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Sample records for meningoencefalitis por neisseria

  1. Crystallographic analysis of Neisseria meningitidis PorB extracellular loops potentially implicated in TLR2 recognition.

    PubMed

    Kattner, Christof; Toussi, Deana N; Zaucha, Jan; Wetzler, Lee M; Rüppel, Nadine; Zachariae, Ulrich; Massari, Paola; Tanabe, Mikio

    2014-03-01

    Among all Neisseriae species, Neisseria meningitidis and Neisseria gonorrhoeae are the only human pathogens, causative agents of bacterial meningitis and gonorrhoea, respectively. PorB, a pan-Neisseriae trimeric porin that mediates diffusive transport of essential molecules across the bacterial outer membrane, is also known to activate host innate immunity via Toll-like receptor 2 (TLR2)-mediated signaling. The molecular mechanism of PorB binding to TLR2 is not known, but it has been hypothesized that electrostatic interactions contribute to ligand/receptor binding. Strain-specific sequence variability in the surface-exposed loops of PorB which are potentially implicated in TLR2 binding, may explain the difference in TLR2-mediated cell activation in vitro by PorB homologs from the commensal Neisseriae lactamica and the pathogen N. meningitidis. Here, we report a comparative structural analysis of PorB from N. meningitidis serogroup B strain 8765 (63% sequence homology with PorB from N. meningitidis serogroup W135) and a mutant in which amino acid substitutions in the extracellular loop 7 lead to significantly reduced TLR2-dependent activity in vitro. We observe that this mutation both alters the loop conformation and causes dramatic changes of electrostatic surface charge, both of which may affect TLR2 recognition and signaling. PMID:24361688

  2. Sequence evolution of the porB gene of Neisseria gonorrhoeae and Neisseria meningitidis: evidence of positive Darwinian selection.

    PubMed

    Smith, N H; Maynard Smith, J; Spratt, B G

    1995-05-01

    Protein 1 (PI) is a major porin of Neisseria gonorrhoeae and Neisseria meningitidis and is encoded by a single locus, porB. Alleles of the porB locus of N. gonorrhoeae are assigned to two homology groups, PI(A) and PI(B), on the basis of immunological and structural similarity. In a like manner, alleles of the porB locus of the closely related bacterium, N. meningitidis, are allocated into class 2 and class 3 homology groups. An individual strain of N. gonorrhoeae or N. meningitidis expresses either one or other of these porin homology groups but never both, and the antigenic reactions of these highly diverse outer membrane proteins form part of the N. gonorrhoeae and N. meningitidis serotyping schemes. A comparison of the number of synonymous and nonsynonymous substitutions per site between the two most divergent alleles of each of these four groups of porB alleles shows that PI(A) alleles have accumulated significantly more nonsynonymous substitutions per site than synonymous substitutions. In contrast the distribution of synonymous and nonsynonymous substitutions between alleles of class 2 and class 3 porins are not significantly different from random. We localize the regions of the PI(A) alleles with an excess of amino acid changes to the surface-exposed loops of these outer membrane proteins and suggest that positive Darwinian selection for diversity, driven by the human immune system, can most easily explain the allelic polymorphism and the pattern of synonymous and nonsynonymous substitutions.

  3. VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways.

    PubMed

    Müller, Anne; Rassow, Joachim; Grimm, Jan; Machuy, Nikolaus; Meyer, Thomas F; Rudel, Thomas

    2002-04-15

    The human pathogen Neisseria gonorrhoeae induces host cell apoptosis during infection by delivering the outer membrane protein PorB to the host cell's mitochondria. PorB is a pore-forming beta-barrel protein sharing several features with the mitochondrial voltage-dependent anion channel (VDAC), which is involved in the regulation of apoptosis. Here we show that PorB of pathogenic Neisseria species produced by host cells is efficiently targeted to mitochondria. Imported PorB resides in the mitochondrial outer membrane and forms multimers with similar sizes as in the outer bacterial membrane. The mitochondria completely lose their membrane potential, a characteristic previously observed in cells infected with gonococci or treated with purified PorB. Closely related bacterial porins of non-pathogenic Neisseria mucosa or Escherichia coli remain in the cytosol. Import of PorB into mitochondria in vivo is independent of a linear signal sequence. Insertion of PorB into the mitochondrial outer membrane in vitro depends on the activity of Tom5, Tom20 and Tom40, but is independent of Tom70. Our data show that human VDAC and bacterial PorB are imported into mitochondria by a similar mechanism. PMID:11953311

  4. Typing and surface charges of the variable loop regions of PorB from Neisseria meningitidis.

    PubMed

    Stefanelli, Paola; Neri, Arianna; Tanabe, Mikio; Fazio, Cecilia; Massari, Paola

    2016-06-01

    PorB is a pan-Neisserial major outer membrane protein with a trimeric β-barrel structure. Each monomer presents eight periplasmic turns and eight surface exposed loop regions with sequence variability. PorB induces activation of host cell responses via a TLR2-dependent mechanism likely mediated by electrostatic interactions between TLR2 and PorB surface exposed loops. Variability in the loop amino acid sequence is known to influence cell responses to PorB in vitro, particularly for the residues in L5 and L7. In this work, the sequence of the porB gene and the electrostatic surface charges of PorB from 35 invasive meningococcal isolates belonging to the main clonal complexes identified in Italy and from five carriage genomes available on the website http://pubmlst.org/neisseria/ were examined. Analysis of the porB encoding regions from the invasive meningococci has identified four new alleles and a potential association between porB alleles, serogroup, and clonal complexes. Through computer-based modeling and analysis of the electrostatic surface charges of PorB from these strains, loop charge segregation between PorB from invasive serogroups B and C was observed. Specifically, loops 1, 4, and 7 were negatively charged and L2 and L8 were mostly neutral in serogroup B isolates, while an overall homogeneous positive surface charge was present in PorB from invasive serogroup C strains. A higher PorB sequence variability was observed among carriage genomes, and a general prevalence of negative loop surface charges. The surface charge differences in PorB from serogroups B and C invasive and carriage strains may, in part, influence the outcomes of Neisseriae interactions with host cells. © 2016 IUBMB Life, 68(6):488-495, 2016. PMID:27156582

  5. Typing and surface charges of the variable loop regions of PorB from Neisseria meningitidis.

    PubMed

    Stefanelli, Paola; Neri, Arianna; Tanabe, Mikio; Fazio, Cecilia; Massari, Paola

    2016-06-01

    PorB is a pan-Neisserial major outer membrane protein with a trimeric β-barrel structure. Each monomer presents eight periplasmic turns and eight surface exposed loop regions with sequence variability. PorB induces activation of host cell responses via a TLR2-dependent mechanism likely mediated by electrostatic interactions between TLR2 and PorB surface exposed loops. Variability in the loop amino acid sequence is known to influence cell responses to PorB in vitro, particularly for the residues in L5 and L7. In this work, the sequence of the porB gene and the electrostatic surface charges of PorB from 35 invasive meningococcal isolates belonging to the main clonal complexes identified in Italy and from five carriage genomes available on the website http://pubmlst.org/neisseria/ were examined. Analysis of the porB encoding regions from the invasive meningococci has identified four new alleles and a potential association between porB alleles, serogroup, and clonal complexes. Through computer-based modeling and analysis of the electrostatic surface charges of PorB from these strains, loop charge segregation between PorB from invasive serogroups B and C was observed. Specifically, loops 1, 4, and 7 were negatively charged and L2 and L8 were mostly neutral in serogroup B isolates, while an overall homogeneous positive surface charge was present in PorB from invasive serogroup C strains. A higher PorB sequence variability was observed among carriage genomes, and a general prevalence of negative loop surface charges. The surface charge differences in PorB from serogroups B and C invasive and carriage strains may, in part, influence the outcomes of Neisseriae interactions with host cells. © 2016 IUBMB Life, 68(6):488-495, 2016.

  6. Structural basis for solute transport, nucleotide regulation, and immunological recognition of Neisseria meningitidis PorB

    SciTech Connect

    Tanabe, Mikio; Nimigean, Crina M.; Iverson, T.M.

    2010-06-25

    PorB is the second most prevalent outer membrane protein in Neisseria meningitidis. PorB is required for neisserial pathogenesis and can elicit a Toll-like receptor mediated host immune response. Here, the x-ray crystal structure of PorB has been determined to 2.3 {angstrom} resolution. Structural analysis and cocrystallization studies identify three putative solute translocation pathways through the channel pore: One pathway transports anions nonselectively, one transports cations nonselectively, and one facilitates the specific uptake of sugars. During infection, PorB likely binds host mitochondrial ATP, and cocrystallization with the ATP analog AMP-PNP suggests that binding of nucleotides regulates these translocation pathways both by partial occlusion of the pore and by restricting the motion of a putative voltage gating loop. PorB is located on the surface of N. meningitidis and can be recognized by receptors of the host innate immune system. Features of PorB suggest that Toll-like receptor mediated recognition outer membrane proteins may be initiated with a nonspecific electrostatic attraction.

  7. Structure and function of the PorB porin from disseminating Neisseria gonorrhoeae.

    PubMed

    Zeth, Kornelius; Kozjak-Pavlovic, Vera; Faulstich, Michaela; Fraunholz, Martin; Hurwitz, Robert; Kepp, Oliver; Rudel, Thomas

    2013-02-01

    The outer membrane of Gram-negative bacteria contains a large number of channel-forming proteins, porins, for the uptake of small nutrient molecules. Neisseria gonorrhoeae PorBIA (PorB of serotype A) are associated with disseminating diseases and mediate a rapid bacterial invasion into host cells in a phosphate-sensitive manner. To gain insights into this structure-function relationship we analysed PorBIA by X-ray crystallography in the presence of phosphate and ATP. The structure of PorBIA in the complex solved at a resolution of 3.3 Å (1 Å=0.1 nm) displays a surplus of positive charges inside the channel. ATP ligand-binding in the channel is co-ordinated by the positively charged residues of the channel interior. These residues ligate the aromatic, sugar and pyrophosphate moieties of the ligand. Two phosphate ions were observed in the structure, one of which clamped by two arginine residues (Arg92 and Arg124) localized at the extraplasmic channel exit. A short β-bulge in β2-strand together with the long L3 loop narrow the barrel diameter significantly and further support substrate specificity through hydrogen bond interactions. Interestingly the structure also comprised a small peptide as a remnant of a periplasmic protein which physically links porin molecules to the peptidoglycan network. To test the importance of Arg92 on bacterial invasion the residue was mutated. In vivo assays of bacteria carrying a R92S mutation confirmed the importance of this residue for host-cell invasion. Furthermore systematic sequence and structure comparisons of PorBIA from Neisseriaceae indicated Arg92 to be unique in disseminating N. gonorrhoeae thereby possibly distinguishing invasion-promoting porins from other neisserial porins.

  8. Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB

    SciTech Connect

    Tanabe, Mikio; Iverson, Tina M.

    2010-01-28

    The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P6{sub 3}. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P6{sub 3} crystal forms contained one PorB monomer in the asymmetric unit. Of the three, the P6{sub 3} crystal form had the best diffraction quality, yielding data extending to 2.3 {angstrom} resolution.

  9. Neisseria meningitidis Lacking the Major Porins PorA and PorB Is Viable and Modulates Apoptosis and the Oxidative Burst of Neutrophils.

    PubMed

    Peak, Ian R; Chen, Adrienne; Jen, Freda E-C; Jennings, Courtney; Schulz, Benjamin L; Saunders, Nigel J; Khan, Arshad; Seifert, H Steven; Jennings, Michael P

    2016-08-01

    The bacterial pathogen Neisseria meningitidis expresses two major outer-membrane porins. PorA expression is subject to phase-variation (high frequency, random, on-off switching), and both PorA and PorB are antigenically variable between strains. PorA expression is variable and not correlated with meningococcal colonisation or invasive disease, whereas all naturally-occurring strains express PorB suggesting strong selection for expression. We have generated N. meningitidis strains lacking expression of both major porins, demonstrating that they are dispensable for bacterial growth in vitro. The porAB mutant strain has an exponential growth rate similar to the parental strain, as do the single porA or porB mutants, but the porAB mutant strain does not reach the same cell density in stationary phase. Proteomic analysis suggests that the double mutant strain exhibits compensatory expression changes in proteins associated with cellular redox state, energy/nutrient metabolism, and membrane stability. On solid media, there is obvious growth impairment that is rescued by addition of blood or serum from mammalian species, particularly heme. These porin mutants are not impaired in their capacity to inhibit both staurosporine-induced apoptosis and a phorbol 12-myristate 13-acetate-induced oxidative burst in human neutrophils suggesting that the porins are not the only bacterial factors that can modulate these processes in host cells.

  10. Population genetics of the porB gene of Neisseria gonorrhoeae: different dynamics in different homology groups.

    PubMed

    Posada, D; Crandall, K A; Nguyen, M; Demma, J C; Viscidi, R P

    2000-03-01

    The porB locus codes for the major outer membrane protein of Neisseria gonorrhoeae. Alleles of this locus have been assigned to two homology groups based on close sequence and immunological relationships and are designated as either PIA or PIB. Several population parameters were estimated and compared among these two groups using a data set of 22 PIA sequences and 91 PIB sequences obtained from diverse geographic localities and from time periods spanning approximately 50 years. Recombination appears to be extensive in the porB gene. While the recombination rates are similar for the PIA and PIB sequences, the relative contribution of recombination to genetic diversity is higher for the PIA sequences. Alleles belonging to the PIB group show greater genetic diversity than do those in the PIA group. Although phylogenetic analysis did not reveal temporal or geographic clustering of sequences, estimates of gene flow and the fixation index suggested that PIB sequences exhibit population substructure based on geographic locality. Selection acts in these homology groups in a different way. While positive Darwinian selection is the dominant force driving the evolution of the PIA sequences, purifying selection operates also on the PIB sequences. These differences may be attributable to the greater propensity of PIA strains, as compared with PIB strains, to cause disseminated gonococcal infection, which would expose the former to intense selection pressure from the host immune system. The molecular evolution of Neisseria gonorrhoeae seems to be driven by the simultaneous action of selection and recombination, but under different rates and selection pressures for the PIA and PIB homology groups.

  11. A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

    PubMed Central

    Hjelmevoll, Stig Ove; Olsen, Merethe Elise; Sollid, Johanna U. Ericson; Haaheim, Håkon; Unemo, Magnus; Skogen, Vegard

    2006-01-01

    Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection. PMID:17065426

  12. Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

    PubMed Central

    Vasquez, Abel E; Manzo, Ricardo A; Soto, Daniel A; Barrientos, Magaly J; Maldonado, Aurora E; Mosqueira, Macarena; Avila, Anastasia; Touma, Jorge; Bruce, Elsa; Harris, Paul R; Venegas, Alejandro

    2015-01-01

    The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant. PMID:25750999

  13. Saturating mutagenesis of an essential gene: a majority of the Neisseria gonorrhoeae major outer membrane porin (PorB) is mutable.

    PubMed

    Chen, Adrienne; Seifert, H Steven

    2014-02-01

    The major outer membrane porin (PorB) of Neisseria gonorrhoeae is an essential protein that mediates ion exchange between the organism and its environment and also plays multiple roles in human host pathogenesis. To facilitate structure-function studies of porin's multiple roles, we performed saturating mutagenesis at the porB locus and used deep sequencing to identify essential versus mutable residues. Random mutations in porB were generated in a plasmid vector, and mutant gene pools were transformed into N. gonorrhoeae to select for alleles that maintained bacterial viability. Deep sequencing of the input plasmid pools and the output N. gonorrhoeae genomic DNA pools identified mutations present in each, and the mutations in both pools were compared to determine which changes could be tolerated by the organism. We examined the mutability of 328 amino acids in the mature PorB protein and found that 308 of them were likely to be mutable and that 20 amino acids were likely to be nonmutable. A subset of these predictions was validated experimentally. This approach to identifying essential amino acids in a protein of interest introduces an additional application for next-generation sequencing technology and provides a template for future studies of both porin and other essential bacterial genes.

  14. Binding of complement factor H to PorB3 and NspA enhances resistance of Neisseria meningitidis to anti-factor H binding protein bactericidal activity.

    PubMed

    Giuntini, Serena; Pajon, Rolando; Ram, Sanjay; Granoff, Dan M

    2015-04-01

    Among 25 serogroup B Neisseria meningitidis clinical isolates, we identified four (16%) with high factor H binding protein (FHbp) expression that were resistant to complement-mediated bactericidal activity of sera from mice immunized with recombinant FHbp vaccines. Two of the four isolates had evidence of human FH-dependent complement downregulation independent of FHbp. Since alternative complement pathway recruitment is critical for anti-FHbp bactericidal activity, we hypothesized that in these two isolates binding of FH to ligands other than FHbp contributes to anti-FHbp bactericidal resistance. Knocking out NspA, a known meningococcal FH ligand, converted both resistant isolates to anti-FHbp susceptible isolates. The addition of a nonbactericidal anti-NspA monoclonal antibody to the bactericidal reaction also increased anti-FHbp bactericidal activity. To identify a role for FH ligands other than NspA or FHbp in resistance, we created double NspA/FHbp knockout mutants. Mutants from both resistant isolates bound 10-fold more recombinant human FH domains 6 and 7 fused to Fc than double knockout mutants prepared from two sensitive meningococcal isolates. In light of recent studies showing functional FH-PorB2 interactions, we hypothesized that PorB3 from the resistant isolates recruited FH. Allelic exchange of porB3 from a resistant isolate to a sensitive isolate increased resistance of the sensitive isolate to anti-FHbp bactericidal activity (and vice versa). Thus, some PorB3 variants functionally bind human FH, which in the presence of NspA enhances anti-FHbp resistance. Combining anti-NspA antibodies with anti-FHbp antibodies can overcome resistance. Meningococcal vaccines that target both NspA and FHbp are likely to confer greater protection than either antigen alone.

  15. Population genetics of Neisseria gonorrhoeae in a high-prevalence community using a hypervariable outer membrane porB and 13 slowly evolving housekeeping genes.

    PubMed

    Pérez-Losada, Marcos; Viscidi, Raphael P; Demma, James C; Zenilman, Jonathan; Crandall, Keith A

    2005-09-01

    Baltimore, Md., is an urban community with a high prevalence of Neisseria gonorrhoeae. Due to partially protective immune responses, introduction of new strains from other host populations, and exposure of N. gonorrhoeae to antibiotics, the phenotypic and genotypic characteristics of the circulating strains can fluctuate over time. Understanding the overall genetic diversity and population structure of N. gonorrhoeae is essential for informing public health interventions to eliminate this pathogen. We studied gonococci population genetics in Baltimore by analyzing a hypervariable and strongly selected outer membrane porB gene and 13 slowly evolving and presumably neutral housekeeping genes (abcZ, adk, aroE, fumC, gdh, glnA, gnd, pdhC, pgm, pilA, ppk, pyrD, and serC) in 204 isolates collected in 1991, 1996, and 2001 from male and female patients of two public sexually transmitted diseases clinics. Genetic diversity (), recombination (C), growth (g), population structure, and adaptive selection under codon-substitution and amino acid property models were estimated and compared between these two gene classes. Estimates of the F(ST) fixation index and the chi(2) test of sequence absolute frequencies revealed significant temporal substructuring for both gene types. Baltimore's N. gonorrhoeae populations have increased since 1991 as indicated by consistent positive values of g. Female patients showed similar or lower levels of and C than male patients. Within the MLST housekeeping genes, levels of and C ranged from 0.001-0.013 and 0.000-0.018, respectively. Overall recombination seems to be the dominant force driving evolution in these populations. All loci showed amino acid sites and physicochemical properties under adaptive (or positive-destabilizing) selection, rejecting the generally assumed hypothesis of stabilizing selection for these MLST genes. Within the porB gene, protein I B showed higher and C values than protein I A. Directional positive selection possibly

  16. One-step purification and porin transport activity of the major outer membrane proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis.

    PubMed

    Kattner, Christof; Pfennig, Sabrina; Massari, Paola; Tanabe, Mikio

    2015-03-01

    Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.

  17. Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA.

    PubMed

    Lindberg, Robert; Fredlund, Hans; Nicholas, Robert; Unemo, Magnus

    2007-06-01

    The recent emergence and transmission of Neisseria gonorrhoeae isolates with reduced susceptibility to expanded-spectrum cephalosporins such as cefixime and ceftriaxone have been reported. The aim of this study was to determine the correlation of different polymorphisms in the penA, mtrR, porB1b (penB), and ponA genes of N. gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone. Eighteen gonococcal isolates with reduced cefixime and ceftriaxone susceptibility (Cef(i)) and two susceptible isolates were characterized using serovar determination, antibiograms, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and sequencing of penA, mtrR, porB1b, and ponA alleles. For the Cef(i) isolates (n = 18), the MICs of cefixime and ceftriaxone ranged between 0.032 to 0.38 mug/ml and 0.064 to 0.125 mug/ml, respectively. These isolates were assigned five different serovars and six divergent NG-MAST sequence types. Eleven isolates (61%) with higher MICs of cefixime and ceftriaxone contained a nearly identical penA mosaic allele and previously described polymorphisms in mtrR (a single nucleotide [A] deletion in the promoter), penB (mutations in porB1b encoding loop 3 of PorB1b), and ponA (ponA1 polymorphism). The remaining seven Cef(i) isolates (39%), which had somewhat lower MICs of cefixime and ceftriaxone, contained an aspartic acid insertion (Asp-345a) in PBP 2 in conjunction with alterations of 4 to 10 amino acid residues in the C-terminal region of the transpeptidase domain of penA. In conclusion, an unambiguous association between penA mosaic alleles, in conjunction with genetic polymorphisms in mtrR, porB1b, and ponA, and greater reduced susceptibility to cefixime and ceftriaxone was identified.

  18. HRM confirmation of Neisseria gonorrhoeae in clinical specimens by G→A (position 857) mutation detection in the 16S rRNA gene before sequencing and after porA confirmation.

    PubMed

    Gurtler, Volker; Mayall, Barrie C; Wang, Jenny

    2012-05-01

    A total of 2273 specimens submitted to the Austin Hospital Pathology Service for Neisseria gonorrhoeae screening between September 1, 2009 and May 11, 2011 were used in this study. Specimens were simultaneously screened and confirmed with a previously published real time PCR assay for the opa gene (extra primers were included to increase sensitivity) and the porA gene respectively. The opa gene screen and initial porA gene confirmation yielded an N. gonorrhoeae positivity rate of 0.88% (20/2273) and 0.49% (11/2191) for specimens and patients respectively. A 16S rDNA High Resolution Melt confirmatory PCR was developed subsequently; this reduced the N. gonorrhoeae positivity rate to 0.35% (8/2273) and 0.27% (6/2191) for specimens and patients respectively (not altered by 16S sequencing). The higher rate of secondary confirmation (16S HRM) in patients compared with samples was due to the detection of species other than N. gonorrhoeae detected by the initial screening and confirmation test. This underlines the importance of performing the secondary confirmatory test that has been developed in this study.

  19. Neisseria gonorrhoeae antimicrobial susceptibility in Barcelona: penA, ponA, mtrR, and porB mutations and NG-MAST sequence types associated with decreased susceptibility to cephalosporins.

