Cultured Rat and Purified Human Pneumocystis carinii Stimulate Intra-but not Extracellular Free Radical Production in Human Neutrophils

The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation. It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils. 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intra-cellular response of superoxide production, and 3) opsonized P. carinii. purified from human lung also stimulated human neutrophils to produce intracellular free radicals.     

The Obligate Human Pathogen, Neisseria gonorrhoeae, Is Polyploid

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.     

Sensitivity of Neisseria gonorrhoeae to antibiotics]

Since 1976 strains of Neisseria gonorrhoeae producing beta-lactamase have been isolated in many countries. Strains with high level resistance to tetracycline have been also described. The appearance of resistant strains implies a constant surveillance of the isolated gonococcus and the study of their antibiotics sensitivity.     

Generation of Human CEACAM1 Transgenic Mice and Binding of Neisseria Opa Protein to Their Neutrophils

Background

Human CEACAM1 is a cell-cell adhesion molecule with multiple functions including insulin clearance in the liver, vasculogenesis in endothelial cells, lumen formation in the mammary gland, and binding of certain human pathogens.

Principal Findings

Three genomic BAC clones containing the human CEACAM1 gene were microinjected into pronuclei of fertilized FVB mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by PCR for the presence of the human CEACAM1 gene. Feces of the PCR positive offspring screened for expression of human CEACAM1. Using this assay, one out of five PCR positive lines was positive for human CEACAM1 expression and showed stable transmission to the F1 generation with the expected transmission frequency (0.5) for heterozygotes. Liver, lung, intestine, kidney, mammary gland, and prostate were strongly positive for the dual expression of both murine and human CEACAM1 and mimic that seen in human tissue. Peripheral blood and bone marrow granulocytes stained strongly for human CEACAM1 and bound Neisseria Opa proteins similar to that in human neutrophils.

Conclusion

These transgenic animals may serve as a model for the binding of human pathogens to human CEACAM1.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

Ribosomal Resistance to Streptomycin and Spectinomycin in Neisseria gonorrhoeae

A cell-free protein synthesizing system was used to study the mechanism of resistance to streptomycin (Str) and spectinomycin (Spc) in laboratory mutants and clinical isolates of Neisseria gonorrhoeae. The 70S ribosomes from sensitive strains were sensitive to the effects of Str and Spc on synthesis directed by several synthetic polynucleotide messengers, whereas 70S ribosomes from resistant strains were resistant to these same effects. In each case, the alteration was localized to the 30S ribosomal subunit by studying antibiotic sensitivities of hybrid 70S ribosomes formed by combining subunits from sensitive and resistant strains. No evidence was found for streptomycin- or spectinomycin-inactivating enzymes.     

Cultivation and Survival Studies of Neisseria gonorrhoeae in a Human Diploid Cell Strain

Neisseria gonorrhoeae was cultivated in a human diploid cell strain (WI-38). Eighty percent of the cultures contained viable gonococci for at least 4 m at 36°C, as evidenced by subculture to brain heart infusion broth. Monthly subcultures of bacteria could be made to fresh WI-38 cultures for at least 11 monthly passages with a 69% survival rate. The identity of gonococci was confirmed by morphology, gram staining, oxidase testing and fermentation reactions. Viability in brain heart infusion broth, minimum essential medium (Eagle), and WI-38 spent fluid was of much shorter duration. The organisms grown in WI-38 cultures appeared to orient largely in the vicinity of the WI-38 cells as well as within the cytoplasm of the cells.     

Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance

Sparling, Philip F. (Communicable Disease Center, Atlanta, Ga.). Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J. Bacteriol. 92:1364-1371. 1966.-Eight strains of Neisseria gonorrhoeae were transformed to streptomycin resistance by deoxyribonucleic acid (DNA) extracted from a streptomycin-resistant strain of N. gonorrhoeae. In all strains, competence was greatest in the naturally occurring, virulent clonal types 1 and 2, which gave transformation frequencies up to 1%. Clonal types 3 and 4, which arise on laboratory transfer and are avirulent, gave maximal transformation frequencies of 0.00005%. Competence was maximal in lag and early log phases of growth, but was maintained throughout the growth cycle. A complex broth was required for the physiological expression of competence. The kinetics of DNA uptake, dose-response curve of DNA versus transformants, time required for phenotypic expression, and other features were similar to those in other bacterial transformation systems.     

Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants

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Highlights

• •Quantitative proteomes of the 2016 WHO Neisseria gonorrhoeae reference strains.
• •Novel gonorrhea vaccine candidates and potential global proteomic AMR markers.
• •First large-scale proteomic profiling of gonorrhea vaccine candidates and AMR.
• •A reference proteomics databank for gonococcal vaccine and AMR research endeavors.

     

Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile

Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies.     

Isolation of an Antigen of Neisseria gonorrhoeae Involved in the Human Immune Response to Gonococcal Infection

Ion-exchange chromatography was used to isolate a fraction from disrupted gonococci which reacts with sera from patients with gonococcal disease in complement-fixation and gel-diffusion tests. This antigenic fraction was shown to be the same as that previously described as having been isolated by gel filtration. The method reported here has the advantage of greater rapidity of isolation together with some improvement in purity.     

Pilin independent binding of Neisseria gonorrhoeae to immobilized glycolipids

The adherence process in pathogenesis involves the attachment of bacteria to structures present on eukaryotic cell surfaces. To investigate components necessary for this interaction, we have characterized the binding of N. gonorrhoeae to eukaryotic glycolipids immobilized on thin layer chromatograms. The gonococci specifically bind to a subset of glycolipids consisting of lactosylceramide, gangliotriosylceramide, and gangliotetraosylceramide. This binding was identified in both piliated and nonpiliated cells, and is postulated to be mediated by a nonpilin lectin-like adhesin protein.     

Human factor H interacts selectively with Neisseria gonorrhoeae and results in species-specific complement evasion

Complement forms a key arm of innate immune defenses against gonococcal infection. Sialylation of gonococcal lipo-oligosaccharide, or expression of porin 1A (Por1A) protein, enables Neisseria gonorrhoeae to bind the alternative pathway complement inhibitor, factor H (fH), and evade killing by human complement. Using recombinant fH fragment-murine Fc fusion proteins, we localized two N. gonorrhoeae Por1A-binding regions in fH: one in complement control protein domain 6, the other in complement control proteins 18-20. The latter is similar to that reported previously for sialylated Por1B gonococci. Upon incubation with human serum, Por1A and sialylated Por1B strains bound full-length human fH (HufH) and fH-related protein 1. In addition, Por1A strains bound fH-like protein 1 weakly. Only HufH, but not fH from other primates, bound directly to gonococci. Consistent with direct HufH binding, unsialylated Por1A gonococci resisted killing only by human complement, but not complement from other primates, rodents or lagomorphs; adding HufH to these heterologous sera restored serum resistance. Lipo-oligosaccharide sialylation of N. gonorrhoeae resulted in classical pathway regulation as evidenced by decreased C4 binding in human, chimpanzee, and rhesus serum but was accompanied by serum resistance only in human and chimpanzee serum. Direct-binding specificity of HufH only to gonococci that prevents serum killing is restricted to humans and may in part explain species-specific restriction of natural gonococcal infection. Our findings may help to improve animal models for gonorrhea while also having implications in the choice of complement sources to evaluate neisserial vaccine candidates.     

Antibiotic susceptibility of Neisseria gonorrhoeae in relation to serogroups

Susceptibility testings, by means of the agar-dilution method, were performed on nine antibiotics of 138 non-PPNG (beta-lactamase-producing = PPNG) strains, 17 of which originated from Thailand, and 88 PPNG isolates. The gonococcal strains were sero-grouped by the co-agglutination method and classified among the sero-groups W I, W II or W III. Statistically significant differences in antibiotic susceptibility between non-PPNG strains of the three sero-groups could be demonstrated with strains belonging to sero-group W I as the most sensitive and W III isolates as the most resistant. Non-PPNG strains from Thailand were significantly more resistant than the other non-PPNG isolates of the same sero-group. There was, however, no significant difference in resistance between non-PPNG strains from Thailand and PPNG isolates of the same sero-group. PPNG strains of sero-group W I were significantly more sensitive than PPNG strains of sero-group W II, whereas there was no significant difference between PPNG isolates of sero-groups W II and W III. Non-PPNG strains, not from Thailand, of sero-groups W I and W II were significantly more susceptible to non beta-lactam antibiotics than PPNG strains of corresponding sero-groups, while no such difference could be demonstrated for non-PPNG and PPNG isolates of sero-group W III.     

