The surface properties of Neisseria gonorrhoeae: topographical distribution of the outer membrane protein antigens.

Gonococci were labelled with 125I using the lactoperoxidase system. The amount of label incorporated was similar with all strains including those which appeared capsulated. Electrophoresis on sodium dodecyl sulphate-polyacrylamide gels revealed that the major proteins labelled were those found in outer membrane preparations. Comparison of variants of one strain showed that the major outer membrane protein (protein I) was always present and heavily labelled. The second major protein (protein II) was present in variable amounts but labelling was proportional to the amount present. A third protein (III) was only present in outer membranes from a freshly isolated variant but was present in whole cells of each strain. Protein III was not labelled in whole cells but was labelled in outer membrane preparations suggesting that many membranes have their inner surface exposed. The labelling of a strain adapted to growth in guinea-pig chambers failed to reveal any new major surface proteins. The results demonstrate the variation in surface topography possible with variants of one strain of gonococcus but show that one major protein antigen is always expressed on the surface.     

Cross-linking analysis of Neisseria gonorrhoeae outer membrane proteins.

The arrangement of proteins in the outer membrane of Neisseria gonorrhoeae was investigated through the use of cleavable chemical cross-linking reagents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-linking of isolated outer membranes yielded dimers and trimers of the major outer membrane protein. In addition, data were obtained suggesting that a stable interaction exists between the major protein I and protein II, the second most prevalent protein in the gonococcal outer membrane.     

Expression of the Neisseria gonorrhoeae major outer membrane protein PI in Escherichia coli

Summary To actively express an outer membrane protein, protein I (PI), from different strains of Neisseria gonorrhoeae in E.␣coli, PI gene fragments from two reference strains and four clinical isolates of Neisseria gonorrhoeae were obtained with PCR amplification. They were cloned into the PCR cloning vector pBS-T to form pBS-T-PI and sequenced. Subsequently, they were cloned into an expression vector pET-30b (+) to generate pET-PI recombinants. After inducing with isopropyl-β-d-thiogalactopyranoside (IPTG), the expressed PI proteins were analysed by SDS-PAGE, Western blotting and ELISA. The results implied that we had successfully constructed the PI gene recombinants from both reference strains and clinical isolates and obtained the recombinant proteins expressed in E. coli at relatively high levels, and the expressed proteins had the immunological activity with the corresponding antibodies. This research will be very helpful for the further study of these proteins in generating preventive vaccines on Neisseria gonorrhoeae infection and clinical diagnosis.     

Structural comparison of Neisseria gonorrhoeae outer membrane proteins.

Outer membranes from opaque colonia variants of Neisseria gonorrhoeae P9 contain a major outer membrane protein (protein I) together with one or more of a series of heat-modifiable proteins (proteins II). Proteins I. II, and IIa have been isolated by detergent extraction of outer membranes. Amino acid analysis showed proteins II and IIa to have a very similar composition. Cyanogen bromide cleavage of proteins II and IIa produced a pair of fragments with identical molecular weight and a pari which differed by an amount (0.5K) equivalent to the difference between the intact proteins. Tryptic peptide maps of 125I-labeled proteins II, IIa, and IIb showed many similarities, with only a few peptides unique to any one protein. Peptide maps of protein IIa from cells which had been surface labeled showed that the unique peptides were exposed on the surface. The heat-modifiable proteins thus appear to form a family of proteins with closely related structure probably differing in that part which is exposed on the bacterial surface.     

A dominant sulfhydryl-containing protein in the outer membrane of Neisseria gonorrhoeae.

By using a method that labels sulfhydryl-containing proteins in situ, we have detected a major outer membrane protein of Neisseria gonorrhoeae at 41 kDa. A protein of this molecular mass has not previously been shown to be a major outer membrane protein in gonococci. In addition, a minor protein rich in cysteinyl residues was detected at 31.5 kDa.     