    PubMed

    Serra-Pladevall, J; Barberá, M J; Rodriguez, S; Bartolomé-Comas, R; Roig, G; Juvé, R; Andreu, A

    2016-09-01

    The aims of this study were to determine the antimicrobial susceptibility of Neisseria gonorrhoeae (NG) in our area, to analyze the molecular mechanisms involved in cephalosporins resistance, and to undertake molecular typing of our NG strains. Antimicrobial susceptibility was determined using the Etest. The genes penA, mtrR, penB, and ponA were studied. Molecular typing was performed by N. gonorrhoeae multiantigen sequence typing. Of 329 strains analyzed in 2013, none showed high-level cephalosporin resistance, but 8.2 % had resistance to cefixime [minimum inhibitory concentration (MIC) > 0.125 μg/mL] and 0.6 % to ceftriaxone (MIC > 0.125 μg/mL). Azithromycin resistance was documented in 4.3 % and ciprofloxacin resistance in 49.2 %. Among 48 strains with an MIC ≥ 0.125 μg/mL to cefixime, 58.3 % showed the penA mosaic pattern XXXIV, 98 % a Leu → Pro substitution at position 421 of the ponA gene, 100 % amino acid changes at positions 101 and 102 of the PorB1b porin, and 87.5 % of strains an adenine deletion in the promoter region of the MtrC-D-E efflux pump. A significant difference between strains with and without decreased cephalosporin susceptibility (MIC ≥ 0.125 μg/mL) was observed for these four genes. Of the 48 strains with an MIC ≥ 0.125 μg/mL to cefixime, 43.8 % belonged to the genogroup G1407 and 27.1 % belonged to the genogroup G2400. A significant association of G1407 with decreased susceptibility (MIC ≥ 0.125 μg/mL) and G2992 with susceptibility was found, and also between G1407 and mosaic pattern XXXIV and between G2400 and A501T substitution in penA. The NG resistance rate in our area is higher than the median of Europe. We have detected the emergence of G2400, which may be a source of antimicrobial resistance. PMID:27255221

  20. The Biology of Neisseria Adhesins

    PubMed Central

    Hung, Miao-Chiu; Christodoulides, Myron

    2013-01-01

    Members of the genus Neisseria include pathogens causing important human diseases such as meningitis, septicaemia, gonorrhoea and pelvic inflammatory disease syndrome. Neisseriae are found on the exposed epithelia of the upper respiratory tract and the urogenital tract. Colonisation of these exposed epithelia is dependent on a repertoire of diverse bacterial molecules, extending not only from the surface of the bacteria but also found within the outer membrane. During invasive disease, pathogenic Neisseriae also interact with immune effector cells, vascular endothelia and the meninges. Neisseria adhesion involves the interplay of these multiple surface factors and in this review we discuss the structure and function of these important molecules and the nature of the host cell receptors and mechanisms involved in their recognition. We also describe the current status for recently identified Neisseria adhesins. Understanding the biology of Neisseria adhesins has an impact not only on the development of new vaccines but also in revealing fundamental knowledge about human biology. PMID:24833056

  1. Neisseria meningitidis B vaccines.

    PubMed

    Panatto, Donatella; Amicizia, Daniela; Lai, Piero Luigi; Gasparini, Roberto

    2011-09-01

    Invasive infections caused by Neisseria meningitidis are a serious public health problem worldwide and have a heavy economic impact. The incidence of invasive disease due to Neisseria meningitidis is highly variable according to geographical area and serogroup distribution. Since the introduction of vaccination programs with conjugated vaccine C in children and adolescents, most cases of invasive meningococcal disease in developed countries have been caused by meningococcus B. It is important to underline that invasive meningococcal disease will not be controlled until safe and effective vaccines for meningococcal B are available and widely used. The aims of this article are to describe the most recent developments in meningococcal B vaccines and to discuss how these vaccines can contribute to containing meningococcal disease.

  2. Neisseria-Avoiding the Jump to Conclusions

    ERIC Educational Resources Information Center

    Spivey, Maria I.; Paschall, Robert T.; Ferrett, Rhonda; Alexander, Randell

    2011-01-01

    "Neisseria gonorrhoeae" infection in a prepubertal child is virtually diagnostic of sexual abuse, provided perinatal infection has been excluded. Therefore, it is imperative that "Neisseria gonorrhoeae" be correctly identified. We present two cases of false positive "Neisseria gonorrhoeae" meningitis encountered at two different children's…

  3. The genetics of Neisseria species.

    PubMed

    Rotman, Ella; Seifert, H Steven

    2014-01-01

    Neisseria gonorrhoeae and Neisseria meningitidis are closely related organisms that cause the sexually transmitted infection gonorrhea and serious bacterial meningitis and septicemia, respectively. Both species possess multiple mechanisms to alter the expression of surface-exposed proteins through the processes of phase and antigenic variation. This potential for wide variability in surface-exposed structures allows the organisms to always have subpopulations of divergent antigenic types to avoid immune surveillance and to contribute to functional variation. Additionally, the Neisseria are naturally competent for DNA transformation, which is their main means of genetic exchange. Although bacteriophages and plasmids are present in this genus, they are not as effective as DNA transformation for horizontal genetic exchange. There are barriers to genetic transfer, such as restriction-modification systems and CRISPR loci, that limit particular types of exchange. These host-restricted pathogens illustrate the rich complexity of genetics that can help define the similarities and differences of closely related organisms.

  4. Mobile DNA in the Pathogenic Neisseria.

    PubMed

    Obergfell, Kyle P; Seifert, H Steven

    2015-02-01

    The genus Neisseria contains two pathogenic species of prominant public health concern: Neisseria gonorrhoeae and Neisseria meningitidis. These pathogens display a notable ability to undergo frequent programmed recombination events. The recombination-mediated pathways of transformation and pilin antigenic variation in the Neisseria are well-studied systems that are critical for pathogenesis. Here we will detail the conserved and unique aspects of transformation and antigenic variation in the Neisseria. Transformation will be followed from initial DNA binding through recombination into the genome with consideration to the factors necessary at each step. Additional focus is paid to the unique type IV secretion system that mediates donation of transforming DNA in the pathogenic Neisseria. The pilin antigenic variation system uses programmed recombinations to alter a major surface determinant, which allows immune avoidance and promotes infection. We discuss the trans- and cis- acting factors which facilitate pilin antigenic variation and present the current understanding of the mechanisms involved in the process.

  5. The laboratory diagnosis of Neisseria gonorrhoeae

    PubMed Central

    Ng, Lai-King; Martin, Irene E

    2005-01-01

    The present article describes the laboratory diagnosis of Neisseria gonorrhoeae by culturing of the organism from different types of clinical specimens followed by confirmatory tests. The success of culture methods requires good quality collection and transport of clinical specimens. The present guide describes the media requirements and cultural conditions for N gonorrhoeae growth and the characteristics for a presumptive identification of N gonorrhoeae. Confirmatory tests include biochemical tests, chromogenic enzyme substrate tests, immunoassays and nucleic acid methods. Nucleic acid detection methods include either amplification-based methods or nonamplification tests, and are increasingly used in clinical laboratories where a viable culture is not possible to obtain. Nucleic acid methods can also be used to detect the presence of low numbers in a specimen. Nucleic acid detection methods need confirmation with another amplification method or gene target. Controls must be included to ensure true positive and negative results, and to rule out nucleic acid contamination. Monitoring of antimicrobial susceptibilities of N gonorrhoeae is important to investigate treatment failure and to evaluate the efficacy of currently recommended therapies. Many methods for the characterization of N gonorrhoeae require cultures. The useful typing methods for determining strain relatedness include auxotyping, serotyping, plasmid profile analysis, DNA sequencing of the porB gene and pulsed-field gel electrophoresis. Quality assurance programs for diagnostic testing and antimicrobial susceptibility testing is reviewed. PMID:18159523

  6. Antimicrobial peptide resistance in Neisseria meningitidis.

    PubMed

    Tzeng, Yih-Ling; Stephens, David S

    2015-11-01

    Antimicrobial peptides (AMPs) play an important role as a host defense against microbial pathogens and are key components of the human innate immune response. Neisseria meningitidis frequently colonizes the human nasopharynx as a commensal but also is a worldwide cause of epidemic meningitis and rapidly fatal sepsis. In the human respiratory tract, the only known reservoir of N. meningitidis, meningococci are exposed to human endogenous AMPs. Thus, it is not surprising that meningococci have evolved effective mechanisms to confer intrinsic and high levels of resistance to the action of AMPs. This article reviews the current knowledge about AMP resistance mechanisms employed by N. meningitidis. Two major resistance mechanisms employed by meningococci are the constitutive modification of the lipid A head groups of lipooligosaccharides by phosphoethanolamine and the active efflux pump mediated excretion of AMPs. Other factors influencing AMP resistance, such as the major porin PorB, the pilin biogenesis apparatus, and capsular polysaccharides, have also been identified. Even with an inherently high intrinsic resistance, several AMP resistance determinants can be further induced upon exposure to AMPs. Many well-characterized AMP resistance mechanisms in other Gram-negative bacteria are not found in meningococci. Thus, N. meningitidis utilizes a limited but highly effective set of molecular mechanisms to mediate antimicrobial peptide resistance. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

  7. Genetic Manipulation of Neisseria gonorrhoeae.

    PubMed

    Dillard, Joseph P

    2011-11-01

    The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and to the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Techniques are described for genetic manipulation of N. gonorrhoeae, as well as for growth of this fastidious organism.

  8. Genetic manipulation of Neisseria gonorrhoeae.

    PubMed

    Dillard, Joseph P

    2006-01-01

    The sexually-transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and to the panmictic structure of the gonococcal population. However, high frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Techniques are described for genetic manipulation of N. gonorrhoeae, as well as for growth of this fastidious organism.

  9. Emerging resistance in Neisseria meningitidis and Neisseria gonorrhoeae.

    PubMed

    Stefanelli, Paola

    2011-02-01

    The value of monitoring antimicrobial resistance is particularly significant for Neisseria meningitidis and Neisseria gonorrhoeae diseases, even if it is for different reasons. Although there is no global alert for the spread of resistant meningococcal strains, the emergence of resistance is correlated to the outcome of treatment and the successful prophylaxis of close contacts. Few cases of resistance among meningococci have been recorded worldwide; it remains unclear what intriguing mechanism is responsible for maintaining resistance in these cases in the absence of significant antibiotic selective pressure, as in the case of penicillin; on the contrary, although rifampicin is the antibiotic of choice in the prophylaxis of close contacts, there is a very low rate of resistance. The emergence of multidrug-resistant N. gonorrhoeae is a great challenge in controlling gonorrhea as one of the main sexually transmitted bacterial diseases. International surveillance programs permit the monitoring of the susceptibility of the pathogen and allow the revision of the standardized treatment regimen when the situation changes. PMID:21342071

  10. Genetic distribution of noncapsular meningococcal group B vaccine antigens in Neisseria lactamica.

    PubMed

    Lucidarme, Jay; Gilchrist, Stefanie; Newbold, Lynne S; Gray, Stephen J; Kaczmarski, Edward B; Richardson, Lynne; Bennett, Julia S; Maiden, Martin C J; Findlow, Jamie; Borrow, Ray

    2013-09-01

    The poor immunogenicity of the meningococcal serogroup B (MenB) capsule has led to the development of vaccines targeting subcapsular antigens, in particular the immunodominant and diverse outer membrane porin, PorA. These vaccines are largely strain specific; however, they offer limited protection against the diverse MenB-associated diseases observed in many industrialized nations. To broaden the scope of its protection, the multicomponent vaccine (4CMenB) incorporates a PorA-containing outer membrane vesicle (OMV) alongside relatively conserved recombinant protein components, including factor H-binding protein (fHbp), Neisseria adhesin A (NadA), and neisserial heparin-binding antigen (NHBA). The expression of PorA is unique to meningococci (Neisseria meningitidis); however, many subcapsular antigens are shared with nonpathogenic members of the genus Neisseria that also inhabit the nasopharynx. These organisms may elicit cross-protective immunity against meningococci and/or occupy a niche that might otherwise accommodate pathogens. The potential for 4CMenB responses to impact such species (and vice versa) was investigated by determining the genetic distribution of the primary 4CMenB antigens among diverse members of the common childhood commensal, Neisseria lactamica. All the isolates possessed nhba but were devoid of fhbp and nadA. The nhba alleles were mainly distinct from but closely related to those observed among a representative panel of invasive MenB isolates from the same broad geographic region. We made similar findings for the immunogenic typing antigen, FetA, which constitutes a major part of the 4CMenB OMV. Thus, 4CMenB vaccine responses may impact or be impacted by nasopharyngeal carriage of commensal neisseriae. This highlights an area for further research and surveillance should the vaccine be routinely implemented.

  11. Genome sequence analyses show that Neisseria oralis is the same species as 'Neisseria mucosa var. heidelbergensis'.

    PubMed

    Bennett, Julia S; Jolley, Keith A; Maiden, Martin C J

    2013-10-01

    Phylogenies generated from whole genome sequence (WGS) data provide definitive means of bacterial isolate characterization for typing and taxonomy. The species status of strains recently defined with conventional taxonomic approaches as representing Neisseria oralis was examined by the analysis of sequences derived from WGS data, specifically: (i) 53 Neisseria ribosomal protein subunit (rps) genes (ribosomal multi-locus sequence typing, rMLST); and (ii) 246 Neisseria core genes (core genome MLST, cgMLST). These data were compared with phylogenies derived from 16S and 23S rRNA gene sequences, demonstrating that the N. oralis strains were monophyletic with strains described previously as representing 'Neisseria mucosa var. heidelbergensis' and that this group was of equivalent taxonomic status to other well-described species of the genus Neisseria. Phylogenetic analyses also indicated that Neisseria sicca and Neisseria macacae should be considered the same species as Neisseria mucosa and that Neisseria flavescens should be considered the same species as Neisseria subflava. Analyses using rMLST showed that some strains currently defined as belonging to the genus Neisseria were more closely related to species belonging to other genera within the family; however, whole genome analysis of a more comprehensive selection of strains from within the family Neisseriaceae would be necessary to confirm this. We suggest that strains previously identified as representing 'N. mucosa var. heidelbergensis' and deposited in culture collections should be renamed N. oralis. Finally, one of the strains of N. oralis was able to ferment lactose, due to the presence of β-galactosidase and lactose permease genes, a characteristic previously thought to be unique to Neisseria lactamica, which therefore cannot be thought of as diagnostic for this species; however, the rMLST and cgMLST analyses confirm that N. oralis is most closely related to N. mucosa.

  12. Construction and Functional Activities of Chimeric Mouse-Human Immunoglobulin G and Immunoglobulin M Antibodies against the Neisseria meningitidis PorA P1.7 and P1.16 Epitopes

    PubMed Central

    Michaelsen, Terje E.; Ihle, Øistein; Beckstrøm, Karen Johanne; Herstad, Tove K.; Kolberg, Jan; Høiby, E. Arne; Aase, Audun

    2003-01-01

    We studied the in vitro protective activities of human immunoglobulin G1 (IgG1), IgG3, and IgM antibodies against group B meningococci by constructing sets of chimeric mouse-human antibodies (chIgG1, chIgG3, and chIgM, respectively) with identical binding regions against the P1.7 and P1.16 epitopes on PorA. This was done by cloning the V genes of three mouse hybridoma antibodies and subsequently transfecting vectors containing the homologous heavy- and light-chain genes into NSO cells. Cell clones secreting intact human chIgG1, chIgG3, or chIgM antibodies originating from three parent mouse antibodies were isolated. The functional affinities appeared to be similar for all human isotypes and surprisingly also for the pentameric chIgM antibody. chIgG1 exhibited greater serum bactericidal activity (SBA) than chIgG3, while chIgG3 was more efficient in inducing a respiratory burst (RB) associated with opsonophagocytosis than chIgG1 was. On the other hand, chIgM exhibited SBA similar to that of chIgG1, but it exhibited much higher RB activity than chIgG3 and chIgG1 exhibited. The antibodies against the P1.16 epitope were more efficient in terms of SBA than the antibodies against the P1.7 epitope were; thus, 10- to 40-fold-lower concentrations of antibodies against P1.16 than of antibodies against P1.7 were needed to induce SBA. On the other hand, antibodies against these epitopes were equally effective in inducing RB. Our results revealed differences in the functional activities of human chIgG1, chIgG3, and chIgM antibodies against meningococci, which might influence their protective effects against meningococcal disease. PMID:14500492

  13. Molecular typing of Neisseria perflava clinical isolates.

    PubMed

    Mechergui, Arij; Achour, Wafa; Giorgini, Dario; Baaboura, Rekaya; Taha, Muhamed-Kheir; Hassen, Assia Ben

    2013-09-01

    Multilocus sequence typing and pulsed-field gel electrophoresis were used to type 22 commensal isolates of Neisseria perflava collected by swabbing from neutropenic patients. High genetic diversity was found among our N. perflava clinical isolates.

  14. Neisseria Base: a comparative genomics database for Neisseria meningitidis.

    PubMed

    Katz, Lee S; Humphrey, Jay C; Conley, Andrew B; Nelakuditi, Viswateja; Kislyuk, Andrey O; Agrawal, Sonia; Jayaraman, Pushkala; Harcourt, Brian H; Olsen-Rasmussen, Melissa A; Frace, Michael; Sharma, Nitya V; Mayer, Leonard W; Jordan, I King

    2011-01-01

    Neisseria meningitidis is an important pathogen, causing life-threatening diseases including meningitis, septicemia and in some cases pneumonia. Genomic studies hold great promise for N. meningitidis research, but substantial database resources are needed to deal with the wealth of information that comes with completely sequenced and annotated genomes. To address this need, we developed Neisseria Base (NBase), a comparative genomics database and genome browser that houses and displays publicly available N. meningitidis genomes. In addition to existing N. meningitidis genome sequences, we sequenced and annotated 19 new genomes using 454 pyrosequencing and the CG-Pipeline genome analysis tool. In total, NBase hosts 27 complete N. meningitidis genome sequences along with their associated annotations. The NBase platform is designed to be scalable, via the underlying database schema and modular code architecture, such that it can readily incorporate new genomes and their associated annotations. The front page of NBase provides user access to these genomes through searching, browsing and downloading. NBase search utility includes BLAST-based sequence similarity searches along with a variety of semantic search options. All genomes can be browsed using a modified version of the GBrowse platform, and a plethora of information on each gene can be viewed using a customized details page. NBase also has a whole-genome comparison tool that yields single-nucleotide polymorphism differences between two user-defined groups of genomes. Using the virulent ST-11 lineage as an example, we demonstrate how this comparative genomics utility can be used to identify novel genomic markers for molecular profiling of N. meningitidis. PMID:21930505

  15. Vaccine potential of the Neisseria meningitidis 2086 lipoprotein.

    PubMed

    Fletcher, Leah D; Bernfield, Liesel; Barniak, Vicki; Farley, John E; Howell, Alan; Knauf, Melissa; Ooi, Peggy; Smith, Robert P; Weise, Paige; Wetherell, Mike; Xie, Xiaoling; Zagursky, Robert; Zhang, Ying; Zlotnick, Gary W

    2004-04-01

    A novel antigen that induces cross-reactive bactericidal antibodies against a number of Neisseria meningitidis strains is described. This antigen, a approximately 28-kDa lipoprotein called LP2086, was first observed within a complex mixture of soluble outer membrane proteins (sOMPs) following a series of fractionation, protein purification, and proteomics steps. Approximately 95 different neisserial isolates tested positive by Western blotting and PCR screening methods for the presence of the protein and the gene encoding LP2086. The strains tested included isolates of N. meningitidis serogroups A, B, C, W135, and Y, Neisseria gonorrhoeae, and Neisseria lactamica. To better understand the microheterogeneity of this protein, the 2086 genes from 63 neisserial isolates were sequenced. Two different subfamilies of LP2086 were identified based on deduced amino acid sequence homology. A high degree of amino acid sequence similarity exists within each 2086 subfamily. The highest degree of genetic diversity was seen between the two subfamilies which share approximately 60 to 75% homology at the nucleic acid level. Flow cytometry (fluorescence-activated cell sorting) analyses and electron microscopy indicated that the LP2086 is localized on the outer surface of N. meningitidis. Antiserum produced against a single protein variant was capable of eliciting bactericidal activity against strains expressing different serosubtype antigens. Combining one recombinant lipidated 2086 (rLP2086) variant from each subfamily with two rPorA variants elicited bactericidal activity against all strains tested. The rLP2086 family of antigens are candidates worthy of further vaccine development. PMID:15039331

  16. Prosthetic valve endocarditis due to Neisseria skkuensis, a novel Neisseria species.

    PubMed

    Park, So Yeon; Kang, Seung Ji; Joo, Eun-Jeong; Ha, Young Eun; Baek, Jin Yang; Wi, Yu Mi; Kang, Cheol-In; Chung, Doo Ryeon; Peck, Kyong Ran; Lee, Nam Young; Song, Jae-Hoon

    2012-08-01

    We describe the first reported case of endocarditis due to Neisseria skkuensis. The organism from the blood cultures taken on admission day was identified initially as unidentified Gram-negative cocci by Vitek2. Finally, it was identified as Neisseria skkuensis by 16 rRNA gene sequence analysis.

  17. Neisseria gonorrhoeae: a versatile pathogen.

    PubMed Central

    Easmon, C S; Ison, C A

    1987-01-01

    Neisseria gonorrhoeae is one of the most important causes of sexually transmitted disease. We do not fully understand the pathogenesis of infection with this organism, although recent improvements in immunological and molecular techniques have brought us closer to an answer. These techniques are now also being used to detect and identify N gonorrhoeae and to analyse the epidemiology of gonorrhoea. Plasmid and chromosomal mediated antibiotic resistance increases the difficulty of controlling gonorrhoea. Resistant strains occur all over the world and new patterns of resistance are still emerging. A better understanding of gonococcal pathogenicity is necessary for the development of an effective vaccine. Despite work on pili and outer membrane proteins no vaccine yet exists. The control of gonorrhoea still depends on diagnosis, treatment, and epidemiological control, facilities that are not widely available in many of those parts of the world where gonorrhoea is a major problem. PMID:3117850

  18. NeisseriaBase: a specialised Neisseria genomic resource and analysis platform

    PubMed Central

    Zheng, Wenning; Mutha, Naresh V.R.; Heydari, Hamed; Dutta, Avirup; Siow, Cheuk Chuen; Jakubovics, Nicholas S.; Wee, Wei Yee; Tan, Shi Yang; Ang, Mia Yang; Wong, Guat Jah

    2016-01-01

    Background. The gram-negative Neisseria is associated with two of the most potent human epidemic diseases: meningococcal meningitis and gonorrhoea. In both cases, disease is caused by bacteria colonizing human mucosal membrane surfaces. Overall, the genus shows great diversity and genetic variation mainly due to its ability to acquire and incorporate genetic material from a diverse range of sources through horizontal gene transfer. Although a number of databases exist for the Neisseria genomes, they are mostly focused on the pathogenic species. In this present study we present the freely available NeisseriaBase, a database dedicated to the genus Neisseria encompassing the complete and draft genomes of 15 pathogenic and commensal Neisseria species. Methods. The genomic data were retrieved from National Center for Biotechnology Information (NCBI) and annotated using the RAST server which were then stored into the MySQL database. The protein-coding genes were further analyzed to obtain information such as calculation of GC content (%), predicted hydrophobicity and molecular weight (Da) using in-house Perl scripts. The web application was developed following the secure four-tier web application architecture: (1) client workstation, (2) web server, (3) application server, and (4) database server. The web interface was constructed using PHP, JavaScript, jQuery, AJAX and CSS, utilizing the model-view-controller (MVC) framework. The in-house developed bioinformatics tools implemented in NeisseraBase were developed using Python, Perl, BioPerl and R languages. Results. Currently, NeisseriaBase houses 603,500 Coding Sequences (CDSs), 16,071 RNAs and 13,119 tRNA genes from 227 Neisseria genomes. The database is equipped with interactive web interfaces. Incorporation of the JBrowse genome browser in the database enables fast and smooth browsing of Neisseria genomes. NeisseriaBase includes the standard BLAST program to facilitate homology searching, and for Virulence Factor

  19. NeisseriaBase: a specialised Neisseria genomic resource and analysis platform.

    PubMed

    Zheng, Wenning; Mutha, Naresh V R; Heydari, Hamed; Dutta, Avirup; Siow, Cheuk Chuen; Jakubovics, Nicholas S; Wee, Wei Yee; Tan, Shi Yang; Ang, Mia Yang; Wong, Guat Jah; Choo, Siew Woh

    2016-01-01

    Background. The gram-negative Neisseria is associated with two of the most potent human epidemic diseases: meningococcal meningitis and gonorrhoea. In both cases, disease is caused by bacteria colonizing human mucosal membrane surfaces. Overall, the genus shows great diversity and genetic variation mainly due to its ability to acquire and incorporate genetic material from a diverse range of sources through horizontal gene transfer. Although a number of databases exist for the Neisseria genomes, they are mostly focused on the pathogenic species. In this present study we present the freely available NeisseriaBase, a database dedicated to the genus Neisseria encompassing the complete and draft genomes of 15 pathogenic and commensal Neisseria species. Methods. The genomic data were retrieved from National Center for Biotechnology Information (NCBI) and annotated using the RAST server which were then stored into the MySQL database. The protein-coding genes were further analyzed to obtain information such as calculation of GC content (%), predicted hydrophobicity and molecular weight (Da) using in-house Perl scripts. The web application was developed following the secure four-tier web application architecture: (1) client workstation, (2) web server, (3) application server, and (4) database server. The web interface was constructed using PHP, JavaScript, jQuery, AJAX and CSS, utilizing the model-view-controller (MVC) framework. The in-house developed bioinformatics tools implemented in NeisseraBase were developed using Python, Perl, BioPerl and R languages. Results. Currently, NeisseriaBase houses 603,500 Coding Sequences (CDSs), 16,071 RNAs and 13,119 tRNA genes from 227 Neisseria genomes. The database is equipped with interactive web interfaces. Incorporation of the JBrowse genome browser in the database enables fast and smooth browsing of Neisseria genomes. NeisseriaBase includes the standard BLAST program to facilitate homology searching, and for Virulence Factor

  20. NeisseriaBase: a specialised Neisseria genomic resource and analysis platform.

    PubMed

    Zheng, Wenning; Mutha, Naresh V R; Heydari, Hamed; Dutta, Avirup; Siow, Cheuk Chuen; Jakubovics, Nicholas S; Wee, Wei Yee; Tan, Shi Yang; Ang, Mia Yang; Wong, Guat Jah; Choo, Siew Woh