Susceptibility of Neisseria gonorrhoeae to seven antibiotics in vitro

One hundred consecutive isolates of N. gonorrhoeae were tested for susceptibility to penicillin, ampicillin, tetracycline, erythromycin, kanamycin, cephaloridine and cephalexin by an agar dilution method. Relative resistance to penicillin was frequent. For 39% of isolates the minimum inhibitory concentration (MIC) of penicillin was 0.05 U./ml. or less; in 55% the MIC was 0.5 to 2.0 U./ml. Ampicillin was slightly more active than penicillin G: all isolates were inhibited by 0.5μg./ml. or less. Resistance to tetracycline and erythromycin was frequent with MIC of 1 μg./ml. or greater observed in 32 and 24% of isolates respectively. The MIC of kanamycin for all gonococci was 8 μg./ml. or greater. Cephalexin was slightly more active than cephaloridine, though each drug exhibited a wide range of MIC values. Gonococcus isolates resistant to penicillin (MIC of 1.0 U./ml. or greater) tended to be resistant to the other antibiotics tested.     

Resistance of Russian isolates of Neisseria gonorrhoeae to fluoroquinolones

Fluoroquinolones still belong to the drugs of choice in the treatment of uncomplicated gonorrhea. At the same time, there have been more data on the spreading N. gonorrhoeae strains resistant to fluoroquinolones. A variety of mechanisms, like modification of the target of antibiotic's action (point mutations in genes gyrA and parC), a decreasing permeability of the bacterial cell membrane (amino-acid changes Por protein) and a growing efflux of antibiotic (mutations in the promoter or in the coding region of mtrR) mediate in the shaping resistance of the drugs. The MIC values for four fluoroquinolone-series antibiotics were determined and the gyrA, parC, por and mtrR genes were examined for resistance-responsible mutations in 32 studied clinical strains of N. gonorrhoeae. Strains with high resistance to fluoroquinolones were detected; 3 of them had no common changes in GyrA or ParC, however, amino acid changes and mutations were detected in Por protein and promoter or gene mtrR encoding region, respectively. The paper contains priority data on the detection (in Russia) of N. gonorrhoeae strains with high resistance to fluoroquinolones. Involvement of different mechanisms in the process of resistance shaping is discussed. The results are of practical importance for planning the antibacterial therapy of gonorrhoeae; they point out the need in regional testing of resistance in the N. gonorrhoeae population encountered in Russia.     

In Vitro Susceptibility of Neisseria gonorrhoeae to Nine Antimicrobial Agents

The in vitro action of nine antibiotics was tested by the agar streak method against 45 gonococcal strains isolated from penicillin-therapy failures. The penicillin susceptibility range of these strains was 0.003 to 1.32 μg/ml, and the tetracycline susceptibility range was 0.125 to 2.0 μg/ml. Minimal inhibitory concentrations of minocycline and doxycycline paralleled the activity of tetracycline and ranged from 0.125 to 1.0 μg/ml and 0.125 to 2.0 μg/ml, respectively. Rifampicin, with a narrow range of 0.5 to 1.0 μg/ml, inhibited 75% of the strains at 0.5 μg/ml. The range for cephaloridine and cephaloglycine was 0.5 to 20.0 μg/ml, but another cephalosporium derivative, cephalexin, exhibited greater activity in its range of 0.25 to 20.0 μg/ml. A semisynthetic penicillin, carbenicillin, with a range of 0.025 to 0.75 μg/ml, displayed more activity against the lower susceptible penicillin G gonococcal strains.