were highly immunogenic in rabbits without additional adjuvant. The rPor antisera were specific for Por by Western blotting and whole-cell radioimmunoprecipitation and were broadly cross-reactive within serogroups. Post-immune, but not pre-immune, sera bound to intact gonococci, induced deposition of complement components C3 and C9 onto gonococcal membranes and increased association with and activation of human neutrophils. Gonococci were not killed in bactericidal assays, and there was no phagocytic killing with gonococci opsonized with recombinant antisera. Lack of killing in bactericidal assays was not caused by the presence of blocking antibodies to the outer-membrane protein Rmp.

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Gonococcal protein III. Purification and chemical characterization of the protein, and the DNA sequence of the structural gene

Cited by 7 publications

References 20 publications

Interaction of the Gonococcal Porin P.IB with G- and F-Actin

Interaction of the Gonococcal Porin P.IB with G- and F-Actin

Neisseria gonorrhoeae porin, P.IB, causes release of ATP from yeast actin

Immunobiology of purified recombinant outer membrane porin protein I of Neisseria gonorrhoeae

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