    2016-01-01

    Background. The gram-negative Neisseria is associated with two of the most potent human epidemic diseases: meningococcal meningitis and gonorrhoea. In both cases, disease is caused by bacteria colonizing human mucosal membrane surfaces. Overall, the genus shows great diversity and genetic variation mainly due to its ability to acquire and incorporate genetic material from a diverse range of sources through horizontal gene transfer. Although a number of databases exist for the Neisseria genomes, they are mostly focused on the pathogenic species. In this present study we present the freely available NeisseriaBase, a database dedicated to the genus Neisseria encompassing the complete and draft genomes of 15 pathogenic and commensal Neisseria species. Methods. The genomic data were retrieved from National Center for Biotechnology Information (NCBI) and annotated using the RAST server which were then stored into the MySQL database. The protein-coding genes were further analyzed to obtain information such as calculation of GC content (%), predicted hydrophobicity and molecular weight (Da) using in-house Perl scripts. The web application was developed following the secure four-tier web application architecture: (1) client workstation, (2) web server, (3) application server, and (4) database server. The web interface was constructed using PHP, JavaScript, jQuery, AJAX and CSS, utilizing the model-view-controller (MVC) framework. The in-house developed bioinformatics tools implemented in NeisseraBase were developed using Python, Perl, BioPerl and R languages. Results. Currently, NeisseriaBase houses 603,500 Coding Sequences (CDSs), 16,071 RNAs and 13,119 tRNA genes from 227 Neisseria genomes. The database is equipped with interactive web interfaces. Incorporation of the JBrowse genome browser in the database enables fast and smooth browsing of Neisseria genomes. NeisseriaBase includes the standard BLAST program to facilitate homology searching, and for Virulence Factor

  1. Cephalosporin Resistance in Neisseria gonorrhoeae

    PubMed Central

    Bala, Manju; Sood, Seema

    2010-01-01

    Gonorrhea, a disease of public health importance, not only leads to high incidence of acute infections and complications but also plays a major role in facilitating human immunodeficiency virus (HIV) acquisition and transmission. One of the major public health needs for gonorrhea control is appropriate, effective treatment. However, treatment options for gonorrhea are diminishing as Neisseria gonorrhoeae have developed resistance to several antimicrobial drugs such as sulfonamides, penicillin, tetracyclines and quinolones. Antimicrobial resistance (AMR) surveillance of N. gonorrhoeae helps establish and maintain the efficacy of standard treatment regimens. AMR surveillance should be continuous to reveal the emergence of new resistant strains, monitor the changing patterns of resistance, and be able to update treatment recommendations so as to assist in disease control. Current treatment guidelines recommend the use of single dose injectable or oral cephalosporins. The emergence and spread of cephalosporin resistant and multi drug resistant N. gonorrhoeae strains, represents a worrying trend that requires monitoring and investigation. Routine clinical laboratories need to be vigilant for the detection of such strains such that strategies for control and prevention could be reviewed and revised from time to time. It will be important to elucidate the genetic mechanisms responsible for decreased susceptibility and future resistance. There is also an urgent need for research of safe, alternative anti-gonococcal compounds that can be administered orally and have effective potency, allowing high therapeutic efficacy (greater than 95.0% cure rate). PMID:20927291

  2. Bacterial hemagglutination by Neisseria gonorrhoeae.

    PubMed Central

    Koransky, J R; Scales, R W; Kraus, S J

    1975-01-01

    Direct bacterial hemagglutination was investigated with 20 clinical isolates of Neisseria gonorrhoeae. The hemagglutination tests were performed by both a macrotechnique with glass slides and a microtechnique with autotrays. Only organisms from form type 1 or 2 colonies caused hemagglutination. There was no statistical difference at a 10% or higher level in hemagglutination powers of type 1 and type 2 organisms, of male urethral and female cervical isolates, and of the eight major human blood types (ABO-Rh). Of seven erythrocyte species tested, only human cells were agglutinated. D-Mannose did not prevent the agglutination. Rabbit antigonococcal serum and high-titer antigonococcal human sera inhibited the hemagglutination. The results suggest the pili are the mediators of hemagglutination and that their specific agglutination of human erythrocytes may be a correlate of their adherence to human mucosal cells in natural infection. Also, although the procedure is presently insensitive, it is possible to detect human antigonococcal antibody by inhibition of direct bacterial hemagglutination. Images PMID:809353

  3. Vaccine development against Neisseria meningitidis

    PubMed Central

    Vogel, Ulrich; Claus, Heike

    2011-01-01

    Summary Meningococcal disease is communicable by close contact or droplet aerosols. Striking features are high case fatality rates and peak incidences of invasive disease in infants, toddlers and adolescents. Vaccine development is hampered by bacterial immune evasion strategies including molecular mimicry. As for Haemophilus influenzae and Streptococcus pneumoniae, no vaccine has therefore been developed that targets all serogroups of Neisseria meningitidis. Polysaccharide vaccines available both in protein conjugated and non‐conjugated form, have been introduced against capsular serogroups A, C, W‐135 and Y, but are ineffective against serogroup B meningococci, which cause a significant burden of disease in many parts of the world. Detoxified outer membrane vesicles are used since decades to elicit protection against epidemic serogroup B disease. Genome mining and biochemical approaches have provided astounding progress recently in the identification of immunogenic, yet reasonably conserved outer membrane proteins. As subcapsular proteins nevertheless are unlikely to immunize against all serogroup B variants, thorough investigation by surrogate assays and molecular epidemiology approaches are needed prior to introduction and post‐licensure of protein vaccines. Research currently addresses the analysis of life vaccines, meningococcus B polysaccharide modifications and mimotopes, as well as the use of N. lactamicaouter membrane vesicles. PMID:21255369

  4. Production of UC-labeled gas in BACTEC Neisseria Differentiation kits by Neisseria cinerea

    SciTech Connect

    Boyce, J.M.; Mitchell, E.B. Jr.; Knapp, J.S.; Buttke, T.M.

    1985-09-01

    Six strains of Neisseria cinerea were tested in BACTEC Neisseria Differentiation kits (Johnston Laboratories, Inc., Towson, Md.), and all yielded positive glucose growth indices and negative maltose and fructose growth indices. These results were similar to those achieved with Neisseria gonorrhoeae. However, most of the N. cinerea isolates tested yielded 3-h glucose growth indices that were lower than those obtained with gonococci. UC-labeled gas was produced significantly faster by N. gonorrhoeae than by N. cinerea. Additional studies suggested that the UC-labeled gas produced by N. cinerea was carbon dioxide. N. cinerea strains were similar to Branhamella catarrhalis strains because both species failed to produce detectable acid from glucose, maltose, sucrose, fructose, and lactose in cysteine-tryptic agar media. However, in contrast to N. cinerea strains, B. catarrhalis strains did not metabolize glucose in BACTEC Neisseria Differentiation kits.

  5. Analytical specificity and sensitivity of the novel dual-target GeneProof Neisseria gonorrhoeae PCR kit for detection of N. gonorrhoeae.

    PubMed

    Golparian, Daniel; Hellmark, Bengt; Unemo, Magnus

    2015-11-01

    Detection of Neisseria gonorrhoeae relies increasingly on nucleic acid amplification tests (NAATs). The specificity of many gonococcal NAATs has been suboptimal and supplementary testing remains recommended in Europe and several additional countries. The novel dual-target GeneProof Neisseria gonorrhoeae PCR kit, targeting porA pseudogene and 16S rRNA gene, showed a high specificity and sensitivity when isolates of non-gonococcal Neisseria and related species (n = 144), and gonococci (n = 104) were tested. However, rare gonococcal porA mutants were only detected in the 16S rRNA gene target and two non-gonococcal isolates showed a low-level cross-reactivity in the 16S rRNA gene target. The detection limit for both targets was 1.5 copies per reaction.

  6. Lipopolysaccharide Engineering in Neisseria meningitidis

    PubMed Central

    Pupo, Elder; Hamstra, Hendrik-Jan; Meiring, Hugo; van der Ley, Peter

    2014-01-01

    Engineering the lipopolysaccharide (LPS) biosynthetic pathway offers the potential to obtain modified derivatives with optimized adjuvant properties. Neisseria meningitidis strain H44/76 was modified by expression of the pagL gene encoding lipid A 3-O-deacylase from Bordetella bronchiseptica and by inactivation of the lgtB gene encoding the terminal oligosaccharide galactosyltransferase. Mass spectrometry analysis of purified mutant LPS was used for detailed compositional analysis of all present molecular species. This determined that the modified LPS was mainly pentaacylated, demonstrating high efficiency of conversion from the hexaacyl to the 3-O-deacylated form by heterologous lipid A 3-O-deacylase (PagL) expression. MS analyses also provided evidence for expression of only one major oligosaccharide glycoform, which lacked the terminal galactose residue as expected from inactivation of the lgtB gene. The immunomodulatory properties of PagL-deacylated LPS were compared with another pentaacyl form obtained from an lpxL1− mutant, which lacks the 2′ secondary acyl chain. Although both LPS mutants displayed impaired capacity to induce production of the pro-inflammatory cytokine IL-6 in the monocytic cell line Mono Mac 6, induction of the Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β-dependent chemokine interferon-γ-induced protein 10 was largely retained only for the lgtB−/pagL+ mutant. Removal of remaining hexaacyl species exclusively present in lgtB−/pagL+ LPS demonstrated that these minor species potentiate but do not determine the activity of this LPS. These results are the first to indicate a qualitatively different response of human innate cells to pentaacyl lpxL1− and pagL+ LPS and show the importance of detailed structure-function analysis when working with modified lipid A structures. The pagL+ LPS has significant potential as immune modulator in humans. PMID:24492609

  7. Cervical spondylitis due to Neisseria meningitidis.

    PubMed

    Mendes, Stéphanie; Bémer, Pascale; Corvec, Stéphane; Faure, Alexis; Redon, Hervé; Drugeon, Henri B

    2006-05-01

    The diverse clinical spectrum of meningococcal infections includes frequent clinical forms, such as meningitis or septicemia, and uncommon manifestations, such as septic arthritis. Neisseria meningitidis is not generally considered to be a causative agent of osteoarticular infections. We report the first case of acute primary cervical spondylitis in a 48-year-old man.

  8. Cervical spondylitis due to Neisseria meningitidis.

    PubMed

    Mendes, Stéphanie; Bémer, Pascale; Corvec, Stéphane; Faure, Alexis; Redon, Hervé; Drugeon, Henri B

    2006-05-01

    The diverse clinical spectrum of meningococcal infections includes frequent clinical forms, such as meningitis or septicemia, and uncommon manifestations, such as septic arthritis. Neisseria meningitidis is not generally considered to be a causative agent of osteoarticular infections. We report the first case of acute primary cervical spondylitis in a 48-year-old man. PMID:16618455

  9. Neisseria proteomics for antigen discovery and vaccine development.

    PubMed

    Christodoulides, Myron

    2014-10-01

    Neisseria meningitidis (meningococcus) is a major causative organism of meningitis and sepsis and Neisseria gonorrhoeae (gonococcus) is the causative organism of the sexually transmitted disease gonorrhea. Infections caused by meningococci are vaccine-preventable, whereas gonococcal vaccine research and development has languished for decades and the correlates of protection are still largely unknown. In the past two decades, complementary 'omic' platforms have been developed to interrogate Neisseria genomes and gene products. Proteomic techniques applied to whole Neisseria bacteria, outer membranes and outer membrane vesicle vaccines have generated protein maps and also allowed the examination of environmental stresses on protein expression. In particular, immuno-proteomics has identified proteins whose expression is correlated with the development of human natural immunity to meningococcal infection and colonization and following vaccination. Neisseria proteomic techniques have produced a catalog of potential vaccine antigens and investigating the functional and biological properties of these proteins could finally provide 'universal' Neisseria vaccines.

  10. Nucleotide sequence composition and method for detection of neisseria gonorrhoeae

    SciTech Connect

    Lo, A.; Yang, H.L.

    1990-02-13

    This patent describes a composition of matter that is specific for {ital Neisseria gonorrhoeae}. It comprises: at least one nucleotide sequence for which the ratio of the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria gonorrhoeae} to the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria meningitidis} is greater than about five. The ratio being obtained by a method described.

  11. Neisseria wadsworthii sp. nov. and Neisseria shayeganii sp. nov., isolated from clinical specimens.

    PubMed

    Wolfgang, William J; Carpenter, Andrea N; Cole, Jocelyn A; Gronow, Sabine; Habura, Andrea; Jose, Sherly; Nazarian, Elizabeth J; Kohlerschmidt, Donna J; Limberger, Ronald; Schoonmaker-Bopp, Dianna; Spröer, Cathrin; Musser, Kimberlee A

    2011-01-01

    An analysis of 16S rRNA gene sequences from archived clinical reference specimens has identified two novel Neisseria species. For each species, two strains from independent sources were identified. Amongst species with validly published names, the closest species to the newly identified organisms were Neisseria canis, N. dentiae, N. zoodegmatis, N. animaloris and N. weaveri. DNA-DNA hybridization studies demonstrated that the newly identified isolates represent species that are distinct from these nearest neighbours. Analysis of partial 23S rRNA gene sequences for the newly identified strains and their nearest neighbours provided additional support for the species designation. Bayesian analysis of 16S rRNA gene sequences suggested that the newly identified isolates belong to distinct but related species of the genus Neisseria, and are members of a clade that includes N. dentiae, N. bacilliformis and N. canis. The predominant cellular fatty acids [16 : 0, summed feature 3 (16 : 1ω7c and/or iso-15 : 0 2-OH) and 18 : 1ω7c], as well as biochemical and morphological analyses further support the designation of Neisseria wadsworthii sp. nov. (type strain 9715(T) =DSM 22247(T) =CIP 109934(T)) and Neisseria shayeganii sp. nov. (type strain 871(T) =DSM 22246(T) =CIP 109933(T)).

  12. Neisseria mucosus septicemia after near-drowning.

    PubMed

    Manser, T J; Warner, J F

    1987-10-01

    Neisseria mucosus is a normal inhabitant of the upper respiratory tract which may rarely cause serious infection. A 21-year-old woman had N mucosus bacteremia and the adult respiratory distress syndrome (ARDS) after a near-drowning episode. Despite appropriate antibiotic therapy and intensive respiratory support, the patient died. Bacteremia with this organism is rare, and we believe it has not previously been reported in association with near-drowning.

  13. Identification of Regions of the Chromosome of Neisseria meningitidis and Neisseria gonorrhoeae Which Are Specific to the Pathogenic Neisseria Species

    PubMed Central

    Perrin, Agnes; Nassif, Xavier; Tinsley, Colin

    1999-01-01

    Neisseria meningitidis and Neisseria gonorrhoeae give rise to dramatically different diseases. Their interactions with the host, however, do share common characteristics: they are both human pathogens which do not survive in the environment and which colonize and invade mucosa at their port of entry. It is therefore likely that they have common properties that might not be found in nonpathogenic bacteria belonging to the same genetically related group, such as Neisseria lactamica. Their common properties may be determined by chromosomal regions found only in the pathogenic Neisseria species. To address this issue, we used a previously described technique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109–11114, 1996) to identify sequences of DNA specific for pathogenic neisseriae and not found in N. lactamica. Sequences present in N. lactamica were physically subtracted from the N. meningitidis Z2491 sequence and also from the N. gonorrhoeae FA1090 sequence. The clones obtained from each subtraction were tested by Southern blotting for their reactivity with the three species, and only those which reacted with both N. meningitidis and N. gonorrhoeae (i.e., not specific to either one of the pathogens) were further investigated. In a first step, these clones were mapped onto the chromosomes of both N. meningitidis and N. gonorrhoeae. The majority of the clones were arranged in clusters extending up to 10 kb, suggesting the presence of chromosomal regions common to N. meningitidis and N. gonorrhoeae which distinguish these pathogens from the commensal N. lactamica. The sequences surrounding these clones were determined from the N. meningitidis genome-sequencing project. Several clones corresponded to previously described factors required for colonization and survival at the port of entry, such as immunoglobulin A protease and PilC. Others were homologous to virulence-associated proteins in other bacteria, demonstrating that the subtractive clones are

  14. Antimicrobial Resistance and Neisseria gonorrhoeae Multiantigen Sequence Typing Profile of Neisseria gonorrhoeae in New Delhi, India.

    PubMed

    Mahajan, Neeraj; Sood, Seema; Singh, Rajendra; Kapil, Arti; Das, Bimal Kumar; Sreenivas, Vishnubhatla; Kar, Hemanta Kumar; Sharma, Vinod Kumar

    2016-08-01

    Molecular epidemiology of 100 consecutive gonococcal isolates collected between April 2010 and October 2013 from New Delhi was investigated using Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) along with its association with antimicrobial resistance profiles. Neisseria gonorrhoeae isolates were assigned into 60 different sequence types and 43 (71.6%) were novel. Sole representation was seen in 76.6% sequence types. There was significant association between ST6058 and resistance to penicillin (P = 0.00) and tetracycline (P = 0.002). PMID:27414684

  15. Mechanisms of iron acquisition by the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae.

    PubMed

    Rohde, Kyle H; Dyer, David W

    2003-09-01

    It is well established that bacterial pathogenesis is dependent on the ability to acquire iron within the host. The success of the highly adapted obligate human pathogens Neisseria meningitidis (NM) and Neisseria gonorrhoeae (NG) can be attributed in part to the efficient utilization of multiple host iron (Fe) sources, allowing replication on mucosal surfaces, in the bloodstream, and intracellularly. Most Gram-negative bacterial strategies for scavenging iron from the human host rely on the TonB protein to energize active iron transport across the outer membrane. Pathogenic Neisseria express multiple high-affinity iron transporters including a family of two-component TonB-dependent receptors as well as multiple single-component TonB-dependent Fe transporters. This review describes our current understanding of the mechanisms Neisseria have evolved to utilize various iron sources encountered during infection of the human host. Recent studies have provided insight into the interaction of neisserial outer membrane receptors with host iron carrier proteins. Emerging structural information on neisserial iron transporters will be compared with the crystal structures and biochemical data available for homologous Escherichia coli TonB-dependent Fe-siderophore receptors. In the process, we will highlight the aspects of the iron transport process that are unique and those that remain to be experimentally demonstrated in Neisseria. These include receptor structure/function, the mechanism of iron removal from protein ligands, the fate of Fe and heme-Fe after traversing the outer membrane, and the role of TonB-associated energy in receptor functions. Finally, we will discuss regulatory mechanisms that control the expression of iron scavenging systems. The investigation of iron metabolism in NM and NG is important for understanding the biochemistry of this virulence factor, the development of vaccines targeted at outer membrane iron receptors, and therapeutic interventions

  16. Historical perspectives and identification of Neisseria and related species.

    PubMed Central

    Knapp, J S

    1988-01-01

    The pathogenic Neisseria spp., N. gonorrhoeae and N. meningitidis, have been studied extensively and rapid identification procedures have been designed to distinguish these species from the commensal Neisseria and related species that are normal flora of the oro- and nasopharynx. The commensal Neisseria spp. have been largely ignored except for isolated studies. It is important that we know about these species, however, because not only may some be misidentified as pathogenic species if identified with inappropriate procedures, but also they may occasionally be isolated from unusual sites and must be correctly identified to the species level for clinical purposes. PMID:3069201

  17. Resistance to peroxynitrite in Neisseria gonorrhoeae.

    PubMed

    Barth, Kenneth R; Isabella, Vincent M; Wright, Lori F; Clark, Virginia L

    2009-08-01

    Neisseria gonorrhoeae encodes a number of important genes that aid in survival during times of oxidative stress. The same immune cells capable of oxygen-dependent killing mechanisms also have the capacity to generate reactive nitrogen species (RNS) that may function antimicrobially. F62 and eight additional gonococcal strains displayed a high level of resistance to peroxynitrite, while Neisseria meningitidis and Escherichia coli showed a four- to seven-log and a four-log decrease in viability, respectively. Mutation of gonococcal orthologues that are known or suspected to be involved in RNS defence in other bacteria (ahpC, dnrN and msrA) resulted in no loss of viability, suggesting that N. gonorrhoeae has a novel mechanism of resistance to peroxynitrite. Whole-cell extracts of F62 prevented the oxidation of dihydrorhodamine, and decomposition of peroxynitrite was not dependent on ahpC, dnrN or msrA. F62 grown in co-culture with E. coli strain DH10B was shown to protect E. coli viability 10-fold. Also, peroxynitrite treatment of F62 did not result in accumulation of nitrated proteins, suggesting that an active peroxynitrite reductase is responsible for peroxynitrite decomposition rather than a protein sink for amino acid modification. PMID:19406894

  18. Neisseria Prophage Repressor Implicated in Gonococcal Pathogenesis

    PubMed Central

    Daou, Nadine; Yu, Chunxiao; Mcclure, Ryan; Gudino, Cynthia; Reed, George W.

    2013-01-01

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can infect and colonize multiple mucosal sites in both men and women. The ability to cope with different environmental conditions requires tight regulation of gene expression. In this study, we identified and characterized a gonococcal transcriptional regulatory protein (Neisseria phage repressor [Npr]) that was previously annotated as a putative gonococcal phage repressor protein. Npr was found to repress transcription of NGNG_00460 to NGNG_00463 (NGNG_00460-00463), an operon present within the phage locus NgoΦ4. Npr binding sites within the NGNG_00460-00463 promoter region were found to overlap the −10 and −35 promoter motifs. A gonococcal npr mutant demonstrated increased adherence to and invasion of human endocervical epithelial cells compared to a wild-type gonococcal strain. Likewise, the gonococcal npr mutant exhibited enhanced colonization in a gonococcal mouse model of mucosal infection. Analysis of the gonococcal npr mutant using RNA sequence (RNA-seq) analysis demonstrated that the Npr regulon is limited to the operon present within the phage locus. Collectively, our studies have defined a new gonococcal phage repressor protein that controls the transcription of genes implicated in gonococcal pathogenesis. PMID:23876804

  19. Neisseria meningitidis serogroup A capsular polysaccharide acetyltransferase, methods and compositions

    DOEpatents

    Stephens, David S.; Gudlavalleti, Seshu K.; Tzeng, Yih-Ling; Datta, Anup K.; Carlson, Russell W.

    2011-02-08

    Provided are methods for recombinant production of an O-acetyltransferase and methods for acetylating capsular polysaccharides, especially those of a Serogroup A Neisseria meningitidis using the recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.

  20. Defenses against oxidative stress in Neisseria gonorrhoeae and Neisseria meningitidis: distinctive systems for different lifestyles.

    PubMed

    Seib, Kate L; Tseng, Hsing-Ju; McEwan, Alastair G; Apicella, Michael A; Jennings, Michael P

    2004-07-01

    Defenses against oxidative stress are crucial for the survival of the pathogens Neisseria meningitidis and Neisseria gonorrhoeae. An Mn(II) uptake system is involved in manganese (Mn)-dependent resistance to superoxide radicals in N. gonorrhoeae. Here, we show that accumulation of Mn also confers resistance to hydrogen peroxide killing via a catalase-independent mechanism. An mntC mutant of N. meningitidis is susceptible to oxidative killing, but supplementation of growth media with Mn does not enhance the organism's resistance to oxidative killing. N. meningitidis is able to grow in the presence of millimolar levels of Mn ion, in contrast to N. gonorrhoeae, whose growth is retarded at Mn concentrations >100 micromol/L, indicating that Mn homeostasis in the 2 species is probably quite different. N. meningitidis superoxide dismutase B plays a role in protection against oxidative killing. However, a sodC mutant of N. meningitidis is no more sensitive to oxidative killing than is the wild type. A cytochrome c peroxidase (Ccp) is present in N. gonorrhoeae but not in N. meningitidis. Investigations of a ccp mutant revealed a role for Ccp in protection against hydrogen peroxide killing. These differences in oxidative defenses in the pathogenic Neisseria are most likely a result of their localization in different ecological niches. PMID:15195253

  1. Neisseria lactamica and Neisseria polysaccharea as possible sources of meningococcal beta-lactam resistance by genetic transformation.

    PubMed Central

    Saez-Nieto, J A; Lujan, R; Martinez-Suarez, J V; Berron, S; Vazquez, J A; Viñas, M; Campos, J

    1990-01-01

    We studied the susceptibilities of relatively penicillin G-resistant and -susceptible strains of Neisseria meningitidis, as well as Neisseria lactamica and Neisseria polysaccharea, to penicillin, ampicillin, and several cephalosporins. The MICs of penicillin, ampicillin, cephalothin, and cefuroxime for moderately resistant meningococci have increased two- to sixfold in relation to MICs for susceptible strains. For these strains of meningococci, N. lactamica, and N. polysaccharea, penicillin, ampicillin, cephalothin, and cefuroxime MICs for 50 and 90% of strains were similar. By genetic transformation of a penicillin-susceptible strain of N. meningitidis to low-level penicillin resistance with DNA from penicillin-resistant strains of N. meningitidis, N. lactamica, N. polysaccharea, and N. gonorrhoeae, isogenic strains with the same pattern of resistance to beta-lactams were obtained, suggesting that these commensal Neisseria spp. could be the source of meningococcal resistance genes. PMID:2127349

  2. Update on Quinolone Resistance in Neisseria gonorrhoeae.

    PubMed

    Zenilman, Jonathan M.