Isolation and characterization of the outer membrane of Neisseria gonorrhoeae

The cell envelope of Neisseria gonorrhoeae strain 2686, colonial type 4, was isolated from spheroplasts formed by the action of ethylenediaminetetraacetic acid and lysozyme. Isopycnic centrifugation of osmotically ruptured spheroplasts resolved the cell envelope into two main membrane fractions. Chemical and enzymatic analyses were used to characterize these isolated membranes. Succinic dehydrogenase, reduced nicotinamide adenine dinucleotide oxidase, and d-lactate dehydrogenase were localized in the membrane fraction of buoyant density, rho degrees = 1.141 g/cm(3). Lipopolysaccharide and over half of the cell envelope protein were associated with the membrane that banded in sucrose at rho degrees = 1.219 g/cm(3). These fractions were consequently designated cytoplasmic and outer or L-membrane, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis of isolated membranes demonstrated the relative simplicity of the protein spectrum of the outer membrane. The majority of the protein in this membrane could be accounted for by proteins of molecular weights 34,500, 22,000, and 11,500. The protein of molecular weight 34,500 accounted for 66% of the total protein of the L-membrane. Isoelectric precipitation at pH 4.6 with 10% acetic acid selectively removed this protein from a 150 mM NaCl in 10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4, extract of purified outer membrane. At pH 4.0, the other proteins of the L-membrane were precipitated. It was concluded that the membrane components of the cell envelope of N. gonorrhoeae were similar to those of other gram-negative bacteria. The cell envelope fractions described here, in particular the outer membrane, are sufficiently well defined to provide a valuable tool for future biochemical and immunological studies on N. gonorrhoeae.     

Purification of outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae

A system of protein purification, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, that results in purified outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae is described. The proteins, which ranged in apparent molecular mass from approximately 31,000 to approximately 92,000 Da, were located by naphthol blue black staining, eluted from nitrocellulose membranes using 88% formic acid, and precipitated by the addition of concentrated ammonium hydroxide. Up to 65% of the original protein present was recovered by this procedure. The resultant purified protein could then be resuspended in aqueous buffer by brief sonication, making it available for further structural and in vivo immunological analyses. Proteins purified in this manner retain their original antigenicity when probed with polyclonal and monoclonal antibodies, and are structurally unaltered by the purification process. This procedure makes it possible to acquire easily usable quantities of highly insoluble outer membrane proteins of gram-negative bacteria.     

Altered outer membrane protein in different colonial types of Neisseria gonorrhoeae.

Dark-colored colony types of Neisseria gonorrhoeae (T3 and dark variants of T1 and T2) had markedly increased amounts of an approximately 28,000-dalton outer membrane protein, as compared with light-colored colony types (T4 and light variants of T1 and T2). The presence of this protein appeared to be unrelated to piliation. The apparent molecular weight of this protein on sodium dodecyl sulfate-polyacrylamide gels varied, depending on methods used to solubilize envelope proteins. In view of the location of this protein on the outer membrane, this protein could be important to the pathogenicity or antigenicity of the organism as well as to colonial characteristics in vitro.     

The effect of iron starvation on the outer membrane protein composition of Neisseria gonorrhoeae

Abstract: A dipeptidase activity on ms -A₂pm- d -Ala and l -Lys- d -Ala was found in sporulating cells of Bacillus sphaericus 9602. The specific activity of this enzyme was low during the growth cycle and showed a rapid increase throughout sporulation. Values in the dormant spores were 1.3-fold higher than those found in cells late in sporulation and 20-fold higher than those found in log-phase cells.     

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

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 outermembrane protein Rmp.     

Genetic locus (nmp-1) affecting the principal outer membrane protein of Neisseria gonorrhoeae.

An increase in the apparent molecular weight of the principal outer membrane protein (POMP) of Neisseria gonorrhoeae is associated with introduction of the penB2 genetic marker, which results in low-level, relatively nonspecific antibiotic resistance. Limited proteolysis of the two forms of POMP showed that they had few if any peptides in common. The nonspecific antibiotic resistance of penB2 was separated from the change in POMP by genetic transformation and by isolation of spontaneous penB mutants that showed no change in POMP. The genetic locus involved in the change from one POMP to another, which we have designated nmp-1, is closely linked to, but not identical with, penB2.     

Acid stress upregulated outer membrane proteins in clinical isolates of Neisseria gonorrhoeae, but not most commensal Neisseria.