    2002-04-01

    Quinolones are widely used for treating gonococcal infections, typically in single-dose, oral regimens. However, in the 1990s, quinolone-resistant Neisseria gonorrhoeae emerged, potentially compromising the utility of this drug class. In the past year, these strains have widely disseminated, accounting for over half of isolates in parts of Southeast Asia. The molecular mechanism of resistance has been localized to multiple mutations in genes coding for the bacterial DNA gyrase and topoisomerase enzymes. These mutations accumulate until the minimum inhibitory concentration is 4.0 g/mL or more, which in clinical studies appears to be the threshold for clinical treatment failure. Quinolone-resistant N. gonorrhoeae is independent from other plasmid- and chromosomally-mediated resistance determinants; nearly all isolates to date have been sensitive to cephalosporins and spectinomycin. Nevertheless, designing public health strategies to contain quinolone-resistant N. gonorrhoeae will be difficult. PMID:11927047

  3. Neisseria meningitidis infection: who, when and where?

    PubMed

    Gianchecchi, Elena; Torelli, Alessandro; Piccini, Giulia; Piccirella, Simona; Montomoli, Emanuele

    2015-01-01

    Neisseria meningitidis is a Gram-negative β-proteobacterium responsible for an endemic worldwide infection. The epidemiology and serogroup distribution can change very quickly. The incidence of meningitis infection varies from very rare to more than 1000 cases per 100,000 of the population yearly. The carriage of N. meningitidis, which represents an exclusive human commensal, is asymptomatic, but in rare cases bacteria proliferate in the CNS and rapidly lead to the death of the affected subjects. Host genetic factors, such as single nucleotide polymorphisms, can promote meningococcal disease, being able to influence the individual predisposition to the pathology. Although a reduction in meningococcal disease has been observed in Europe, a continuous surveillance is necessary to control any possible outbreaks of new hypervirulent clones into populations that could modify the epidemiology of meningococcal infections and the clinical spectrum of affected subjects. PMID:26190347

  4. Neisseria meningitidis; clones, carriage, and disease.

    PubMed

    Read, R C

    2014-05-01

    Neisseria meningitidis, the cause of meningococcal disease, has been the subject of sophisticated molecular epidemiological investigation as a consequence of the significant public health threat posed by this organism. The use of multilocus sequence typing and whole genome sequencing classifies the organism into clonal complexes. Extensive phenotypic, genotypic and epidemiological information is available on the PubMLST website. The human nasopharynx is the sole ecological niche of this species, and carrier isolates show extensive genetic diversity as compared with hyperinvasive lineages. Horizontal gene exchange and recombinant events within the meningococcal genome during residence in the human nasopharynx result in antigenic diversity even within clonal complexes, so that individual clones may express, for example, more than one capsular polysaccharide (serogroup). Successful clones are capable of wide global dissemination, and may be associated with explosive epidemics of invasive disease. PMID:24766477

  5. Peptidoglycan fragment release from Neisseria meningitidis.

    PubMed

    Woodhams, Katelynn L; Chan, Jia Mun; Lenz, Jonathan D; Hackett, Kathleen T; Dillard, Joseph P

    2013-09-01

    Neisseria meningitidis (meningococcus) is a symbiont of the human nasopharynx. On occasion, meningococci disseminate from the nasopharynx to cause invasive disease. Previous work showed that purified meningococcal peptidoglycan (PG) stimulates human Nod1, which leads to activation of NF-κB and production of inflammatory cytokines. No studies have determined if meningococci release PG or activate Nod1 during infection. The closely related pathogen Neisseria gonorrhoeae releases PG fragments during normal growth. These fragments induce inflammatory cytokine production and ciliated cell death in human fallopian tubes. We determined that meningococci also release PG fragments during growth, including fragments known to induce inflammation. We found that N. meningitidis recycles PG fragments via the selective permease AmpG and that meningococcal PG recycling is more efficient than gonococcal PG recycling. Comparison of PG fragment release from N. meningitidis and N. gonorrhoeae showed that meningococci release less of the proinflammatory PG monomers than gonococci and degrade PG to smaller fragments. The decreased release of PG monomers by N. meningitidis relative to N. gonorrhoeae is partly due to ampG, since replacement of gonococcal ampG with the meningococcal allele reduced PG monomer release. Released PG fragments in meningococcal supernatants induced significantly less Nod1-dependent NF-κB activity than released fragments in gonococcal supernatants and tended to induce less interleukin-8 (IL-8) secretion in primary human fallopian tube explants. These results support a model in which efficient PG recycling and extensive degradation of PG fragments lessen inflammatory responses and may be advantageous for maintaining meningococcal carriage in the nasopharynx.

  6. Conservation of meningococcal antigens in the genus Neisseria.

    PubMed

    Muzzi, Alessandro; Mora, Marirosa; Pizza, Mariagrazia; Rappuoli, Rino; Donati, Claudio

    2013-06-11

    Neisseria meningitidis, one of the major causes of bacterial meningitis and sepsis, is a member of the genus Neisseria, which includes species that colonize the mucosae of many animals. Three meningococcal proteins, factor H-binding protein (fHbp), neisserial heparin-binding antigen (NHBA), and N. meningitidis adhesin A (NadA), have been described as antigens protective against N. meningitidis of serogroup B, and they have been employed as vaccine components in preclinical and clinical studies. In the vaccine formulation, fHbp and NHBA were fused to the GNA2091 and GNA1030 proteins, respectively, to enhance protein stability and immunogenicity. To determine the possible impact of vaccination on commensal neisseriae, we determined the presence, distribution, and conservation of these antigens in the available genome sequences of the genus Neisseria, finding that fHbp, NHBA, and NadA were conserved only in species colonizing humans, while GNA1030 and GNA2091 were conserved in many human and nonhuman neisseriae. Sequence analysis showed that homologous recombination contributed to shape the evolution and distribution of both NHBA and fHbp, three major variants of which have been defined. fHbp variant 3 was probably the ancestral form of meningococcal fHbp, while fHbp variant 1 from N. cinerea was introduced into N. meningitidis by a recombination event. fHbp variant 2 was the result of a recombination event inserting a stretch of 483 bp from variant 1 into the variant 3 background. These data indicate that a high rate of exchange of genetic material between neisseriae that colonize the human upper respiratory tract exists. IMPORTANCE The upper respiratory tract of healthy individuals is a complex ecosystem colonized by many bacterial species. Among these, there are representatives of the genus Neisseria, including Neisseria meningitidis, a major cause of bacterial meningitis and sepsis. Given the close relationship between commensal and pathogenic species, a protein

  7. Neisseria lactamica selectively induces mitogenic proliferation of the naive B cell pool via cell surface Ig.

    PubMed

    Vaughan, Andrew T; Brackenbury, Louise S; Massari, Paola; Davenport, Victoria; Gorringe, Andrew; Heyderman, Robert S; Williams, Neil A

    2010-09-15

    Neisseria lactamica is a commensal bacteria that colonizes the human upper respiratory tract mucosa during early childhood. In contrast to the closely related opportunistic pathogen Neisseria meningitidis, there is an absence of adaptive cell-mediated immunity to N. lactamica during the peak age of carriage. Instead, outer membrane vesicles derived from N. lactamica mediate a B cell-dependent proliferative response in mucosal mononuclear cells that is associated with the production of polyclonal IgM. We demonstrate in this study that this is a mitogenic human B cell response that occurs independently of T cell help and any other accessory cell population. The ability to drive B cell proliferation is a highly conserved property and is present in N. lactamica strains derived from diverse clonal complexes. CFSE staining of purified human tonsillar B cells demonstrated that naive IgD(+) and CD27(-) B cells are selectively induced to proliferate by outer membrane vesicles, including the innate CD5(+) subset. Neither purified lipooligosaccharide nor PorB from N. lactamica is likely to be responsible for this activity. Prior treatment of B cells with pronase to remove cell-surface Ig or treatment with BCR-specific Abs abrogated the proliferative response to N. lactamica outer membrane vesicles, suggesting that this mitogenic response is dependent upon the BCR.

  8. Meningitis and Bacteremia Due to Neisseria cinerea following a Percutaneous Rhizotomy of the Trigeminal Ganglion

    PubMed Central

    Richter, H.; Bruderer, T.; Goldenberger, D.; Emonet, S.; Strahm, C.

    2015-01-01

    Neisseria cinerea is a human commensal. The first known case of meningitis and bacteremia due to Neisseria cinerea following percutaneous glycerol instillation of the trigeminal ganglion is reported. Conventional phenotypic methods and complete 16S RNA gene sequencing accurately identified the pathogen. Difficulties in differentiation from pathogenic neisseriae are discussed. PMID:26511743

  9. Meningitis and Bacteremia Due to Neisseria cinerea following a Percutaneous Rhizotomy of the Trigeminal Ganglion.

    PubMed

    von Kietzell, M; Richter, H; Bruderer, T; Goldenberger, D; Emonet, S; Strahm, C

    2016-01-01

    Neisseria cinerea is a human commensal. The first known case of meningitis and bacteremia due to Neisseria cinerea following percutaneous glycerol instillation of the trigeminal ganglion is reported. Conventional phenotypic methods and complete 16S RNA gene sequencing accurately identified the pathogen. Difficulties in differentiation from pathogenic neisseriae are discussed.

  10. Structural insight into the lactoferrin receptors from pathogenic Neisseria.

    PubMed

    Noinaj, Nicholas; Cornelissen, Cynthia Nau; Buchanan, Susan K

    2013-10-01

    Neisseria are pathogenic bacteria that cause gonorrhea, septicemia, and meningitis. Like other pathogenic bacteria, Neisseria must acquire iron for survival from their local environment within the human host. Instead of secreting siderophores to scavenge iron, Neisseria steal iron from human iron binding proteins such as hemoglobin, transferrin and lactoferrin for survival. Recently we reported the crystal structures of the Neisseria meningitidis transferrin receptors TbpA and TbpB, as well as the structures of apo and holo human transferrin. We also analyzed these proteins using small angle X-ray scattering and electron microscopy to provide the molecular details explaining how Neisseria are able to interact with and extract iron from transferrin. Here, we utilize the structural reports, as well as the recently reported structure of the N-lobe of LbpB from Moraxella bovis, to assemble improved 3D homology models for the neisserial lactoferrin import receptors LbpA and LbpB, both of which are important vaccine targets against N. meningitidis. We then analyzed these models to gain structural insights into the lactoferrin-iron import system and form a mechanistic model fashioned in parallel to the homologous transferrin-iron import system.

  11. Additive and Synergistic Bactericidal Activity of Antibodies Directed against Minor Outer Membrane Proteins of Neisseria meningitidis▿

    PubMed Central

    Weynants, Vincent E.; Feron, Christiane M.; Goraj, Karine K.; Bos, Martine P.; Denoël, Philippe A.; Verlant, Vincent G.; Tommassen, Jan; Peak, Ian R. A.; Judd, Ralph C.; Jennings, Michael P.; Poolman, Jan T.

    2007-01-01

    Neisseria meningitidis serogroup B is a major cause of bacterial meningitis in younger populations. The available vaccines are based on outer membrane vesicles obtained from wild-type strains. In children less than 2 years old they confer protection only against strains expressing homologous PorA, a major, variable outer membrane protein (OMP). We genetically modified a strain in order to eliminate PorA and to overproduce one or several minor and conserved OMPs. Using a mouse model mimicking children's PorA-specific bactericidal activity, it was demonstrated that overproduction of more than one minor OMP is required to elicit antibodies able to induce complement-mediated killing of strains expressing heterologous PorA. It is concluded that a critical density of bactericidal antibodies needs to be reached at the surface of meningococci to induce complement-mediated killing. With minor OMPs, this threshold is reached when more than one antigen is targeted, and this allows cross-protection. PMID:17664268

  12. How clonal are Neisseria species? The epidemic clonality model revisited.

    PubMed

    Tibayrenc, Michel; Ayala, Francisco J

    2015-07-21

    The three species Neisseria meningitidis, Neisseria gonorrheae, and Neisseria lactamica are often regarded as highly recombining bacteria. N. meningitidis has been considered a paradigmatic case of the "semiclonal model" or of "epidemic clonality," demonstrating occasional bouts of clonal propagation in an otherwise recombining species. In this model, occasional clonality generates linkage disequilibrium in the short term. In the long run, however, the effects of clonality are countered by recombination. We show that many data are at odds with this proposal and that N. meningitidis fits the criteria that we have proposed for predominant clonal evolution (PCE). We point out that (i) the proposed way to distinguish epidemic clonality from PCE may be faulty and (ii) the evidence of deep phylogenies by microarrays and whole-genome sequencing is at odds with the predictions of the semiclonal model. Last, we revisit the species status of N. meningitidis, N. gonorrheae, and N. lactamica in the light of the PCE model.

  13. Regulation of capsule in Neisseria meningitidis.

    PubMed

    Tzeng, Yih-Ling; Thomas, Jennifer; Stephens, David S

    2016-09-01

    Neisseria meningitidis, a devastating pathogen exclusive to humans, expresses capsular polysaccharides that are the major meningococcal virulence determinants and the basis for successful meningococcal vaccines. With rare exceptions, the expression of capsule (serogroups A, B, C, W, X, Y) is required for systemic invasive meningococcal disease. Changes in capsule expression or structure (e.g. hypo- or hyper-encapsulation, capsule "switching", acetylation) can influence immunologic diagnostic assays or lead to immune escape. The loss or down-regulation of capsule is also critical in meningococcal biology facilitating meningococcal attachment, microcolony formation and the carriage state at human mucosal surfaces. Encapsulated meningococci contain a cps locus with promoters located in an intergenic region between the biosynthesis and the conserved capsule transport operons. The cps intergenic region is transcriptionally regulated (and thus the amount of capsule expressed) by IS element insertion, by a two-component system, MisR/MisS and through sequence changes that result in post-transcriptional RNA thermoregulation. Reversible on-off phase variation of capsule expression is controlled by slipped strand mispairing of homo-polymeric tracts and by precise insertion and excision of IS elements (e.g. IS1301) in the biosynthesis operon. Capsule structure can be altered by phase-variable expression of capsular polymer modification enzymes or "switched" through transformation and homologous recombination of different polymerases. Understanding the complex regulation of meningococcal capsule has important implications for meningococcal biology, pathogenesis, diagnostics, current and future vaccine development and vaccine strategies.

  14. Temperature triggers immune evasion by Neisseria meningitidis

    PubMed Central

    Loh, Edmund; Kugelberg, Elisabeth; Tracy, Alexander; Zhang, Qian; Gollan, Bridget; Ewles, Helen; Chalmers, Ronald; Pelicic, Vladimir; Tang, Christoph M.

    2013-01-01

    Neisseria meningitidis has multiple strategies to evade complement-mediated killing, which contribute to its ability to cause septicaemic disease and meningitis. However, the meningococcus is primarily an obligate commensal of the human nasopharynx, and it is unclear why the bacterium has evolved exquisite mechanisms to avoid host immunity. Here we demonstrate that mechanisms of meningococcal immune evasion and resistance against complement increase in response to an elevation in ambient temperature. We have identified three independent RNA thermosensors located in the 5′-UTRs of genes necessary for capsule biosynthesis, the expression of factor H binding protein, and sialylation of lipopolysaccharide, which are essential for meningococcal resistance against immune killing1,2. Therefore increased temperature (which occurs during inflammation) acts as a ‘danger signal’ for the meningococcus which enhances defence against human immune killing. Infection with viral pathogens, such as influenza, leads to inflammation in the nasopharynx with an elevated temperature and recruitment of immune effectors3,4. Thermoregulation of immune defence could offer an adaptive advantage to the meningococcus during co-infection with other pathogens, and promote the emergence of virulence in an otherwise commensal bacterium. PMID:24067614

  15. Metabolism and virulence in Neisseria meningitidis.

    PubMed

    Schoen, Christoph; Kischkies, Laura; Elias, Johannes; Ampattu, Biju Joseph

    2014-01-01

    A longstanding question in infection biology addresses the genetic basis for invasive behavior in commensal pathogens. A prime example for such a pathogen is Neisseria meningitidis. On the one hand it is a harmless commensal bacterium exquisitely adapted to humans, and on the other hand it sometimes behaves like a ferocious pathogen causing potentially lethal disease such as sepsis and acute bacterial meningitis. Despite the lack of a classical repertoire of virulence genes in N. meningitidis separating commensal from invasive strains, molecular epidemiology suggests that carriage and invasive strains belong to genetically distinct populations. In recent years, it has become increasingly clear that metabolic adaptation enables meningococci to exploit host resources, supporting the concept of nutritional virulence as a crucial determinant of invasive capability. Here, we discuss the contribution of core metabolic pathways in the context of colonization and invasion with special emphasis on results from genome-wide surveys. The metabolism of lactate, the oxidative stress response, and, in particular, glutathione metabolism as well as the denitrification pathway provide examples of how meningococcal metabolism is intimately linked to pathogenesis. We further discuss evidence from genome-wide approaches regarding potential metabolic differences between strains from hyperinvasive and carriage lineages and present new data assessing in vitro growth differences of strains from these two populations. We hypothesize that strains from carriage and hyperinvasive lineages differ in the expression of regulatory genes involved particularly in stress responses and amino acid metabolism under infection conditions. PMID:25191646

  16. Regulation of capsule in Neisseria meningitidis.

    PubMed

    Tzeng, Yih-Ling; Thomas, Jennifer; Stephens, David S

    2016-09-01

    Neisseria meningitidis, a devastating pathogen exclusive to humans, expresses capsular polysaccharides that are the major meningococcal virulence determinants and the basis for successful meningococcal vaccines. With rare exceptions, the expression of capsule (serogroups A, B, C, W, X, Y) is required for systemic invasive meningococcal disease. Changes in capsule expression or structure (e.g. hypo- or hyper-encapsulation, capsule "switching", acetylation) can influence immunologic diagnostic assays or lead to immune escape. The loss or down-regulation of capsule is also critical in meningococcal biology facilitating meningococcal attachment, microcolony formation and the carriage state at human mucosal surfaces. Encapsulated meningococci contain a cps locus with promoters located in an intergenic region between the biosynthesis and the conserved capsule transport operons. The cps intergenic region is transcriptionally regulated (and thus the amount of capsule expressed) by IS element insertion, by a two-component system, MisR/MisS and through sequence changes that result in post-transcriptional RNA thermoregulation. Reversible on-off phase variation of capsule expression is controlled by slipped strand mispairing of homo-polymeric tracts and by precise insertion and excision of IS elements (e.g. IS1301) in the biosynthesis operon. Capsule structure can be altered by phase-variable expression of capsular polymer modification enzymes or "switched" through transformation and homologous recombination of different polymerases. Understanding the complex regulation of meningococcal capsule has important implications for meningococcal biology, pathogenesis, diagnostics, current and future vaccine development and vaccine strategies. PMID:26089023

  17. Diagnosis of Neisseria gonorrhoeae using molecular beacon.

    PubMed

    Sachdev, Divya; Patel, Achchhe Lal; Sonkar, Subash Chandra; Kumari, Indu; Saluja, Daman

    2015-01-01

    Neisseria gonorrhoeae is an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit and rmp gene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries.

  18. Biodegradation of cellulose acetate by Neisseria sicca.

    PubMed

    Sakai, K; Yamauchi, T; Nakasu, F; Ohe, T

    1996-10-01

    Bacteria capable of assimilating cellulose acetate, strains SB and SC, were isolated from soil on a medium containing cellulose acetate as a carbon source, and identified as Neisseria sicca. Both strains degraded cellulose acetate membrane filters (degree of substitution, DS, mixture of 2.8 and 2.0) and textiles (DS, 2.34) in a medium containing cellulose acetate (DS, 2.34) or its oligomer, but were not able to degrade these materials in a medium containing cellobiose octaacetate. Biodegradation of cellulose acetate (DS, 1.81 and 2.34) on the basis of biochemical oxygen demand reached 51 and 40% in the culture of N. sicca SB and 60 and 45% in the culture of N. sicca SC within 20 days. A decrease in the acetyl content of degraded cellulose acetate films and powder was confirmed by infrared and nuclear magnetic resonance analyses. After 10-day cultivation of N. sicca SB and SC, the number-average molecular weight of residual cellulose acetate decreased by 9 and 5%, respectively. Activities of enzymes that released acetic acid and produced reducing sugars from cellulose acetate were mainly present in the culture supernatant. Reactivity of enzymes for cellulose acetate (DS, 1.81) was higher than that for cellulose acetate (DS, 2.34).

  19. Non-pathogenic Neisseria: members of an abundant, multi-habitat, diverse genus.

    PubMed

    Liu, Guangyu; Tang, Christoph M; Exley, Rachel M

    2015-07-01

    The genus Neisseria contains the important pathogens Neisseria meningitidis and Neisseria gonorrhoeae. These Gram-negative coccoid bacteria are generally thought to be restricted to humans and inhabit mucosal surfaces in the upper respiratory and genito-urinary tracts. While the meningococcus and gonococcus have been widely studied, far less attention has been paid to other Neisseria species. Here we review current knowledge of the distribution of commensal Neisseria in humans and other hosts. Analysis of the microbiome has revealed that Neisseria is an abundant member of the oropharyngeal flora, and we review its potential impact on health and disease. Neisseria also exhibit remarkable diversity, exhibiting both coccoid and rod-shaped morphologies, as well as environmental strains which are capable of degrading complex organic molecules.

  20. Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species.

    PubMed

    Mechergui, Arij; Achour, Wafa; Hassen, Assia Ben

    2014-05-01

    In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains.

  1. Isolation and characterization of a Neisseria strain from the liver of a Chinese Peking duck.

    PubMed

    Wang, Hanqing; Chen, Zongyan; Wang, Xiaoxu; Li, Ning; Wei, Jianzhong; Sun, Pei; Wang, Guijun; Liu, Guangqing

    2014-07-01

    A Neisseria strain, Neisseria sp. AH-N10, was isolated from liver of a Chinese Peking duck and characterized using a number of phenotypic and genotypic approaches. Based on scanning electron microscopy examination, the isolated strain has the typical structure of Neisseria species. Sequence comparison of 16S rRNA gene and phylogenetic analysis suggest that Neisseria sp. AH-N10 is closely related to Neisseria canis, which was previously isolated from a human dog bite wound. Animal infection experiments demonstrated that the isolated Neisseria sp. AH-N10 is pathogenic in ducks and mice. The pathogenicity to humans and evolutional origin of this Neisseria strain should be further investigated.

  2. Neisseria weaveri Andersen et al. 1993 is a later heterotypic synonym of Neisseria weaveri Holmes et al. 1993.

    PubMed

    Yi, Hana; Chun, Jongsik

    2015-02-01

    Two species of the genus Neisseria, namely Neisseria weaveri Andersen et al. 1993 and Neisseria weaveri Holmes et al. 1993, were simultaneously proposed and described in the same volume of International Journal of Systematic Bacteriology, and have been maintained as heterotypic homonyms. However, the identical 16S rRNA gene sequence and high (99.1 %) average nucleotide identity (ANI) between the genome sequences of the two type strains implied that these two taxa should be united as a single genomic species. To clarify their taxonomic status, phenotypic properties including enzymic activities and substrate-utilization profiles were investigated. The results demonstrated that the two taxa have no pronounced differences and should constitute a single species. Therefore, the reclassification of N. weaveri Andersen et al. 1993 as a later heterotypic synonym of N. weaveri Holmes et al. 1993 is proposed.

  3. An evaluation of the role of properdin in alternative pathway activation on Neisseria meningitidis and Neisseria gonorrhoeae.

    PubMed

    Agarwal, Sarika; Ferreira, Viviana P; Cortes, Claudio; Pangburn, Michael K; Rice, Peter A; Ram, Sanjay

    2010-07-01

    Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against invasive neisserial infections. Recently, commercially available unfractionated properdin was shown to bind to certain biological surfaces, including Neisseria gonorrhoeae, which facilitated C3 deposition. Unfractionated properdin contains aggregates or high-order oligomers, in addition to its physiological "native" (dimeric, trimeric, and tetrameric) forms. We examined the role of properdin in AP activation on diverse strains of Neisseria meningitidis and N. gonorrhoeae specifically using native versus unfractionated properdin. C3 deposition on Neisseria decreased markedly when properdin function was blocked using an anti-properdin mAb or when properdin was depleted from serum. Maximal AP-mediated C3 deposition on Neisseriae even at high (80%) serum concentrations required properdin. Consistent with prior observations, preincubation of bacteria with unfractionated properdin, followed by the addition of properdin-depleted serum resulted in higher C3 deposition than when bacteria were incubated with properdin-depleted serum alone. Unexpectedly, none of 10 Neisserial strains tested bound native properdin. Consistent with its inability to bind to Neisseriae, preincubating bacteria with native properdin followed by the addition of properdin-depleted serum did not cause detectable increases in C3 deposition. However, reconstituting properdin-depleted serum with native properdin a priori enhanced C3 deposition on all strains of Neisseria tested. In conclusion, the physiological forms of properdin do not bind directly to either N. meningitidis or N. gonorrhoeae but play a crucial role in augmenting AP-dependent C3 deposition on the bacteria through the "conventional" mechanism of stabilizing AP C3 convertases. PMID:20530262

  4. Epidemiological characterization of Neisseria gonorrhoeae by lectins.

    PubMed Central

    Schalla, W O; Whittington, W L; Rice, R J; Larsen, S A

    1985-01-01

    A total of 101 isolates of penicillinase-producing and non-penicillinase-producing Neisseria gonorrhoeae with known nutritional requirements, plasmid content, and serovars, were examined for lectin agglutination patterns. These isolates were from outbreaks in Georgia, California, Hawaii, and Pennsylvania. Cell suspensions made from 16- to 18-h cultures were mixed with 14 different lectins, and the resultant agglutination patterns were classified as agglutination groups. Among the 101 isolates tested, 24 different agglutination groups were demonstrated. Of the organisms tested, 55% were located in 3 of the 24 groups, and 86% of the isolates reacted with the lectins Trichosanthes kinlowii, Griffonia simplicifolia I, peanut agglutinin, soybean agglutinin, potato agglutinin, and wheat germ agglutinin. One isolate did not react with peanut or potato agglutinin, five isolates lacked reactivity with potato agglutinin, and six isolates did not react with wheat germ agglutinin. Of the wheat germ-negative isolates, four were from Pennsylvania and were identical with regard to auxotype, plasmid content, serovar, and lectin group. The other two wheat germ-negative isolates were from California and were unrelated by the same criteria to the four Pennsylvania isolates and to each other. Among the isolates tested, there were no differences in lectin groups with regard to the sex of the patient. In the Georgia collection, agglutination with one lectin group was confined to isolates of serogroup IA. This association was not observed for the other geographic areas. Some isolates showing identical auxotype, plasmid content, and serovars could be differentiated based on lectin agglutination patterns, whereas other isolates were identical by all testing criteria. PMID:3930560

  5. Species-specificity of Neisseria gonorrhoeae infection: do human complement regulators contribute?

    PubMed

    Ngampasutadol, Jutamas; Tran, Connie; Gulati, Sunita; Blom, Anna M; Jerse, E Ann; Ram, Sanjay; Rice, Peter A

    2008-12-30

    Neisseria gonorrhoeae is the causative agent of gonorrhea, a disease restricted to humans. Complement forms a key arm of the innate immune system that combats gonococcal infections. N. gonorrhoeae uses its outer membrane porin (Por) molecules to bind complement down-regulatory proteins, C4b-binding protein (C4BP) and factor H (fH), to evade killing by human complement. In addition, sialylation of gonococcal lipooligosaccharide (LOS) also enables N. gonorrhoeae to bind fH. Strains of N. gonorrhoeae that resist killing by human serum complement are killed by serum from rodent, lagomorph and primate species, which cannot be readily infected experimentally with this organism and whose C4BP and/or fH molecules do not bind toN. gonorrhoeae. Serum resistance of gonococci is restored in these sera by human C4BP and/or fH. Direct binding specificity of human and chimpanzee C4BP and human fH to gonococci may explain, in part, species-specific restriction of natural gonococcal infection and address why Por1B, but not Por1A containing gonococcal strains, have been successful in experimental chimpanzee 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.

  6. Unusual soft tissue manifestations of Neisseria meningitidis infections.

    PubMed

    Gupta, Ruchi; Levent, Fatma; Healy, C Mary; Edwards, Morven S

    2008-05-01

    Soft tissue involvement is an unusual presenting feature for children with meningococcal infection. We describe 2 children, 1 with conjunctivitis and another with a thyroglossal duct cyst abscess associated with Neisseria meningitidis, and review previous reports of these entities to emphasize the broad spectrum of meningococcal disease and pertinent aspects of treatment and of prophylaxis of contacts.

  7. Complete Genome Sequence of Neisseria weaveri Strain NCTC13585.

    PubMed

    Alexander, Sarah; Fazal, Mohammed-Abbas; Burnett, Edward; Deheer-Graham, Ana; Oliver, Karen; Holroyd, Nancy; Parkhill, Julian; Russell, Julie E

    2016-01-01

    Neisseria weaveri is a commensal organism of the canine oral cavity and an occasional opportunistic human pathogen which is associated with dog bite wounds. Here we report the first complete genomic sequence of the N. weaveri NCTC13585 (CCUG30381) strain, which was originally isolated from a patient with a canine bite wound. PMID:27563039

  8. Recovery of Neisseria gonorrhoeae from 4 commercially available transport systems.

    PubMed

    Papp, John R; Henning, Tara; Khubbar, Manjeet; Kalve, Valdis; Bhattacharyya, Sanjib; Travanty, Emily; Xavier, Karen; Jones, Kelly; Rudrik, James T; Gaynor, Anne; Hagan, Celia

    2016-10-01

    Four commercial transport systems for the recovery of Neisseria gonorrhoeae were evaluated in support of the need to obtain culture isolates for the detection of antimicrobial resistance. Bacterial recovery from the InTray GC system was superior with minimal loss of viability in contrast to non-nutritive transport systems. PMID:27489119

  9. Isolated pharyngeal Neisseria gonorrhoeae in heterosexual male contacts.

    PubMed

    Thomson-Glover, Rebecca; Brown, Ruth; Edirisinghe, Damitha N

    2013-12-01

    Pharyngeal infection with Neisseria gonorrhoeae (NG) in heterosexual men is thought to be of low prevalence and the value of routinely testing this group of patients is uncertain. We present two cases of NG, isolated only in the pharynx, in asymptomatic heterosexual male contacts. The presence of pharyngeal NG was found irrespective of direct oropharyngeal sexual exposure.

  10. DNA stability of Chlamydia trachomatis and Neisseria gonorrhoeae in urine.

    PubMed

    Le Guern, Rémi; Miaux, Brigitte; Pischedda, Patricia; Herwegh, Stéphanie; Courcol, René

    2016-07-01

    We evaluated the DNA stability of Chlamydia trachomatis and Neisseria gonorrhoeae in 55 urine samples. Crossing threshold (Ct) values were highly similar after 3 to 14 days at room temperature (+0.002, P = 0.99). Consequently, it does not seem necessary to transfer urine specimens into a transport medium in less than 24 hours as recommended by manufacturers. PMID:27130478

  11. The transferrin-iron import system from pathogenic Neisseria species.

    PubMed

    Noinaj, Nicholas; Buchanan, Susan K; Cornelissen, Cynthia Nau

    2012-10-01

    Two pathogenic species within the genus Neisseria cause the diseases gonorrhoea and meningitis. While vaccines are available to protect against four N. meningitidis serogroups, there is currently no commercial vaccine to protect against serogroup B or against N. gonorrhoeae. Moreover, the available vaccines have significant limitations and with antibiotic resistance becoming an alarming issue, the search for effective vaccine targets to elicit long-lasting protection against Neisseria species is becoming more urgent. One strategy for vaccine development has targeted the neisserial iron import systems. Without iron, the Neisseriae cannot survive and, therefore, these iron import systems tend to be relatively well conserved and are promising vaccine targets, having the potential to offer broad protection against both gonococcal and meningococcal infections. These efforts have been boosted by recent reports of the crystal structures of the neisserial receptor proteins TbpA and TbpB, each solved in complex with human transferrin, an iron binding protein normally responsible for delivering iron to human cells. Here, we review the recent structural reports and put them into perspective with available functional studies in order to derive the mechanism(s) for how the pathogenic Neisseriae are able to hijack human iron transport systems for their own survival and pathogenesis. PMID:22957710

  12. Neisseria meningitidis Serogroup X in Sub-Saharan Africa

    PubMed Central

    Agnememel, Alain; Hong, Eva; Giorgini, Dario; Nuñez-Samudio, Viginia; Deghmane, Ala-Eddine

    2016-01-01

    The epidemiology of meningococcal disease varies by geography and time. Whole-genome sequencing of Neisseria meningitidis serogroup X isolates from sub-Saharan Africa and Europe showed that serogroup X emergence in sub-Saharan Africa resulted from expansion of particular variants within clonal complex 181. Virulence of these isolates in experimental mouse models was high. PMID:26982628

  13. Complete Genome Sequence of Neisseria weaveri Strain NCTC13585

    PubMed Central

    Fazal, Mohammed-Abbas; Burnett, Edward; Deheer-Graham, Ana; Oliver, Karen; Holroyd, Nancy; Russell, Julie E.

    2016-01-01

    Neisseria weaveri is a commensal organism of the canine oral cavity and an occasional opportunistic human pathogen which is associated with dog bite wounds. Here we report the first complete genomic sequence of the N. weaveri NCTC13585 (CCUG30381) strain, which was originally isolated from a patient with a canine bite wound. PMID:27563039

  14. Neisseria meningitis GNA1030 is a ubiquinone-8 binding protein.

    PubMed

    Donnarumma, Danilo; Golfieri, Giacomo; Brier, Sébastien; Castagnini, Marta; Veggi, Daniele; Bottomley, Matthew James; Delany, Isabel; Norais, Nathalie

    2015-06-01

    Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).

  15. The transferrin-iron import system from pathogenic Neisseria species.

    PubMed

    Noinaj, Nicholas; Buchanan, Susan K; Cornelissen, Cynthia Nau

    2012-10-01

    Two pathogenic species within the genus Neisseria cause the diseases gonorrhoea and meningitis. While vaccines are available to protect against four N. meningitidis serogroups, there is currently no commercial vaccine to protect against serogroup B or against N. gonorrhoeae. Moreover, the available vaccines have significant limitations and with antibiotic resistance becoming an alarming issue, the search for effective vaccine targets to elicit long-lasting protection against Neisseria species is becoming more urgent. One strategy for vaccine development has targeted the neisserial iron import systems. Without iron, the Neisseriae cannot survive and, therefore, these iron import systems tend to be relatively well conserved and are promising vaccine targets, having the potential to offer broad protection against both gonococcal and meningococcal infections. These efforts have been boosted by recent reports of the crystal structures of the neisserial receptor proteins TbpA and TbpB, each solved in complex with human transferrin, an iron binding protein normally responsible for delivering iron to human cells. Here, we review the recent structural reports and put them into perspective with available functional studies in order to derive the mechanism(s) for how the pathogenic Neisseriae are able to hijack human iron transport systems for their own survival and pathogenesis.

  16. Transcriptional and functional analysis of the Neisseria gonorrhoeae fur regulon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator senses intracellular iron stores and acting as a repressor, directly regulates transcription of iron-responsive genes by binding to a conserve...

  17. Neisseria meningitidis, pathogenetic mechanisms to overcome the human immune defences.

    PubMed

    Gasparini, R; Amicizia, D; Lai, P L; Panatto, D

    2012-06-01

    Neisseria meningitidis is hosted only by humans and colonizes the nasopharynx; it survives in the human body by reaching an equilibrium with its exclusive host. Indeed, while cases of invasive disease are rare, the number of asymptomatic Neisseria meningitides carriers is far higher. The aim of this paper is to summarize the current knowledge of survival strategies of Neisseria meningitides against the human immune defences. Neisseria meningitidis possesses a variety of adaptive characteristics which enable it to avoid being killed by the immune system, such as the capsule, the lipopolysaccharide, groups of proteins that block the action of the antimicrobial proteins (AMP), proteins that inhibit the complement system, and components that prevent both the maturation and the perfect functioning of phagocytes. The main means of adhesion of Neisseria meningitides to the host cells are Pili, constituted by several proteins of whom the most important is Pilin E. Opacity-associated proteins (Opa) and (Opc) are two proteins that make an important contribution to the process of adhesion to the cell. Porins A and B contribute to neisserial adhesion and penetration into the cells, and also inhibit the complement system. Factor H binding protein (fhbp) binds factor H, allowing the bacteria to survive in the blood. Neisserial adhesin A (NadA) is a minor adhesin that is expressed by 50% of the pathogenic strains. NadA is known to be involved in cell adhesion and invasion and in the induction of proinflammatory cytokines. Neisserial heparin binding antigen (NHBA) binds heparin, thus increasing the resistance of the bacterium in the serum.

  18. Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

    PubMed Central

    Peterson, S. W.; Martin, I.; Demczuk, W.; Bharat, A.; Hoang, L.; Wylie, J.; Allen, V.; Lefebvre, B.; Tyrrell, G.; Horsman, G.; Haldane, D.; Garceau, R.; Wong, T.

    2015-01-01

    The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results. PMID:25878350

  19. Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae.

    PubMed

    Peterson, S W; Martin, I; Demczuk, W; Bharat, A; Hoang, L; Wylie, J; Allen, V; Lefebvre, B; Tyrrell, G; Horsman, G; Haldane, D; Garceau, R; Wong, T; Mulvey, M R

    2015-07-01

    The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

  20. Evaluation of six commercial nucleic acid amplification tests for detection of Neisseria gonorrhoeae and other Neisseria species.

    PubMed

    Tabrizi, Sepehr N; Unemo, Magnus; Limnios, Athena E; Hogan, Tiffany R; Hjelmevoll, Stig-Ove; Garland, Susanne M; Tapsall, John

    2011-10-01

    Molecular detection of Neisseria gonorrhoeae in extragenital samples may result in false-positive results due to cross-reaction with commensal Neisseria species or Neisseria meningitidis. This study examined 450 characterized clinical culture isolates, comprising 216 N. gonorrhoeae isolates and 234 isolates of nongonococcal Neisseria species (n = 218) and 16 isolates of other closely related bacteria, with six commercial nucleic acid amplification tests (NAATs). The six NAATs tested were Gen-Probe APTIMA COMBO 2 and APTIMA GC, Roche COBAS Amplicor CT/NG and COBAS 4800 CT/NG tests, BD ProbeTec GC Qx amplified DNA assay, and Abbott RealTime CT/NG test. All assays except COBAS Amplicor CT/NG test where four (1.9%) isolates were not detected showed a positive result with all N. gonorrhoeae isolates (n = 216). Among the 234 nongonococcal isolates examined, initial results from all assays displayed some false-positive results due to cross-reactions. Specifically, the COBAS Amplicor and ProbeTec tests showed the highest number of false-positive results, detecting 33 (14.1%) and 26 (11%) nongonococcal Neisseria isolates, respectively. On the first testing, APTIMA COMBO 2, APTIMA GC, Abbott RealTime, and Roche COBAS 4800 showed lower level of cross-reactions with five (2.1%), four (1.7%), two (1%), and two (1%) of the isolates showing low-level positivity, respectively. Upon retesting of these nine nongonococcal isolates using freshly cultured colonies, none were positive by the APTIMA COMBO 2, Abbott RealTime, or COBAS 4800 test. In conclusion, the COBAS Amplicor and ProbeTec tests displayed high number of false-positive results, while the remaining NAATs showed only sporadic low-level false-positive results. Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites.

  1. Functional comparison of the binding of factor H short consensus repeat 6 (SCR 6) to factor H binding protein from Neisseria meningitidis and the binding of factor H SCR 18 to 20 to Neisseria gonorrhoeae porin.

    PubMed

    Shaughnessy, Jutamas; Lewis, Lisa A; Jarva, Hanna; Ram, Sanjay

    2009-05-01

    Both Neisseria meningitidis and Neisseria gonorrhoeae recruit the alternative pathway complement inhibitory protein factor H (fH) to their surfaces to evade complement-dependent killing. Meningococci bind fH via fH binding protein (fHbp), a surface-exposed lipoprotein that is subdivided into three variant families based on one classification scheme. Chimeric proteins that comprise contiguous domains of fH fused to murine Fc were used to localize the binding site for all three fHbp variants on fH to short consensus repeat 6 (SCR 6). As expected, fH-like protein 1 (FHL-1), which contains fH SCR 6, also bound to fHbp-expressing meningococci. Using site-directed mutagenesis, we identified histidine 337 and histidine 371 in SCR 6 as important for binding to fHbp. These findings may provide the molecular basis for recent observations that demonstrated human-specific fH binding to meningococci. Differences in the interactions of fHbp variants with SCR 6 were evident. Gonococci bind fH via their porin (Por) molecules (PorB.1A or PorB.1B); sialylation of lipooligosaccharide enhances fH binding. Both sialylated PorB.1B- and (unsialylated) PorB.1A-bearing gonococci bind fH through SCR 18 to 20; PorB.1A can also bind SCR 6, but only weakly, as evidenced by a low level of binding of FHL-1 relative to that of fH. Using isogenic strains expressing either meningococcal fHbp or gonococcal PorB.1B, we discovered that strains expressing gonococcal PorB.1B in the presence of sialylated lipooligosaccharide bound more fH, more effectively limited C3 deposition, and were more serum resistant than their isogenic counterparts expressing fHbp. Differences in fH binding to these two related pathogens may be important for modulating their individual responses to host immune attack.

  2. Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria.

    PubMed

    Barth, Kenneth R; Isabella, Vincent M; Clark, Virginia L

    2009-12-01

    Since Neisseria gonorrhoeae and Neisseria meningitidis are obligate human pathogens, a comparison with commensal species of the same genus could reveal differences important in pathogenesis. The recent completion of commensal Neisseria genome draft assemblies allowed us to perform a comparison of the genes involved in the catalysis, assembly and regulation of the denitrification pathway, which has been implicated in the virulence of several bacteria. All species contained a highly conserved nitric oxide reductase (NorB) and a nitrite reductase (AniA or NirK) that was highly conserved in the catalytic but divergent in the N-terminal lipid modification and C-terminal glycosylation domains. Only Neisseria mucosa contained a nitrate reductase (Nar), and only Neisseria lactamica, Neisseria cinerea, Neisseria subflava, Neisseria flavescens and Neisseria sicca contained a nitrous oxide reductase (Nos) complex. The regulators of the denitrification genes, FNR, NarQP and NsrR, were highly conserved, except for the GAF domain of NarQ. Biochemical examination of laboratory strains revealed that all of the neisserial species tested except N. mucosa had a two- to fourfold lower nitrite reductase activity than N. gonorrhoeae, while N. meningitidis and most of the commensal Neisseria species had a two- to fourfold higher nitric oxide (NO) reductase activity. For N. meningitidis and most of the commensal Neisseria, there was a greater than fourfold reduction in the NO steady-state level in the presence of nitrite as compared with N. gonorrhoeae. All of the species tested generated an NO steady-state level in the presence of an NO donor that was similar to that of N. gonorrhoeae. The greatest difference between the Neisseria species was the lack of a functional Nos system in the pathogenic species N. gonorrhoeae and N. meningitidis.

  3. Structure-function studies of the Neisseria gonorrhoeae major outer membrane porin.

    PubMed

    Chen, Adrienne; Seifert, H Steven

    2013-12-01

    The major outer membrane porin (PorB) expressed by Neisseria gonorrhoeae plays multiple roles during infection, in addition to its function as an outer membrane pore. We have generated a panel of mutants of N. gonorrhoeae strain FA1090 expressing a variety of mutant porB genes that all function as porins. We identified multiple regions of porin that are involved in its binding to the complement regulatory factors C4b-binding protein and factor H and confirmed that the ability to bind at least one factor is required for FA1090 to survive the bactericidal effects of human serum. We tested the ability of these mutants to inhibit both apoptosis and the oxidative burst in polymorphonuclear leukocytes but were unable to identify the porin domains required for either function. This study has identified nonessential porin domains and some potentially essential portions of the protein and has further expanded our understanding of the contribution of the porin domains required for complement regulation.

  4. Sequence Type 4821 Clonal Complex Serogroup B Neisseria meningitidis in China, 1978–2013

    PubMed Central

    Zhu, Bingqing; Xu, Zheng; Du, Pengcheng; Xu, Li; Sun, Xiaofang; Gao, Yuan

    2015-01-01

    Serogroup B Neisseria meningitidis strains belonging to sequence type 4821 clonal complex (CC4821), a hyperinvasive lineage first identified for serogroup C in 2003, have been increasingly isolated in China. We characterized the outer membrane protein genes of 48 serogroup B and 214 serogroup C strains belonging to CC4821 and analyzed the genomic sequences of 22 strains. Four serogroup B strains had porin A (i.e., PorA), PorB, and ferric enterobactin transport (i.e., FetA) genotypes identical to those for serogroup C. Phylogenetic analysis of the genomic sequences showed that the 22 CC4821 strains from patients and healthy carriers were unevenly clustered into 2 closely related groups; each group contained serogroup B and C strains. Serogroup B strains appeared variable at the capsule locus, and several recombination events had occurred at uncertain breakpoints. These findings suggest that CC4821 serogroup C N. meningitidis is the probable origin of highly pathogenic CC4821 serogroup B strains. PMID:25989189

  5. Human C4b-binding protein selectively interacts with Neisseria gonorrhoeae and results in species-specific infection.

    PubMed

    Ngampasutadol, Jutamas; Ram, Sanjay; Blom, Anna M; Jarva, Hanna; Jerse, Ann E; Lien, Egil; Goguen, Jon; Gulati, Sunita; Rice, Peter A

    2005-11-22

    Neisseria gonorrhoeae is the causative agent of gonorrhea, a disease that is restricted to humans. Complement forms a key arm of the innate immune system that combats gonococcal infections. N. gonorrhoeae uses its outer membrane porin (Por) molecules to bind the classical pathway of complement down-regulatory protein C4b-binding protein (C4bp) to evade killing by human complement. Strains of N. gonorrhoeae that resisted killing by human serum complement were killed by serum from rodent, lagomorph, and primate species, which cannot be readily infected experimentally with this organism and whose C4bp molecules did not bind to N. gonorrhoeae. In contrast, we found that Yersinia pestis, an organism that can infect virtually all mammals, bound species-specific C4bp and uniformly resisted serum complement-mediated killing by these species. Serum resistance of gonococci was restored in these sera by human C4bp. An exception was serotype Por1B-bearing gonococcal strains that previously had been used successfully in a chimpanzee model of gonorrhea that simulates human disease. Por1B gonococci bound chimpanzee C4bp and resisted killing by chimpanzee serum, providing insight into the host restriction of gonorrhea and addressing why Por1B strains, but not Por1A strains, have been successful in experimental chimpanzee infection. Our findings may lead to the development of better animal models for gonorrhea and may also have implications in the choice of complement sources to evaluate neisserial vaccine candidates.

  6. Neisseria infection of rhesus macaques as a model to study colonization, transmission, persistence, and horizontal gene transfer.

    PubMed

    Weyand, Nathan J; Wertheimer, Anne M; Hobbs, Theodore R; Sisko, Jennifer L; Taku, Nyiawung A; Gregston, Lindsay D; Clary, Susan; Higashi, Dustin L; Biais, Nicolas; Brown, Lewis M; Planer, Shannon L; Legasse, Alfred W; Axthelm, Michael K; Wong, Scott W; So, Magdalene

    2013-02-19

    The strict tropism of many pathogens for man hampers the development of animal models that recapitulate important microbe-host interactions. We developed a rhesus macaque model for studying Neisseria-host interactions using Neisseria species indigenous to the animal. We report that Neisseria are common inhabitants of the rhesus macaque. Neisseria isolated from the rhesus macaque recolonize animals after laboratory passage, persist in the animals for at least 72 d, and are transmitted between animals. Neisseria are naturally competent and acquire genetic markers from each other in vivo, in the absence of selection, within 44 d after colonization. Neisseria macacae encodes orthologs of known or presumed virulence factors of human-adapted Neisseria, as well as current or candidate vaccine antigens. We conclude that the rhesus macaque model will allow studies of the molecular mechanisms of Neisseria colonization, transmission, persistence, and horizontal gene transfer. The model can potentially be developed further for preclinical testing of vaccine candidates.

  7. Description and nomenclature of Neisseria meningitidis capsule locus.

    PubMed

    Harrison, Odile B; Claus, Heike; Jiang, Ying; Bennett, Julia S; Bratcher, Holly B; Jolley, Keith A; Corton, Craig; Care, Rory; Poolman, Jan T; Zollinger, Wendell D; Frasch, Carl E; Stephens, David S; Feavers, Ian; Frosch, Matthias; Parkhill, Julian; Vogel, Ulrich; Quail, Michael A; Bentley, Stephen D; Maiden, Martin C J

    2013-04-01

    Pathogenic Neisseria meningitidis isolates contain a polysaccharide capsule that is the main virulence determinant for this bacterium. Thirteen capsular polysaccharides have been described, and nuclear magnetic resonance spectroscopy has enabled determination of the structure of capsular polysaccharides responsible for serogroup specificity. Molecular mechanisms involved in N. meningitidis capsule biosynthesis have also been identified, and genes involved in this process and in cell surface translocation are clustered at a single chromosomal locus termed cps. The use of multiple names for some of the genes involved in capsule synthesis, combined with the need for rapid diagnosis of serogroups commonly associated with invasive meningococcal disease, prompted a requirement for a consistent approach to the nomenclature of capsule genes. In this report, a comprehensive description of all N. meningitidis serogroups is provided, along with a proposed nomenclature, which was presented at the 2012 XVIIIth International Pathogenic Neisseria Conference.