Human immune serum recognition of outer membrane components from commensal and pathogenic Neisseria cultured under neutral and acidic conditions was investigated. Acid stress caused no detectable alterations in lipooligosaccharide migration and (or) staining, in outer membrane protein profiles, or in immune serum recognition of outer membrane components from Neisseria mucosa or Neisseria sicca. There was also no difference in the lipoologosaccharide electrophoretic pattern of acid- and neutral-grown Neisseria lactamica, but there were differences in outer membrane protein expression. The outer membrane protein alterations induced by acid stress in N. lactamica were not the same as those seen in isolates from patients with uncomplicated gonococcal infection, pelvic inflammatory disease, and disseminated gonococcal infection. Many differences were detected in the immune serum recognition of outer membrane components from acid- and neutral-cultured N. lactamica and from the clinical isolates of Neisseria gonorrhoeae, and these should be considered in vaccine design.     

Cloning, sequencing, and characterization of the gene encoding FrpB, a major iron-regulated, outer membrane protein of Neisseria gonorrhoeae.

FrpB (for Fe-regulated protein B) is a 76-kDa outer membrane protein that is part of the iron regulon of Neisseria gonorrhoeae and Neisseria meningitidis. The frpB gene from gonococcal strain FA19 was cloned and sequenced. FrpB was homologous to several TonB-dependent outer membrane receptors of Escherichia coli as well as HemR of Yersinia enterocolitica and CopB of Moraxella catarrhalis. An omga insertion into the frpB coding sequence caused a 60% reduction in 55Fe uptake from heme, but careful analysis suggested that this effect was nonspecific. While FrpB was related to the family of TonB-dependent proteins, a function in iron uptake could not be documented.     

Comparative proteome analysis of the outer membrane proteins of in vitro-induced multi-drug resistant Neisseria gonorrhoeae

Antimicrobial-resistant gonococcus has been a major problem in sexually transmitted disease control . Outer membrane proteins (OMPs) of Neisseria gonorrhoeae were suggested to have influence on its resistance to antibiotics. So, in this work, we provide a proteomic analysis tool for examining the OMPs of N. gonorrhoeae and also provide a comparative analysis of the OMPs between the susceptible parent strain (92WT) and the resistance-induced isogenic mutant (92mu13) to determine the OMPs responsible for resistance. The 2-D gel spots of 92mu13 differed from 92WT particularly in porin, pilus secretion protein (PilQ) and enzymes. PilQ expression in 92mu13 was considerably reduced by abrupt termination at nucleotide 2,112. This made it difficult to form a high molecular mass (HMM) pore at the outer membrane; it is suspected that reduction of PilQ serves a role in antibiotic resistance in N. gonorrhoeae. The amount of porin was not changed but its isoelectric point (pI) shifted to a basic region, which is caused by the alteration of an amino acid of porin and it is suggested to relate to the development of antimicrobial resistance . Differential regulation of the enzymes involved in metabolism was found in 92mu13, believed to represent an adaptation of N. gonorrhoeae to the antibiotic environment.     

Five structural classes of major outer membrane proteins in Neisseria meningitidis

Group B Neisseria meningitidis is thus far subdivided into 15 protein serotypes based on antigenically different major outer membrane proteins. Most serotypes have three or four major proteins in their outer membranes. Comparative structural analysis by chymotryptic 125I-peptide mapping was performed on these major proteins from the prototype strains as well as from six non-serotypable strains. The major outer membrane proteins from each of the serotypes were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the Laemmli system. Individual proteins within the gel slices were radioiodinated and digested with chymotrypsin, and then their 125I-peptides were separated by electrophoresis and chromatography on cellulose thin-layer plates. The peptide maps obtained by autoradiography were categorized into five different structural classes which correlated with the apparent molecular weights of proteins, i.e., 46 +/- 1K, 41 +/- 1K, 38 +/- 1K, 33 +/- 1K, and 28 +/- 1K. Each of the major outer membrane proteins within a strain had a distinctly different chymotryptic peptide map, indicating significant differences in the primary structure of these proteins. In contrast, outer membrane proteins of the same or very similar molecular weight from different serotype strains had similar, occasionally identical peptide maps, indicating a high degree of structural homology. The unique peptides from proteins of the same structural classes were often hydrophilic, whereas common peptides were often hydrophobic, suggesting that the serotype determinants reside within the variable hydrophilic regions of major outer membrane proteins.