  8. Neisseria meningitidis B vaccines: recent advances and possible immunization policies.

    PubMed

    Gasparini, Roberto; Amicizia, Daniela; Domnich, Alexander; Lai, Piero Luigi; Panatto, Donatella

    2014-03-01

    Since the development of the first-generation vaccines based on outer membrane vesicles (OMV), which were able to contain strain-specific epidemics, but were not suitable for universal use, enormous steps forward in the prevention of Neisseria meningitidis B have been made. The first multicomponent vaccine, Bexsero(®), has recently been authorized for use; other vaccines, bivalent rLP2086 and next-generation OMV vaccines, are under development. The new vaccines may substantially contribute to reducing invasive bacterial infections as they could cover most Neisseria meningitidis B strains. Moreover, other potentially effective serogroup B vaccine candidates are being studied in preclinical settings. It is therefore appropriate to review what has recently been achieved in the prevention of disease caused by serogroup B.

  9. Strain differentiation of Neisseria gonorrhoeae by reverse passive hemagglutination.

    PubMed Central

    Armstrong, A S; Mathias, J R; DeYoung, M I; Hirata, A A

    1979-01-01

    A reverse passive hemagglutination test that utilizes human erythrocytes coated with antibody to gonococci was developed to distinguish differences among 11 strains of Neisseria gonorrhoeae. Different rabbits were immunized with each strain of gonococcus. Antibody was purified by passing antiserum over an immunoadsorbent column containing homologous cell walls trapped in a cross-linked polyacrylamide gel. Antibody, after absorption with N. meningitidis, was used for coating 11 individual suspensions of erythrocytes, each with antibody to one gonococcal strain. The panel of coated erythrocytes was added to microtiter trays containing dilutions of homologous bacterial lysate and lysates from 10 heterologous strains. Agglutination titers were highest with homologous lysates, although cross-reactions occurred among some heterologous lysates. Lysates of nongonococcal Neisseria species and of other genera did not agglutinate coated erythrocytes. The reverse passive hemagglutination test can be a useful procedure to distinguish differences among strains of N. gonorrhoeae. Images PMID:110695

  10. Conjunctivitis due to Neisseria sicca: a case report.

    PubMed

    Eser, Ilker; Akcali, Alper; Tatman-Otkun, Muserref; Taskiran-Comez, Arzu

    2014-03-01

    We report the first case, in Medline-based literature, of conjunctivitis caused by gram negative diplococcus, Neisseria sicca. Although it is not widely accepted as such, isolation from cultures of repeated eye swab samples suggests that N. sicca may be a pathogen in conjunctival infections. Positive culture for this organism should not be readily dismissed. Such conjunctivitis responded favorably to treatment with netilmicin eye drops.

  11. Pathogenic Neisseria hitchhike on the uropod of human neutrophils.

    PubMed

    Söderholm, Niklas; Vielfort, Katarina; Hultenby, Kjell; Aro, Helena

    2011-01-01

    Polymorphonuclear neutrophils (PMNs) are important components of the human innate immune system and are rapidly recruited at the site of bacterial infection. Despite the effective phagocytic activity of PMNs, Neisseria gonorrhoeae infections are characterized by high survival within PMNs. We reveal a novel type IV pilus-mediated adherence of pathogenic Neisseria to the uropod (the rear) of polarized PMNs. The direct pilus-uropod interaction was visualized by scanning electron microscopy and total internal reflection fluorescence (TIRF) microscopy. We showed that N. meningitidis adhesion to the PMN uropod depended on both pilus-associated proteins PilC1 and PilC2, while N. gonorrhoeae adhesion did not. Bacterial adhesion elicited accumulation of the complement regulator CD46, but not I-domain-containing integrins, beneath the adherent bacterial microcolony. Electrographs and live-cell imaging of PMNs suggested that bacterial adherence to the uropod is followed by internalization into PMNs via the uropod. We also present data showing that pathogenic Neisseria can hitchhike on PMNs to hide from their phagocytic activity as well as to facilitate the spread of the pathogen through the epithelial cell layer.

  12. Clinical evaluation of the Vitek Neisseria-Haemophilus Identification card.

    PubMed

    Janda, W M; Malloy, P J; Schreckenberger, P C

    1987-01-01

    A clinical evaluation of the Vitek Neisseria-Haemophilus Identification (NHI) card (Vitek Systems, Inc., Hazelwood, Mo.) was performed with 480 clinical isolates and stock strains of Neisseria spp., Haemophilus spp., and other fastidious microorganisms included in the data base of the system. Identifications obtained with the NHI card were compared with those determined by conventional methods. The card identified 83.2% of 244 Neisseria spp. and Branhamella catarrhalis, 54.9% of 164 Haemophilus spp., and 84.7% of 72 fastidious gram-negative species with no further testing required. Some isolates produced good confidence-marginal separation identifications, in which the correct identification was listed with one or two other possible identifications and extra tests were required and suggested. When isolates producing good confidence-marginal separation identifications were included, correct identifications of these organism groups increased to 97.1, 92.7, and 94.4%, respectively. Among the commonly isolated microorganisms, the NHI card identified 99.1% of 110 N. gonorrhoeae, 98.5% of 68 N. meningitidis, 93.9% of 98 H. influenzae, and 95.6% of 46 H. parainfluenzae strains. All of these organisms produced excellent to very good confidence level identifications except for H. influenzae biotypes II, III, and VII, for which hemolytic reactions were required for differentiation from H. haemolyticus. The NHI card reliably identified other fastidious gram-negative species, including H. aphrophilus, Eikenella corrodens, Gardnerella vaginalis, and Kingella denitrificans.

  13. Clinical evaluation of the Vitek Neisseria-Haemophilus Identification card.

    PubMed Central

    Janda, W M; Malloy, P J; Schreckenberger, P C

    1987-01-01

    A clinical evaluation of the Vitek Neisseria-Haemophilus Identification (NHI) card (Vitek Systems, Inc., Hazelwood, Mo.) was performed with 480 clinical isolates and stock strains of Neisseria spp., Haemophilus spp., and other fastidious microorganisms included in the data base of the system. Identifications obtained with the NHI card were compared with those determined by conventional methods. The card identified 83.2% of 244 Neisseria spp. and Branhamella catarrhalis, 54.9% of 164 Haemophilus spp., and 84.7% of 72 fastidious gram-negative species with no further testing required. Some isolates produced good confidence-marginal separation identifications, in which the correct identification was listed with one or two other possible identifications and extra tests were required and suggested. When isolates producing good confidence-marginal separation identifications were included, correct identifications of these organism groups increased to 97.1, 92.7, and 94.4%, respectively. Among the commonly isolated microorganisms, the NHI card identified 99.1% of 110 N. gonorrhoeae, 98.5% of 68 N. meningitidis, 93.9% of 98 H. influenzae, and 95.6% of 46 H. parainfluenzae strains. All of these organisms produced excellent to very good confidence level identifications except for H. influenzae biotypes II, III, and VII, for which hemolytic reactions were required for differentiation from H. haemolyticus. The NHI card reliably identified other fastidious gram-negative species, including H. aphrophilus, Eikenella corrodens, Gardnerella vaginalis, and Kingella denitrificans. PMID:3539996

  14. A New Family of Secreted Toxins in Pathogenic Neisseria Species

    PubMed Central

    Jamet, Anne; Jousset, Agnès B.; Euphrasie, Daniel; Mukorako, Paulette; Boucharlat, Alix; Ducousso, Alexia; Charbit, Alain; Nassif, Xavier

    2015-01-01

    The genus Neisseria includes both commensal and pathogenic species which are genetically closely related. However, only meningococcus and gonococcus are important human pathogens. Very few toxins are known to be secreted by pathogenic Neisseria species. Recently, toxins secreted via type V secretion system and belonging to the widespread family of contact-dependent inhibition (CDI) toxins have been described in numerous species including meningococcus. In this study, we analyzed loci containing the maf genes in N. meningitidis and N. gonorrhoeae and proposed a novel uniform nomenclature for maf genomic islands (MGIs). We demonstrated that mafB genes encode secreted polymorphic toxins and that genes immediately downstream of mafB encode a specific immunity protein (MafI). We focused on a MafB toxin found in meningococcal strain NEM8013 and characterized its EndoU ribonuclease activity. maf genes represent 2% of the genome of pathogenic Neisseria, and are virtually absent from non-pathogenic species, thus arguing for an important biological role. Indeed, we showed that overexpression of one of the four MafB toxins of strain NEM8013 provides an advantage in competition assays, suggesting a role of maf loci in niche adaptation. PMID:25569427

  15. Structural basis for iron piracy by pathogenic Neisseria

    PubMed Central

    Noinaj, N.; Easley, N.C.; Oke, M.; Mizuno, N.; Gumbart, J.; Boura, E.; Steere, A.N.; Zak, O.; Aisen, P.; Tajkhorshid, E.; Evans, R.W.; Gorringe, A.R.; Mason, A.B.; Steven, A.C.; Buchanan, S.K.

    2012-01-01

    SUMMARY Neisseria are obligate human pathogens causing bacterial meningitis, septicemia, and gonorrhea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are: 1) how human transferrin is specifically targeted, and 2) how the bacteria liberate iron from transferrin at neutral pH. To address them, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Collectively, our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process. PMID:22327295

  16. Structural basis for iron piracy by pathogenic Neisseria.

    PubMed

    Noinaj, Nicholas; Easley, Nicole C; Oke, Muse; Mizuno, Naoko; Gumbart, James; Boura, Evzen; Steere, Ashley N; Zak, Olga; Aisen, Philip; Tajkhorshid, Emad; Evans, Robert W; Gorringe, Andrew R; Mason, Anne B; Steven, Alasdair C; Buchanan, Susan K

    2012-02-12

    Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small-angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process.

  17. A new family of secreted toxins in pathogenic Neisseria species.

    PubMed

    Jamet, Anne; Jousset, Agnès B; Euphrasie, Daniel; Mukorako, Paulette; Boucharlat, Alix; Ducousso, Alexia; Charbit, Alain; Nassif, Xavier

    2015-01-01

    The genus Neisseria includes both commensal and pathogenic species which are genetically closely related. However, only meningococcus and gonococcus are important human pathogens. Very few toxins are known to be secreted by pathogenic Neisseria species. Recently, toxins secreted via type V secretion system and belonging to the widespread family of contact-dependent inhibition (CDI) toxins have been described in numerous species including meningococcus. In this study, we analyzed loci containing the maf genes in N. meningitidis and N. gonorrhoeae and proposed a novel uniform nomenclature for maf genomic islands (MGIs). We demonstrated that mafB genes encode secreted polymorphic toxins and that genes immediately downstream of mafB encode a specific immunity protein (MafI). We focused on a MafB toxin found in meningococcal strain NEM8013 and characterized its EndoU ribonuclease activity. maf genes represent 2% of the genome of pathogenic Neisseria, and are virtually absent from non-pathogenic species, thus arguing for an important biological role. Indeed, we showed that overexpression of one of the four MafB toxins of strain NEM8013 provides an advantage in competition assays, suggesting a role of maf loci in niche adaptation.

  18. Improving the immunogenicity of a trivalent Neisseria meningitidis native outer membrane vesicle vaccine by genetic modification.

    PubMed

    Zhang, Lan; Wen, Zhiyun; Lin, Jing; Xu, Hui; Herbert, Paul; Wang, Xin-Min; Mehl, John T; Ahl, Patrick L; Dieter, Lance; Russell, Ryann; Kosinski, Mike J; Przysiecki, Craig T

    2016-07-29

    Trivalent native outer membrane vesicles (nOMVs) derived from three genetically modified Neisseria meningitidis serogroup B strains have been previously evaluated immunologically in mice and rabbits. This nOMV vaccine elicited serum bactericidal activity (SBA) against multiple N. meningitidis serogroup B strains as well as strains from serogroups C, Y, W, and X. In this study, we used trivalent nOMVs isolated from the same vaccine strains and evaluated their immunogenicity in an infant Rhesus macaque (IRM) model whose immune responses to the vaccine are likely to be more predictive of the responses in human infants. IRMs were immunized with trivalent nOMV vaccines and sera were evaluated for exogenous human serum complement-dependent SBA (hSBA). Antibody responses to selected hSBA generating antigens contained within the trivalent nOMVs were also measured and we found that antibody titers against factor H binding protein variant 2 (fHbpv2) were very low in the sera from animals immunized with these original nOMV vaccines. To increase the fHbp content in the nOMVs, the vaccine strains were further genetically altered by addition of another fHbp gene copy into the porB locus. Trivalent nOMVs from the three new vaccine strains had higher fHbp antigen levels and generated higher anti-fHbp antibody responses in immunized mice and IRMs. As expected, fHbp insertion into the porB locus resulted in no PorB expression. Interestingly, higher expression of PorA, an hSBA generating antigen, was observed for all three modified vaccine strains. Compared to the trivalent nOMVs from the original strains, higher PorA levels in the improved nOMVs resulted in higher anti-PorA antibody responses in mice and IRMs. In addition, hSBA titers against other strains with PorA as the only hSBA antigen in common with the vaccine strains also increased. PMID:27269057

  19. Improving the immunogenicity of a trivalent Neisseria meningitidis native outer membrane vesicle vaccine by genetic modification.

    PubMed

    Zhang, Lan; Wen, Zhiyun; Lin, Jing; Xu, Hui; Herbert, Paul; Wang, Xin-Min; Mehl, John T; Ahl, Patrick L; Dieter, Lance; Russell, Ryann; Kosinski, Mike J; Przysiecki, Craig T

    2016-07-29

    Trivalent native outer membrane vesicles (nOMVs) derived from three genetically modified Neisseria meningitidis serogroup B strains have been previously evaluated immunologically in mice and rabbits. This nOMV vaccine elicited serum bactericidal activity (SBA) against multiple N. meningitidis serogroup B strains as well as strains from serogroups C, Y, W, and X. In this study, we used trivalent nOMVs isolated from the same vaccine strains and evaluated their immunogenicity in an infant Rhesus macaque (IRM) model whose immune responses to the vaccine are likely to be more predictive of the responses in human infants. IRMs were immunized with trivalent nOMV vaccines and sera were evaluated for exogenous human serum complement-dependent SBA (hSBA). Antibody responses to selected hSBA generating antigens contained within the trivalent nOMVs were also measured and we found that antibody titers against factor H binding protein variant 2 (fHbpv2) were very low in the sera from animals immunized with these original nOMV vaccines. To increase the fHbp content in the nOMVs, the vaccine strains were further genetically altered by addition of another fHbp gene copy into the porB locus. Trivalent nOMVs from the three new vaccine strains had higher fHbp antigen levels and generated higher anti-fHbp antibody responses in immunized mice and IRMs. As expected, fHbp insertion into the porB locus resulted in no PorB expression. Interestingly, higher expression of PorA, an hSBA generating antigen, was observed for all three modified vaccine strains. Compared to the trivalent nOMVs from the original strains, higher PorA levels in the improved nOMVs resulted in higher anti-PorA antibody responses in mice and IRMs. In addition, hSBA titers against other strains with PorA as the only hSBA antigen in common with the vaccine strains also increased.

  20. Neisserial Correia repeat-enclosed elements do not influence the transcription of pil genes in Neisseria gonorrhoeae and Neisseria meningitidis.

    PubMed

    Lin, Ya-Hsun; Ryan, Catherine S; Davies, John K

    2011-10-01

    Two human-specific neisserial pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, require the expression of type IV pili (tfp) for initial attachment to the host during infection. However, the mechanisms controlling the assembly and functionality of tfp are poorly understood. It is known that the gonococcal pilE gene, encoding the major subunit, is positively regulated by IHF, a multifunctional DNA binding protein. A neisserial specific repetitive DNA sequence, termed the Correia repeat-enclosed element (CREE) is situated upstream of three pil loci: pilHIJKX (pilH-X), pilGD, and pilF. CREEs have been shown to contain strong promoters, and some CREE variants contain a functional IHF binding site. CREEs might therefore be involved in the regulation of tfp biogenesis in pathogenic Neisseria. Site-directed and deletion mutagenesis on promoter::cat reporter constructs demonstrated that transcription of pilH-X and pilGD is from a σ(70) promoter and is independent of the CREE. The insertion of a CREE in the pilF promoter region in N. meningitidis generated a functional σ(70) promoter. However, there is also a functional promoter at this position in N. gonorrhoeae, where there is no CREE. These results suggest CREE insertion in these three pil loci does not influence transcription and that IHF does not coordinately regulate tfp biogenesis.

  1. 21 CFR 866.2410 - Culture medium for pathogenic Neisseria spp.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Culture medium for pathogenic Neisseria spp. 866.2410 Section 866.2410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Culture medium for pathogenic Neisseria spp. (a) Identification. A culture medium for pathogenic...

  2. 21 CFR 866.2410 - Culture medium for pathogenic Neisseria spp.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Culture medium for pathogenic Neisseria spp. 866.2410 Section 866.2410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Culture medium for pathogenic Neisseria spp. (a) Identification. A culture medium for pathogenic...

  3. 21 CFR 866.2410 - Culture medium for pathogenic Neisseria spp.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Culture medium for pathogenic Neisseria spp. 866.2410 Section 866.2410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Culture medium for pathogenic Neisseria spp. (a) Identification. A culture medium for pathogenic...

  4. 21 CFR 866.2410 - Culture medium for pathogenic Neisseria spp.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Culture medium for pathogenic Neisseria spp. 866.2410 Section 866.2410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Culture medium for pathogenic Neisseria spp. (a) Identification. A culture medium for pathogenic...

  5. 21 CFR 866.2410 - Culture medium for pathogenic Neisseria spp.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Culture medium for pathogenic Neisseria spp. 866.2410 Section 866.2410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Culture medium for pathogenic Neisseria spp. (a) Identification. A culture medium for pathogenic...

  6. Prevalence and genotyping of commensal Neisseria with reduced susceptibility to penicillin.

    PubMed

    Mechergui, Arij; Achour, Wafa; Ben Hassen, Assia

    2015-01-01

    We analyzed 85 Neisseria spp. strains collected by swabbing from neutropenic patients to determine the prevalence of reduced susceptibility to penicillin and to ascertain the clonal relationship between these strains. High genetic diversity and an elevated level of penicillin resistance were found among commensal Neisseria clinical isolates.

  7. Role of penA polymorphisms for penicillin susceptibility in Neisseria lactamica and Neisseria meningitidis.

    PubMed

    Karch, André; Vogel, Ulrich; Claus, Heike

    2015-10-01

    In meningococci, reduced penicillin susceptibility is associated with five specific mutations in the transpeptidase region of penicillin binding protein 2 (PBP2). We showed that the same set of mutations was present in 64 of 123 Neisseria lactamica strains obtained from a carriage study (MIC range: 0.125-2.0mg/L). The PBP2 encoding penA alleles in these strains were genetically similar to those found in intermediate resistant meningococci suggesting frequent interspecies genetic exchange. Fifty-six N. lactamica isolates with mostly lower penicillin MICs (range: 0.064-0.38mg/L) exhibited only three of the five mutations. The corresponding penA alleles were unique to N. lactamica and formed a distinct genetic clade. PenA alleles with no mutations on the other hand were unique to meningococci. Under penicillin selective pressure, genetic transformation of N. lactamica penA alleles in meningococci was only possible for alleles encoding five mutations, but not for those encoding three mutations; the transfer resulted in MICs comparable to those of meningococci harboring penA alleles that encoded PBP2 with five mutations, but considerably lower than those of the corresponding N. lactamica donor strains. Due to a transformation barrier the complete N. lactamica penA could not be transformed into N. meningitidis. In summary, penicillin MICs in N. lactamica were associated with the number of mutations in the transpeptidase region of PBP2. Evidence for interspecific genetic transfer was only observed for penA alleles associated with higher MICs, suggesting that alleles encoding only three mutations in the transpeptidase region are biologically not effective in N. meningitidis. Factors other than PBP2 seem to be responsible for the high levels of penicillin resistance in N. lactamica. A reduction of penicillin susceptibility in N. meningitidis by horizontal gene transfer from N. lactamica is unlikely to happen.

  8. Genome sequencing reveals widespread virulence gene exchange among human Neisseria species.

    PubMed

    Marri, Pradeep Reddy; Paniscus, Mary; Weyand, Nathan J; Rendón, María A; Calton, Christine M; Hernández, Diana R; Higashi, Dustin L; Sodergren, Erica; Weinstock, George M; Rounsley, Steven D; So, Magdalene

    2010-07-28

    Commensal bacteria comprise a large part of the microbial world, playing important roles in human development, health and disease. However, little is known about the genomic content of commensals or how related they are to their pathogenic counterparts. The genus Neisseria, containing both commensal and pathogenic species, provides an excellent opportunity to study these issues. We undertook a comprehensive sequencing and analysis of human commensal and pathogenic Neisseria genomes. Commensals have an extensive repertoire of virulence alleles, a large fraction of which has been exchanged among Neisseria species. Commensals also have the genetic capacity to donate DNA to, and take up DNA from, other Neisseria. Our findings strongly suggest that commensal Neisseria serve as reservoirs of virulence alleles, and that they engage extensively in genetic exchange.

  9. Cefixime and ceftriaxone susceptibility of Neisseria gonorrhoeae in Italy from 2006 to 2010.

    PubMed

    Carannante, A; Prignano, G; Cusini, M; Matteelli, A; Dal Conte, I; Ghisetti, V; D'Antuono, A; Cavrini, F; Antonetti, R; Stefanelli, P

    2012-06-01

    Neisseria gonorrhoeae resistance to cephalosporins, the currently recommended treatment, and treatment failures with cefixime have been reported worldwide. The purposes of the present study were (i) to examine the susceptibility of N. gonorrhoeae isolates isolated in Italy from 2006 through 2010 to cefixime (n = 293) taking into account both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical And Laboratory Standards Institute (CLSI) criteria for categorization; (ii) to determine the contribution to decreased/resistant susceptibility of mutations in the penA, mtrR, ponA and porB1b genes in a subsample of isolates; and (iii) to genotype the isolates showing decreased susceptibility or resistance to cefixime, by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and by pulsed-field gel electrophoresis (PFGE) to identify the predominant genotypes. Minimum inhibitory concentrations (MICs) were determined by the E-test and agar dilution method on 293 isolates and results were interpreted according to both EUCAST 2010 (MIC R >0.12 mg/L) and CLSI 2008 (MIC R >0.25 mg/L) criteria. All isolates showed full susceptibility to ceftriaxone, whereas those with a MIC for cefixime ≥0.125 mg/L were on the increase from 2008 through 2010. The same penA gene alterations were found among isolates with MICs close to the EUCAST breakpoint as the resistant ones, and they belong to ST1407. Seven isolates, belonging to various sequence types, showed a different por allele, though similar to the por 908 allele present in ST1407. PFGE divided strains ST1407 into two main groups confirming their genetic relationship.

  10. High resolution clear native electrophoresis (hrCNE) allows a detailed analysis of the heterotrimeric structure of recombinant Neisseria meningitidis porins inserted into liposomes.

    PubMed

    Freixeiro, Paula; Diéguez-Casal, Ernesto; Costoya, Liliana; Marzoa, Juan; Ferreirós, Carlos M; Criado, María Teresa; Sánchez, Sandra

    2013-02-01

    Three recombinant proteins of Neisseria meningitidis, rPorB, rPorA, and rRmpM, were purified and incorporated into liposomes prepared by dialysis-extrusion. The protein complexes formed using different combinations of recombinant proteins were studied by high resolution clear native electrophoresis (hrCNE) and 2-D hrCNE/SDS-PAGE, analyzing the influence of the stoichiometry of the two porins in the formation of complexes and comparing them with native porin complexes present in OMVs from five different N. meningitidis strains. Insertion of the recombinant proteins into liposomes allowed a complete refolding of porin complexes, and the electrophoretic analyses showed that, when the three recombinant proteins are present, the pattern of porin complexes obtained is similar to that observed in native OMVs. We could show homocomplexes of each individual porin and PorA/PorB, RmpM/PorB, and PorA/PorB/RmpM heterocomplexes. Our results suggest that RmpM binds only to PorB, confirm the trimeric structure of N. meningitidis pores, and demonstrate that insertion into liposomes restores the native structure of porin complexes.

  11. Antimicrobial susceptibility/resistance and molecular epidemiological characteristics of Neisseria gonorrhoeae in 2009 in Belarus.

    PubMed

    Glazkova, Slavyana; Golparian, Daniel; Titov, Leonid; Pankratova, Nataliya; Suhabokava, Nataliya; Shimanskaya, Irina; Domeika, Marius; Unemo, Magnus

    2011-08-01

    Increased antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global concern, and ultimately gonorrhoea may become untreatable. Nonetheless, AMR data from East-Europe are scarce beyond Russia, and no AMR data or other characteristics of gonococci have been reported from Belarus for more than 20 years. The aim was to describe the prevalence of AMR, and report molecular epidemiological characteristics of gonococci circulating in 2009 in Belarus. In a sample of 80 isolates, resistance prevalences to antimicrobials used for gonorrhoea treatment in Belarus were: Ceftriaxone 0%, spectinomycin 0%, azithromycin 17.3%, tetracycline 25.9%, ciprofloxacin 34.6% and erythromycin 59.2%. The isolates displayed no penA mosaic alleles, 38 porB gene sequences and 35 N. gonorrhoeae multiantigen sequence types, of which 20 have not been described before worldwide. Due to the high levels of antimicrobial resistance, only ceftriaxone and spectinomycin can be recommended for empirical treatment of gonorrhoea in Belarus according to WHO recommendations. Continuous gonococcal AMR surveillance in Eastern Europe is crucial. This is now initiated in Belarus using WHO protocols.

  12. Neisseria adhesin A variation and revised nomenclature scheme.

    PubMed

    Bambini, Stefania; De Chiara, Matteo; Muzzi, Alessandro; Mora, Marirosa; Lucidarme, Jay; Brehony, Carina; Borrow, Ray; Masignani, Vega; Comanducci, Maurizio; Maiden, Martin C J; Rappuoli, Rino; Pizza, Mariagrazia; Jolley, Keith A

    2014-07-01

    Neisseria adhesin A (NadA), involved in the adhesion and invasion of Neisseria meningitidis into host tissues, is one of the major components of Bexsero, a novel multicomponent vaccine licensed for protection against meningococcal serogroup B in Europe, Australia, and Canada. NadA has been identified in approximately 30% of clinical isolates and in a much lower proportion of carrier isolates. Three protein variants were originally identified in invasive meningococci and named NadA-1, NadA-2, and NadA-3, whereas most carrier isolates either lacked the gene or harbored a different variant, NadA-4. Further analysis of isolates belonging to the sequence type 213 (ST-213) clonal complex identified NadA-5, which was structurally similar to NadA-4, but more distantly related to NadA-1, -2, and -3. At the time of this writing, more than 89 distinct nadA allele sequences and 43 distinct peptides have been described. Here, we present a revised nomenclature system, taking into account the complete data set, which is compatible with previous classification schemes and is expandable. The main features of this new scheme include (i) the grouping of the previously named NadA-2 and NadA-3 variants into a single NadA-2/3 variant, (ii) the grouping of the previously assigned NadA-4 and NadA-5 variants into a single NadA-4/5 variant, (iii) the introduction of an additional variant (NadA-6), and (iv) the classification of the variants into two main groups, named groups I and II. To facilitate querying of the sequences and submission of new allele sequences, the nucleotide and amino acid sequences are available at http://pubmlst.org/neisseria/NadA/.

  13. Comparative Proteomics Analysis of Two Strains of Neisseria meningitidis Serogroup B and Neisseria lactamica

    PubMed Central

    Sheikhi, Raheleh; Amin, Mansour; Hamidinia, Maryam; Assarehzadegan, Mohammad Ali; Rostami, Soodabeh; Mojtahedi, Zahra

    2015-01-01

    Background: Antigenic similarities between Neisseria lactamica as a commensal species and N. meningitidis serogroup B (NmB) as an important cause of meningitis infection have been considered for the development of an effective vaccine based on their common proteins to prevent life-threatening bacterial meningitis. Objectives: The main aims of this study were to determine whole proteome profiles of N. lactamica strains and to compare them with whole proteome profile of a reference strain of NmB for identification of some of common proteins between the two species. Materials and Methods: We compared the whole proteomic profiles of N. lactamica strains and a reference strain of NmB. Lysates from bacterial strains were resolved by two-dimensional gel electrophoresis (2-DE), followed by Coomassie Brilliant blue staining. Some of the protein spots were excised from the gel and subjected to matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis. Results: The analysis of Coomassie-stained gels using ImageMaster 2D Platinum software identified approximately 800 reproducible protein spots in the range of pI 4.5 - 9.5 and Mr of 8 - 100 kDa for each 2-DE gel of the studied bacterial strains. By comparing proteome maps of 2-DE gels, more than 200 common protein spots were recognized between the two species. Forty-eight common protein spots between the studied bacterial strains were identified by MALDI-TOF/TOF-MS. The results indicated that among the protein spots identified by MOLDI-TOF/TOF mass spectrometry, the groups of proteins included cell surface, energy metabolism, amino acid transport and metabolism, coenzyme metabolism, defense, multifunctional cellular processes, DNA, RNA and protein synthesis, ribosomal structure, regulatory functions, replication, transcription, translation, unknown and hypothetical proteins with unknown function. We found that N. lactamica strains have a proteome profile somewhat similar to

  14. [Antimicrobal resistance of Neisseria gonorrhoeae strains in Hungary].

    PubMed

    Nemes-Nikodém, Éva; Brunner, Alexandra; Tóth, Béla; Tóth, Veronika; Bánvölgyi, András; Ostorházi, Eszter

    2015-02-01

    Bevezetés: A Neisseria gonorrhoeae-infekciók kezelésére kiadott európai ajánlás elsősorban a nyugat-európai adatok alapján készült, és nem egyértelműen használható a magyarországi helyzet ismeretében. Célkitűzés: A szerzők 2011. január és 2014. június közötti időszakban a Semmelweis Egyetem, Bőr-, Nemikórtani és Bőronkológiai Klinika Országos Szexuális Úton Terjedő Betegségek Centrumában izolált Neisseria gonorrhoeae törzsek rezisztenciaadatait összevetették az izolált törzsek molekuláris tipizálási eredményeivel, azzal a céllal, hogy pontos adatokat kapjanak a hazánkban előforduló Neisseria gonorrhoeae törzsek antimikrobiális rezisztenciájáról. Módszer: Az antibiotikumrezisztencia-meghatározás minimális inhibitorkoncentráció-méréssel, a szekvenciameghatározás a Neisseria gonorrhoeae Multi Antigen Sequence Typing módszerrel történt. Eredmények: A jelenleg terápiának ajánlott széles spektrumú cefalosporinok elleni rezisztencia ritka, az utóbbi években az azithromycinrezisztencia előfordulása viszont rohamosan növekedett. Következtetések: Az új terápiás irányelvek készítésekor figyelembe kell venni, hogy a gyakran fertőzést okozó molekuláris típusba sorolható törzsek között kiemelkedően magas az azithromycinrezisztensek aránya. Orv. Hetil., 2015, 156(6), 226–229.

  15. Using laser tweezers to measure twitching motility in Neisseria.

    PubMed

    Maier, Berenike

    2005-06-01

    Dynamic properties of type IV pili are essential for their function in bacterial infection, twitching motility and gene transfer. Laser tweezers are versatile tools to study the molecular mechanism underlying pilus dynamics at the single molecule level. Recently, these optical tweezers have been used to monitor pilus elongation and retraction in vivo at a resolution of several nanometers. The force generated by type IV pili exceeds 100 pN making pili the strongest linear motors characterized to date. The study of pilus dynamics at the single molecule level sheds light on kinetics, force generation, switching and mechanics of the Neisseria gonorrhoeae pilus motor. PMID:15939360

  16. Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

    PubMed Central

    Hoyne, P A; Haas, R; Meyer, T F; Davies, J K; Elleman, T C

    1992-01-01

    Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell. Images PMID:1358873

  17. Immunoresponses to Neisseria meningitidis epitopes: primary versus secondary antiphosphorylcholine responses.

    PubMed Central

    Faro, J; Seoane, R; Puentes, E; Martínez Ubeira, F; Regueiro, B J

    1985-01-01

    Specific antiphosphorylcholine immune responses were found to be elicited by different Neisseria meningitidis group B M986 preparations. Our results suggest the functional presence of phosphorylcholine in the bacteria. The immune responses, mostly immunoglobulin M, were measured with a plaque-forming cell assay. The secondary phosphorylcholine-specific immune response induced by intact meningococci was significantly lower than the primary phosphorylcholine-specific immune response induced by the same antigens. This suppression is priming time dependent and does not represent an early switching to the expression of other classes of immunoglobulins. PMID:2580791

  18. Dense cultures of Neisseria gonorrhoeae in liquid medium.

    PubMed

    Brookes, R; Hedén, C G

    1967-03-01

    Cultivation of Neisseria gonorrhoeae was effected in a conical glass culture vessel surrounded by a constant-temperature water jacket, and with facilities for stirring, aeration, and pH measurement and control. With the use of an aerated peptone-based medium, containing polypropylene glycol to prevent foam build-up, the yields obtained over the pH range from 5.8 to 7.4 were determined. The greatest yield was obtained at pH 6.4 when the dry weight was 1.5 g/liter. At pH 7.2 to 7.6, lysis was extensive.

  19. Sigma factor RpoN (σ54) regulates pilE transcription in commensal Neisseria elongata.

    PubMed

    Rendón, María A; Hockenberry, Alyson M; McManus, Steven A; So, Magdalene

    2013-10-01

    Human-adapted Neisseria includes two pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, and at least 13 species of commensals that colonize many of the same niches as the pathogens. The Type IV pilus plays an important role in the biology of pathogenic Neisseria. In these species, Sigma factor RpoD (σ(70)), Integration Host Factor, and repressors RegF and CrgA regulate transcription of pilE, the gene encoding the pilus structural subunit. The Type IV pilus is also a strictly conserved trait in commensal Neisseria. We present evidence that a different mechanism regulates pilE transcription in commensals. Using Neisseria elongata as a model, we show that Sigma factor RpoN (σ(54)), Integration Host Factor, and an activator we name Npa regulate pilE transcription. Taken in context with previous reports, our findings indicate pilE regulation switched from an RpoN- to an RpoD-dependent mechanism as pathogenic Neisseria diverged from commensals during evolution. Our findings have implications for the timing of Tfp expression and Tfp-mediated host cell interactions in these two groups of bacteria. PMID:23899162

  20. Sigma factor RpoN (σ54) regulates pilE transcription in commensal Neisseria elongata.

    PubMed

    Rendón, María A; Hockenberry, Alyson M; McManus, Steven A; So, Magdalene

    2013-10-01

    Human-adapted Neisseria includes two pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, and at least 13 species of commensals that colonize many of the same niches as the pathogens. The Type IV pilus plays an important role in the biology of pathogenic Neisseria. In these species, Sigma factor RpoD (σ(70)), Integration Host Factor, and repressors RegF and CrgA regulate transcription of pilE, the gene encoding the pilus structural subunit. The Type IV pilus is also a strictly conserved trait in commensal Neisseria. We present evidence that a different mechanism regulates pilE transcription in commensals. Using Neisseria elongata as a model, we show that Sigma factor RpoN (σ(54)), Integration Host Factor, and an activator we name Npa regulate pilE transcription. Taken in context with previous reports, our findings indicate pilE regulation switched from an RpoN- to an RpoD-dependent mechanism as pathogenic Neisseria diverged from commensals during evolution. Our findings have implications for the timing of Tfp expression and Tfp-mediated host cell interactions in these two groups of bacteria.

  1. Study of the stability of proteoliposomes as vehicles for vaccines against Neisseria meningitidis based on recombinant porin complexes.

    PubMed

    Freixeiro, Paula; Diéguez-Casal, Ernesto; Costoya, Liliana; Seijo, Begoña; Ferreirós, Carlos M; Criado, María Teresa; Sánchez, Sandra

    2013-02-25

    Although effective against epidemic serogroup B Neisseria meningitidis strains, vaccines based on outer membrane vesicles continue to present important limitations, and great efforts are currently being focused in the development of a variety of new vaccine candidates and in the reformulation of currently existing ones. In this work, three N. meningitidis proteins, the PorA and PorB porins and the RmpM protein, were cloned, purified and incorporated into liposomes to build defined systems. The ability of proteoliposomes to allow the refolding porin complexes, and their stability during storage at 4°C and after lyophilization in presence of two cryoprotection agents, glucose and trehalose, were evaluated. This approach allowed to mimic the porin complexes present in natural OMVs, reducing the content of hypervariable protein PorA. During storage at 4°C, our systems showed some changes in the morphology and aggregation after three months, while after lyophilization the systems maintained their properties during the whole nine months of storage checked, with glucose allowing the best preservation of the antigenic properties of the proteins in the proteoliposomes.

  2. Geotemporal Analysis of Neisseria meningitidis Clones in the United States: 2000–2005

    PubMed Central

    Wiringa, Ann E.; Shutt, Kathleen A.; Marsh, Jane W.; Cohn, Amanda C.; Messonnier, Nancy E.; Zansky, Shelley M.; Petit, Susan; Farley, Monica M.; Gershman, Ken; Lynfield, Ruth; Reingold, Arthur; Schaffner, William; Thompson, Jamie; Brown, Shawn T.; Lee, Bruce Y.; Harrison, Lee H.

    2013-01-01

    Background The detection of meningococcal outbreaks relies on serogrouping and epidemiologic definitions. Advances in molecular epidemiology have improved the ability to distinguish unique Neisseria meningitidis strains, enabling the classification of isolates into clones. Around 98% of meningococcal cases in the United States are believed to be sporadic. Methods Meningococcal isolates from 9 Active Bacterial Core surveillance sites throughout the United States from 2000 through 2005 were classified according to serogroup, multilocus sequence typing, and outer membrane protein (porA, porB, and fetA) genotyping. Clones were defined as isolates that were indistinguishable according to this characterization. Case data were aggregated to the census tract level and all non-singleton clones were assessed for non-random spatial and temporal clustering using retrospective space-time analyses with a discrete Poisson probability model. Results Among 1,062 geocoded cases with available isolates, 438 unique clones were identified, 78 of which had ≥2 isolates. 702 cases were attributable to non-singleton clones, accounting for 66.0% of all geocoded cases. 32 statistically significant clusters comprised of 107 cases (10.1% of all geocoded cases) were identified. Clusters had the following attributes: included 2 to 11 cases; 1 day to 33 months duration; radius of 0 to 61.7 km; and attack rate of 0.7 to 57.8 cases per 100,000 population. Serogroups represented among the clusters were: B (n = 12 clusters, 45 cases), C (n = 11 clusters, 27 cases), and Y (n = 9 clusters, 35 cases); 20 clusters (62.5%) were caused by serogroups represented in meningococcal vaccines that are commercially available in the United States. Conclusions Around 10% of meningococcal disease cases in the U.S. could be assigned to a geotemporal cluster. Molecular characterization of isolates, combined with geotemporal analysis, is a useful tool for understanding the spread of virulent meningococcal

  3. Molecular characterization of the interaction between sialylated Neisseria gonorrhoeae and factor H.

    PubMed

    Shaughnessy, Jutamas; Ram, Sanjay; Bhattacharjee, Arnab; Pedrosa, Joana; Tran, Connie; Horvath, Gabor; Monks, Brian; Visintin, Alberto; Jokiranta, T Sakari; Rice, Peter A

    2011-06-24

    Human factor H (HufH), a key inhibitor of the alternative pathway of complement, binds to Neisseria gonorrhoeae and constitutes an important mechanism of human-specific complement evasion. The C-terminal domain 20 of HufH contains the binding site for sialylated gonococci. We exploited differences in amino acid sequences between human and non-binding chimpanzee fH domain 20 to create cross-species mutations to define amino acids important for binding to sialylated gonococci. We used fH/Fc fusion constructs that contained contiguous fH domains 18-20 fused to Fc fragments of murine IgG2a. The Fc region was used both as a tag for detection of each fusion molecule on the bacterial surface and as an indicator for complement-dependent killing. Arg-1203 was critical for binding to both porin (Por) B.1A and PorB.1B strains. Modeling of the R1203N human-to-chimpanzee mutation using the crystal structure of HufH19-20 as a template showed a loss of positive charge that protrudes at the C terminus of domain 20. We tested the functional importance of Arg-1203 by incubating sialylated gonococci with normal human serum, in the presence of wild-type HufH18-20/Fc or its R1203A mutant. Gonococci bound and were killed by wild-type HufH18-20/Fc but not by the R1203A mutant. A recombinant fH/Fc molecule that contained chimpanzee domain 20, humanized only at amino acid 1203 (N1203R) also bound to sialylated gonococci and restored killing. These findings provide further insights into the species specificity of gonococcal infections and proof-of-concept of a novel therapeutic approach against gonorrhea, a disease rapidly becoming resistant to conventional antibiotics. PMID:21531728

  4. Population and Functional Genomics of Neisseria Revealed with Gene-by-Gene Approaches

    PubMed Central

    Harrison, Odile B.

    2016-01-01

    Rapid low-cost whole-genome sequencing (WGS) is revolutionizing microbiology; however, complementary advances in accessible, reproducible, and rapid analysis techniques are required to realize the potential of these data. Here, investigations of the genus Neisseria illustrated the gene-by-gene conceptual approach to the organization and analysis of WGS data. Using the gene and its link to phenotype as a starting point, the BIGSdb database, which powers the PubMLST databases, enables the assembly of large open-access collections of annotated genomes that provide insight into the evolution of the Neisseria, the epidemiology of meningococcal and gonococcal disease, and mechanisms of Neisseria pathogenicity. PMID:27098959

  5. [How fastidious are the so-called non-fastidious neisseriae? (AUTHOR'S TRANSL)].

    PubMed

    Fahr, A M; Berger, U

    1975-01-01

    75 strains of all the so-called non-fastidious Neisseria species were examined for their ability to grow on blood and nutrient agar at temperatures between 22 degrees C and 45 degrees C. The result was that only Neisseria mucosa var. mucosa, N. catarrhalis, N. ovis and N. canis grow sufficiently on nutrient agar at 22 degrees C. N. lactamica has an even narrower range of of growth (30-37 degrees C) than meningococci. Therefore, the statement in Bergey's Mnaual that the saprophytic neisseriae can be separated from the pathogenic species by their minor needs for temperature and media should be corrected.

  6. Inhibition of the alternative pathway of nonhuman infant complement by porin B2 contributes to virulence of Neisseria meningitidis in the infant rat model.

    PubMed

    Lewis, Lisa A; Vu, David M; Granoff, Dan M; Ram, Sanjay

    2014-06-01

    Neisseria meningitidis utilizes capsular polysaccharide, lipooligosaccharide (LOS) sialic acid, factor H binding protein (fHbp), and neisserial surface protein A (NspA) to regulate the alternative pathway (AP) of complement. Using meningococcal mutants that lacked all four of the above-mentioned molecules (quadruple mutants), we recently identified a role for PorB2 in attenuating the human AP; inhibition was mediated by human fH, a key downregulatory protein of the AP. Previous studies showed that fH downregulation of the AP via fHbp or NspA is specific for human fH. Here, we report that PorB2-expressing quadruple mutants also regulate the AP of baby rabbit and infant rat complement. Blocking a human fH binding region on PorB2 of the quadruple mutant of strain 4243 with a chimeric protein that comprised human fH domains 6 and 7 fused to murine IgG Fc enhanced AP-mediated baby rabbit C3 deposition, which provided evidence for an fH-dependent mechanism of nonhuman AP regulation by PorB2. Using isogenic mutants of strain H44/76 that differed only in their PorB molecules, we confirmed a role for PorB2 in resistance to killing by infant rat serum. The PorB2-expressing strain also caused higher levels of bacteremia in infant rats than its isogenic PorB3-expressing counterpart, thus providing a molecular basis for increased survival of PorB2 isolates in this model. These studies link PorB2 expression with infection of infant rats, which could inform the choice of meningococcal strains for use in animal models, and reveals, for the first time, that PorB2-expressing strains of N. meningitidis regulate the AP of baby rabbits and rats.

  7. Uncovering oral Neisseria tropism and persistence using metagenomic sequencing.

    PubMed

    Donati, Claudio; Zolfo, Moreno; Albanese, Davide; Tin Truong, Duy; Asnicar, Francesco; Iebba, Valerio; Cavalieri, Duccio; Jousson, Olivier; De Filippo, Carlotta; Huttenhower, Curtis; Segata, Nicola

    2016-01-01

    Microbial epidemiology and population genomics have previously been carried out near-exclusively for organisms grown in vitro. Metagenomics helps to overcome this limitation, but it is still challenging to achieve strain-level characterization of microorganisms from culture-independent data with sufficient resolution for epidemiological modelling. Here, we have developed multiple complementary approaches that can be combined to profile and track individual microbial strains. To specifically profile highly recombinant neisseriae from oral metagenomes, we integrated four metagenomic analysis techniques: single nucleotide polymorphisms in the clade's core genome, DNA uptake sequence signatures, metagenomic multilocus sequence typing and strain-specific marker genes. We applied these tools to 520 oral metagenomes from the Human Microbiome Project, finding evidence of site tropism and temporal intra-subject strain retention. Although the opportunistic pathogen Neisseria meningitidis is enriched for colonization in the throat, N. flavescens and N. subflava populate the tongue dorsum, and N. sicca, N. mucosa and N. elongata the gingival plaque. The buccal mucosa appeared as an intermediate ecological niche between the plaque and the tongue. The resulting approaches to metagenomic strain profiling are generalizable and can be extended to other organisms and microbiomes across environments. PMID:27572971

  8. Serological classification of Neisseria gonorrhoeae with monoclonal antibodies.

    PubMed Central

    Tam, M R; Buchanan, T M; Sandström, E G; Holmes, K K; Knapp, J S; Siadak, A W; Nowinski, R C

    1982-01-01

    Hybrid cells producing monoclonal antibodies against antigens of Neisseria gonorrhoeae were obtained by the polyethylene glycol-mediated fusion of mouse myeloma cells and lymphocytes from mice immunized with gonococcal protein I or outer membrane proteins. From four fusions, 16 phenotypically stable, independently cloned hybrid cell lines were selected for continued study. Each of the cell lines produced a characteristically different monoclonal antibody which reacted in immunoprecipitation assays with a unique antigenic determinant on protein I of the outer membrane complex of the bacteria. In antibody binding, immunofluorescence, and coagglutination assays these antibodies each reacted with a restricted group of N. gonorrhoeae strains. None of the monoclonal antibodies reacted with 17 other different species of Neisseria or with Branhamella catarrhalis. When tested on 34 N. gonorrhoeae reference serotyping strains, the monoclonal antibodies demonstrated serological relationships between the strains which paralleled those observed with conventional polyvalent antisera. These antibodies now provide standardized reagents for the rapid and precise serological characterization of many strains of N. gonorrhoeae. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 PMID:6807844

  9. Neisseria lactamica antigens complexed with a novel cationic adjuvant

    PubMed Central

    Gaspar, Emanuelle B.; Rosetti, Andreza S.; Lincopan, Nilton; De Gaspari, Elizabeth

    2013-01-01

    Colonization of the nasopharynx by non-pathogenic Neisseria species, including N. lactamica, has been suggested to lead to the acquisition of natural immunity against Neisseria meningitidis in young children. The aim of this study was to identify a model complex of antigens and adjuvant for immunological preparation against N. meningitidis B, based on cross reactivity with N. lactamica outer membrane vesicles (OMV) antigens and the (DDA-BF) adjuvant. Complexes of 25 µg of OMV in 0.1 mM of DDA-BF were colloidally stable, exhibiting a mean diameter and charge optimal for antigen presentation. Immunogenicity tests for these complexes were performed in mice. A single dose of OMV/DDA-BF was sufficient to induce a (DTH) response, while the same result was achieved only after two doses of OMV/alum. In addition, to achieve total IgG levels that are similar to a single immunization with OMV/DDA-BF, it was necessary to give the mice a second dose of OMV/alum. Moreover, the antibodies induced from a single immunization with OMV/DDA-BF had an intermediate avidity, but antibodies with a similar avidity were only induced by OMV/alum after two immunizations. The use of this novel cationic adjuvant for the first time with a N. lactamica OMV preparation revealed good potential for future vaccine design. PMID:23296384

  10. Transcriptional landscape and essential genes of Neisseria gonorrhoeae

    PubMed Central

    Remmele, Christian W.; Xian, Yibo; Albrecht, Marco; Faulstich, Michaela; Fraunholz, Martin; Heinrichs, Elisabeth; Dittrich, Marcus T.; Müller, Tobias; Reinhardt, Richard; Rudel, Thomas

    2014-01-01

    The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes. PMID:25143534

  11. Identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria with the MicroScan Haemophilus-Neisseria identification panel.

    PubMed Central

    Janda, W M; Bradna, J J; Ruther, P

    1989-01-01

    The Haemophilus-Neisseria identification (HNID) panel (American MicroScan, Sacramento, Calif.) is a 4-h microdilution format system for identification of Haemophilus and Neisseria spp., Branhamella (Moraxella) catarrhalis, and Gardnerella vaginalis. The HNID panel was evaluated by using 423 clinical isolates and stock strains of these organisms, and HNID identifications were compared with those obtained by conventional methods. In addition, 32 isolates representing six genera not included in the HNID data base were tested to determine whether these organisms would produce unique biotype numbers for possible inclusion in the data base. The HNID panel correctly identified 95.3% of 86 Neisseria gonorrhoeae strains, 96% of 25 G. vaginalis strains, and 100% of 28 Neisseria lactamica strains and 48 B. catarrhalis strains. Only 64.7% of 68 Neisseria meningitidis isolates were identified correctly owing to false-negative or equivocal carbohydrate and/or aminopeptidase reactions. Among the Haemophilus spp., 98.8% of 83 H. influenzae strains, 97.1% of 34 H. parainfluenzae strains, and 80% of 15 H. aphrophilus and H. paraphrophilus strains were correctly identified. Eight strains of Neisseria cinerea, a species not included in the data base, produced profiles identical with those for B. catarrhalis and N. gonorrhoeae. Isolates of other species not included in the data base, including Eikenella corrodens, Kingella spp., and Cardiobacterium hominis, produced unique biochemical reaction patterns on the panel. Modification of interpretative criteria for certain tests, expansion of the data base to include other species, and suggestions for additional confirmatory tests will increase the accuracy and utility of the HNID panel. PMID:2501351

  12. Pili-taxis: Clustering of Neisseria gonorrhoeae bacteria

    NASA Astrophysics Data System (ADS)

    Taktikos, Johannes; Zaburdaev, Vasily; Biais, Nicolas; Stark, Holger; Weitz, David A.

    2012-02-01

    The first step of colonization of Neisseria gonorrhoeae bacteria, the etiological agent of gonorrhea, is the attachment to human epithelial cells. The attachment of N. gonorrhoeae bacteria to surfaces or other cells is primarily mediated by filamentous appendages, called type IV pili (Tfp). Cycles of elongation and retraction of Tfp are responsible for a common bacterial motility called twitching motility which allows the bacteria to crawl over surfaces. Experimentally, N. gonorrhoeae cells initially dispersed over a surface agglomerate into round microcolonies within hours. It is so far not known whether this clustering is driven entirely by the Tfp dynamics or if chemotactic interactions are needed. Thus, we investigate whether the agglomeration may stem solely from the pili-mediated attraction between cells. By developing a statistical model for pili-taxis, we try to explain the experimental measurements of the time evolution of the mean cluster size, number of clusters, and area fraction covered by the cells.

  13. Purification and characterization of cytochrome c' from Neisseria meningitidis.

    PubMed

    Huston, W M; Lowe, E C; Butler, C S; Moir, J W B

    2005-02-01

    Cytochrome c', a c-type cytochrome with unique spectroscopic and magnetic properties, has been characterized in a variety of denitrifying and photosynthetic bacteria. Cytochrome c' has a role in defence and/or removal of NO but the mechanism of action is not clear. To examine the function of cytochrome c' from Neisseria meningitidis, the protein was purified after heterologous overexpression in Escherichia coli. The electronic spectra of the oxidized c' demonstrated a pH-dependent transition (over the pH range of 6-10) typical of known c'-type cytochromes. Interestingly, the form in which NO is supplied determines the redox state of the resultant haem-nitrosyl complex. Fe(III)-NO complexes were formed when Fe(II) or Fe(III) cytochrome c' was sparged with NO gas, whereas an Fe(II)-NO complex was generated when NO was supplied using DEA NONOate (diazeniumdiolate).

  14. Neisseria gonorrhoeae and fosfomycin: Past, present and future.

    PubMed

    Tesh, Lauren D; Shaeer, Kristy M; Cho, Jonathan C; Estrada, Sandy J; Huang, Vanthida; Bland, Christopher M; DiMondi, V Paul; Potter, Alicia N; Hussein, Gamal; Bookstaver, P Brandon

    2015-09-01

    Drug-resistant Neisseria gonorrhoeae has become a global health concern that requires immediate attention. Due to increasing resistance to cephalosporins, pursuing novel alternatives for treating N. gonorrhoeae infections is paramount. Whilst new drug development is often cumbersome, reviving antiquated antibiotic agents for treatment of modern infections has become prevalent in clinical practice. Fosfomycin exhibits bactericidal activity through a unique mechanism of action, and a variety of organisms including N. gonorrhoeae are susceptible. In vitro studies have demonstrated that fosfomycin can retain activity against ceftriaxone-resistant N. gonorrhoeae; however, it remains unclear whether there is synergy between fosfomycin and other antibiotics. Clinical investigations evaluating fosfomycin for the treatment of N. gonorrhoeae infections are confounded by methodological limitations, none the less they do provide some perspective on its potential role in therapy. Future studies are needed to establish a safe, convenient and effective fosfomycin regimen for treating N. gonorrhoeae infections.

  15. Neisseria meningitidis Serogroup C Causing Primary Arthritis in a Child

    PubMed Central

    Straticiuc, Sergiu; Ignat, Ancuta; Hanganu, Elena; Lupu, Vasile Valeriu; Ciubara, Alexandru Bogdan; Cretu, Roxana

    2016-01-01

    Abstract Introduction: Neisseria meningitidis (N. meningitidis) is associated with severe invasive infections such as meningitis and fulminant septicemia. Septic arthritis due to N. meningitidis is rare and bone infections have been reported exceptionally. We report the case of a 1-year old girl who presented with a painful, swollen right knee, accompanied by fever and agitation. Arthrocentesis of the right knee, while patient was under anesthesia, yielded grossly purulent fluid, so we made arthrotomy and drainage. The culture from synovial fluid revealed N. meningitidis, sensitive to Ceftriaxone. The patient received intravenous antibiotherapy with Ceftriaxone. The status of the patient improved after surgical drainage and intravenous antibiotic therapy. She recovered completely after 1 month. Conclusion: This observation illustrates an unusual presentation of invasive meningococcal infection and the early identification of the bacteria, combined with the correct treatment, prevent the complications and even death. PMID:26844522

  16. Potential Therapy for Neisseria Gonorrhoeae Infections With Human Chorionic Gonadotropin.

    PubMed

    Rao, C V

    2015-12-01

    The scientific evidence suggests that Neisseria gonorrhoeae (NG) infects human fallopian tubes by molecular mimicry in which pathogens act like a ligand to bind to epithelial cell surface human chorionic gonadotrophin (hCG)/luteinizing hormone (LH) receptors. The hCG-like molecule has been identified as ribosomal protein L12 in NG coat surface. Human fallopian tube epithelial cells have been shown to contain functional hCG/LH receptors. As previously shown in human fallopian tube organ and cell culture studies, cellular invasion and infection can be prevented by exposing the cells to excess hCG, which would outnumber and outcompete NG for receptor binding. Based on these data, we suggest testing hCG in clinical trials on infected women.

  17. Neisseria elongata subsp elongata infective endocarditis following endurance exercise.

    PubMed

    Jenkins, Joanne May; Fife, Amanda; Baghai, Max; Dworakowski, Rafal

    2015-12-11

    A 31-year-old Argentinian woman presented with a 3-week history of fever, night sweats, myalgia and lethargy following a work trip to Uganda where she ran a marathon. Malarial screens were negative but C reactive protein, erythrocyte sedimentation rate and neutrophil count were raised and she was anaemic. A new pansystolic murmur was heard over the mitral valve and the transthoracic echocardiogram showed a large vegetation (>1 cm) with at least moderate mitral regurgitation. Blood cultures grew Neisseria elongata, subsp elongata treated initially with ceftriaxone then oral ciprofloxacin to complete 4 weeks of treatment. CT scan revealed a wedge-shaped area of low attenuation in the spleen in keeping with a splenic infarct. Seven days postadmission, the patient underwent a successful mitral valve repair. Recovery was complicated by a likely embolic infarct in the right frontal lobe, but the patient was discharged 12 days postoperative with no neurological sequelae.

  18. Antimicrobial susceptibility pattern of Neisseria gonorrhoeae in western Austria.

    PubMed

    Allerberger, F; Kofler, H; Brezinka, C; Guggenbichler, J P; Dierich, M P

    1993-01-01

    From January to October 1992 24 Neisseria gonorrhoeae isolates from clinical specimens were collected at the Federal Public Health Laboratory in Innsbruck (Austria) and screened for resistance to penicillin G, erythromycin, tetracycline, spectinomycin, ceftriaxone, cefuroxime, ciprofloxacine, and silver nitrate. Patients originated from the Austrian provinces Salzburg, Tirol, and Vorarlberg, and presented with manifest gonorrhoea. Two of 24 isolates were penicillinase-producing N. gonorrhoeae. Both strains were isolated from men who had just returned from Thailand or Kenya. The isolate from Africa was also resistant to tetracycline. Five of 24 infections were acquired abroad, sex tourism being involved in four cases. The antimicrobial resistance pattern found in gonococci in western Austria revealed that topical silver nitrate and erythromycin are equally acceptable for use in prophylaxis of neonatal ophthalmia. Penicillin is still the drug of choice in the treatment of endemic infections. If gonorrhoea has been acquired abroad, especially in Asia or Africa, ceftriaxone, spectinomycin or ciprofloxazine are recommended for therapy. PMID:8333204

  19. Neisseria gonorrhoeae and fosfomycin: Past, present and future.

    PubMed

    Tesh, Lauren D; Shaeer, Kristy M; Cho, Jonathan C; Estrada, Sandy J; Huang, Vanthida; Bland, Christopher M; DiMondi, V Paul; Potter, Alicia N; Hussein, Gamal; Bookstaver, P Brandon

    2015-09-01

    Drug-resistant Neisseria gonorrhoeae has become a global health concern that requires immediate attention. Due to increasing resistance to cephalosporins, pursuing novel alternatives for treating N. gonorrhoeae infections is paramount. Whilst new drug development is often cumbersome, reviving antiquated antibiotic agents for treatment of modern infections has become prevalent in clinical practice. Fosfomycin exhibits bactericidal activity through a unique mechanism of action, and a variety of organisms including N. gonorrhoeae are susceptible. In vitro studies have demonstrated that fosfomycin can retain activity against ceftriaxone-resistant N. gonorrhoeae; however, it remains unclear whether there is synergy between fosfomycin and other antibiotics. Clinical investigations evaluating fosfomycin for the treatment of N. gonorrhoeae infections are confounded by methodological limitations, none the less they do provide some perspective on its potential role in therapy. Future studies are needed to establish a safe, convenient and effective fosfomycin regimen for treating N. gonorrhoeae infections. PMID:26145201

  20. Neisseria gonorrhoeae and humans perform an evolutionary LINE dance.

    PubMed

    Anderson, Mark T; Seifert, H Steven

    2011-05-01

    Horizontal gene transfer is an important mechanism for generating genetic diversity. As the number of sequenced genomes continues to increase, so do the examples of horizontal genetic exchange between both related and divergent organisms. Here we discuss the recent finding that certain strains of the human pathogen Neisseria gonorrhoeae have incorporated a small fragment of human DNA sequence into their genomes. The horizontally acquired sequence exhibits 98-100% nucleotide identity to a 685 bp portion of the highly repetitive retrotransposable element L1 and its presence in the gonococcal genome has been confirmed by multiple molecular techniques. The possibility of similar L1 horizontal gene transfer events having occurred in other bacteria based on genomic sequence evidence is explored. Potential mechanisms of how N. gonorrhoeae was able to acquire and maintain this human sequence are also discussed in addition to the evolutionary implications of such an event. PMID:22016852

  1. Structure of the Neisseria meningitidis Type IV pilus

    PubMed Central

    Kolappan, Subramania; Coureuil, Mathieu; Yu, Xiong; Nassif, Xavier; Egelman, Edward H.; Craig, Lisa

    2016-01-01

    Neisseria meningitidis use Type IV pili (T4P) to adhere to endothelial cells and breach the blood brain barrier, causing cause fatal meningitis. T4P are multifunctional polymers of the major pilin protein, which share a conserved hydrophobic N terminus that is a curved extended α-helix, α1, in X-ray crystal structures. Here we report a 1.44 Å crystal structure of the N. meningitidis major pilin PilE and a ∼6 Å cryo-electron microscopy reconstruction of the intact pilus, from which we built an atomic model for the filament. This structure reveals the molecular arrangement of the N-terminal α-helices in the filament core, including a melted central portion of α1 and a bridge of electron density consistent with a predicted salt bridge necessary for pilus assembly. This structure has important implications for understanding pilus biology. PMID:27698424

  2. Quinolones for the Treatment of Neisseria Gonorrhoeae and Chlamydia Trachomatis

    PubMed Central

    1993-01-01

    The most commonly sexually transmitted bacteria are Neisseria gonorrhoeae and Chlamydia trachomatis. The quinolones ofloxacin and ciprofloxacin have been shown to have activity against both of these bacteria in vitro and in vivo. Ofloxacin is particularly well suited for the treatment of N. gonorrhoeae and C. trachomatis cervical infection, which can be considered the earliest manifestation of pelvic inflammatory disease (PID). Not only can ofloxacin be effectively used as a single agent, it is also useful in treating urinary tract infections caused by Enterobacteriaceae. Although it has moderate activity against anaerobes in general, ofloxacin does have activity against the anaerobes commonly isolated from female patients with soft tissue pelvic infections. Thus, ofloxacin has the potential for being utilized to treat early salpingitis. PMID:18475328

  3. Functional significance of factor H binding to Neisseria meningitidis.

    PubMed

    Schneider, Muriel C; Exley, Rachel M; Chan, Hannah; Feavers, Ian; Kang, Yu-Hoi; Sim, Robert B; Tang, Christoph M

    2006-06-15

    Neisseria meningitidis is an important cause of septicemia and meningitis. To cause disease, the bacterium must successfully survive in the bloodstream where it has to avoid being killed by host innate immune mechanisms, particularly the complement system. A number of pathogenic microbes bind factor H (fH), the negative regulator of the alternative pathway of complement activation, to promote their survival in vivo. In this study, we show that N. meningitidis binds fH to its surface. Binding to serogroups A, B, and C N. meningitidis strains was detected by FACS and Far Western blot analysis, and occurred in the absence of other serum factors such as C3b. Unlike Neisseria gonorrhoeae, binding of fH to N. meningitidis was independent of sialic acid on the bacterium, either as a component of its LPS or its capsule. Characterization of the major fH binding partner demonstrated that it is a 33-kDa protein; examination of insertion mutants showed that porins A and B, outer membrane porins expressed by N. meningitidis, do not contribute significantly to fH binding. We examined the physiological consequences of fH bound to the bacterial surface. We found that fH retains its activity as a cofactor of factor I when bound to the bacterium and contributes to the ability of N. meningitidis to avoid complement-mediated killing in the presence of human serum. Therefore, the recruitment of fH provides another mechanism by which this important human pathogen evades host innate immunity.

  4. Immunologic classification of Neisseria gonorrhoeae with micro-immunofluorescence.

    PubMed

    Wang, S P; Holmes, K K; Knapp, J S; Ott, S; Kyzer, D D

    1977-09-01

    A reproducible immunologic classification of Neisseria gonorrhoeae strains has been achieved by the micro-immunofluorescence (Micro-IF)3 method by using formalinized whole organisms as test antigens and mouse antisera prepared by i.v. immunization with the whole organisms as antibody. Immunologic differences among Neisseria species were also distinct in this test system. Immunologic differences among gonococcal strains were not influenced by gonococcal colony type. Classification of gonococci was facilitated by use of antisera absorbed with an antigenically unique gonococcus strain. Of 180 gonococcal strains, 175 could be classified into three immunotypes: A, B, and C. Each type was further divided into subtypes designated A1, A2, A3, B1, B2, B3, C1, and C2. Minor antigenic differences still exist within each subtype. The two gonococcal isolates from each of 17 pairs of sexual contacts fell into the same subtype. Seventy-one of 73 isolates which required arginine, hypoxanthine, and uracil for growth (Arg-Hyx-Ura-) and seven of 107 other auxotypes belonged to subtypes A2 and A3. Marked geographical differences in distribution of gonococcal immunotypes were observed among those available for testing. Subtypes A2 and A3 were predominant in Seattle whereas types B and C were predominant in Southeast Asia. The only Arg-Hyx-Ura- isolates not belonging to subtypes A2 or A3 were the only two that were serum sensitive. This Micro-IF immunotyping appears potentially useful for future immunologic, epidemiologic, and genetic studies of N. gonorrhoeae. PMID:408415

  5. Seminal plasma initiates a Neisseria gonorrhoeae transmission state.

    PubMed

    Anderson, Mark T; Dewenter, Lena; Maier, Berenike; Seifert, H Steven

    2014-03-04

    Niche-restricted pathogens are evolutionarily linked with the specific biological fluids that are encountered during infection. Neisseria gonorrhoeae causes the genital infection gonorrhea and is exposed to seminal fluid during sexual transmission. Treatment of N. gonorrhoeae with seminal plasma or purified semen proteins lactoferrin, serum albumin, and prostate-specific antigen each facilitated type IV pilus-mediated twitching motility of the bacterium. Motility in the presence of seminal plasma was characterized by high velocity and low directional persistence. In addition, infection of epithelial cells with N. gonorrhoeae in the presence of seminal plasma resulted in enhanced microcolony formation. Close association of multiple pili in the form of bundles was also disrupted after seminal plasma treatment leading to an increase in the number of single pilus filaments on the bacterial surface. Thus, exposure of N. gonorrhoeae to seminal plasma is proposed to alter bacterial motility and aggregation characteristics to influence the processes of transmission and colonization. IMPORTANCE There are greater than 100 million estimated new cases of gonorrhea annually worldwide. Research characterizing the mechanisms of pathogenesis and transmission of Neisseria gonorrhoeae is important for developing new prevention strategies, since antibiotic resistance of the organism is becoming increasingly prevalent. Our work identifies seminal plasma as a mediator of N. gonorrhoeae twitching motility and microcolony formation through functional modification of the type IV pilus. These findings provide insight into motility dynamics and epithelial cell colonization under conditions that are relevant to sexual transmission. Type IV pili are common virulence factors with diverse functions among bacterial pathogens, and this work identifies interactions between type IV pili and the host environment. Finally, this work illustrates the importance of the host environment and niche

  6. Functional significance of factor H binding to Neisseria meningitidis.

    PubMed

    Schneider, Muriel C; Exley, Rachel M; Chan, Hannah; Feavers, Ian; Kang, Yu-Hoi; Sim, Robert B; Tang, Christoph M

    2006-06-15

    Neisseria meningitidis is an important cause of septicemia and meningitis. To cause disease, the bacterium must successfully survive in the bloodstream where it has to avoid being killed by host innate immune mechanisms, particularly the complement system. A number of pathogenic microbes bind factor H (fH), the negative regulator of the alternative pathway of complement activation, to promote their survival in vivo. In this study, we show that N. meningitidis binds fH to its surface. Binding to serogroups A, B, and C N. meningitidis strains was detected by FACS and Far Western blot analysis, and occurred in the absence of other serum factors such as C3b. Unlike Neisseria gonorrhoeae, binding of fH to N. meningitidis was independent of sialic acid on the bacterium, either as a component of its LPS or its capsule. Characterization of the major fH binding partner demonstrated that it is a 33-kDa protein; examination of insertion mutants showed that porins A and B, outer membrane porins expressed by N. meningitidis, do not contribute significantly to fH binding. We examined the physiological consequences of fH bound to the bacterial surface. We found that fH retains its activity as a cofactor of factor I when bound to the bacterium and contributes to the ability of N. meningitidis to avoid complement-mediated killing in the presence of human serum. Therefore, the recruitment of fH provides another mechanism by which this important human pathogen evades host innate immunity. PMID:16751403

  7. NadA diversity and carriage in Neisseria meningitidis.

    PubMed

    Comanducci, Maurizio; Bambini, Stefania; Caugant, Dominique A; Mora, Marirosa; Brunelli, Brunella; Capecchi, Barbara; Ciucchi, Laura; Rappuoli, Rino; Pizza, Mariagrazia

    2004-07-01

    NadA is a novel vaccine candidate recently identified in Neisseria meningitidis and involved in adhesion to host tissues. The nadA gene has been found in approximately 50% of the strains isolated from patients and in three of the four hypervirulent lineages of non-serogroup A strains. Here we investigated the presence of the nadA gene in 154 meningococcal strains isolated from healthy people (carrier strains). Only 25 (16.2%) of the 154 carrier isolates harbored the nadA gene. The commensal species Neisseria lactamica was also found not to have the nadA gene. Eighteen of the carrier strains belonged to the ET-5 and ET-37 hypervirulent clusters, indicating that only the 5.1% of the genuine carrier population actually harbored nadA (7 of 136 strains). Five of the seven strains harbored a novel allele of the nadA gene that was designated nadA4. The NadA4 protein was present on the bacterial surface as heat-stable high-molecular-weight oligomers. Antibodies against the recombinant NadA4 protein were bactericidal against homologous strains, whereas the activity against other NadA alleles was weak. In conclusion, the nadA gene segregates differently in the population of strains isolated from healthy individuals and in the population of strains isolated from patients. The presence of NadA can therefore be used as a tool to study the dynamics of meningococcal infections and understand why this bacterium, which is mostly a commensal, can become a severe pathogen.

  8. Modeling Neisseria meningitidis metabolism: from genome to metabolic fluxes

    PubMed Central

    Baart, Gino JE; Zomer, Bert; de Haan, Alex; van der Pol, Leo A; Beuvery, E Coen; Tramper, Johannes; Martens, Dirk E

    2007-01-01

    Background Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years. Results Using the genomic database of N. meningitidis serogroup B together with biochemical and physiological information in the literature we constructed a genome-scale flux model for the primary metabolism of N. meningitidis. The validity of a simplified metabolic network derived from the genome-scale metabolic network was checked using flux-balance analysis in chemostat cultures. Several useful predictions were obtained from in silico experiments, including substrate preference. A minimal medium for growth of N. meningitidis was designed and tested succesfully in batch and chemostat cultures. Conclusion The verified metabolic model describes the primary metabolism of N. meningitidis in a chemostat in steady state. The genome-scale model is valuable because it offers a framework to study N. meningitidis metabolism as a whole, or certain aspects of it, and it can also be used for the purpose of vaccine process development (for example, the design of growth media). The flux distribution of the main metabolic pathways (that is, the pentose phosphate pathway and the Entner-Douderoff pathway) indicates that the major part of pyruvate (69%) is synthesized through the ED-cleavage, a finding that is in good agreement with literature. PMID:17617894

  9. Genetic Diversity and Carriage Dynamics of Neisseria lactamica in Infants

    PubMed Central

    Bennett, Julia S.; Griffiths, David T.; McCarthy, Noel D.; Sleeman, Karen L.; Jolley, Keith A.; Crook, Derrick W.; Maiden, Martin C. J.

    2005-01-01

    Neisseria lactamica, a harmless human commensal found predominantly in the upper respiratory tracts of infants, is closely related to Neisseria meningitidis, a pathogen of global significance. Colonization with N. lactamica may be responsible for the increase in immunity to meningococcal disease that occurs during childhood, when rates of meningococcal carriage are low. This observation has led to the suggestion that N. lactamica whole cells or components are potential constituents of novel meningococcal vaccines. However, the dynamics of carriage and population diversity of N. lactamica in children are poorly understood, presenting difficulties for the choice of representative isolates for use in vaccine development. This problem was addressed by the multilocus sequence typing of N. lactamica isolates from two longitudinal studies of bacterial carriage in infants. The studies comprised 100 and 216 subjects, with N. lactamica carriage monitored from age 4 weeks until age 96 weeks and from age 2 weeks until age 24 weeks, respectively. The maximum observed carriage rate was 44% at 56 weeks of age, with isolates obtained on multiple visits for the majority (54 of 75, 72%) of carriers. The N. lactamica isolates were genetically diverse, with 69 distinct genotypes recovered from the 75 infants. Carriage was generally long-lived, with an average rate of loss of under 1% per week during the 28 weeks following acquisition. Only 11 of the 75 infants carried more than one genotypically unique isolate during the course of the study. Some participants shared identical isolates with siblings, but none shared identical isolates with their parents. These findings have implications for the design of vaccines based on this organism. PMID:15784588

  10. Human wound infections caused by Neisseria animaloris and Neisseria zoodegmatis, former CDC Group EF-4a and EF-4b

    PubMed Central

    Heydecke, Anna; Andersson, Birgitta; Holmdahl, Torsten; Melhus, Åsa

    2013-01-01

    Background Neisseria animaloris and Neisseria zoodegmatis, former CDC Group EF-4a and -4b, are considered to be rare zoonotic pathogens, usually associated with dog or cat bites. The aim of the study was to phenotypicaly characterize 13 EF-4 isolates from wound infections, determine their antibiotic susceptibility and to follow the clinical outcome of the patients. Methods 13 of the EF-4 isolates were cultured on agar plates. Conventional biochemical tests and the Biolog system were used for phenotypical identification. An arbitrary primed polymerase chain reaction (AP-PCR) was carried out to determine the genetic profiles. Minimum inhibitory concentration (MIC) values were determined for different antibiotics were determined. According to this, clinical data for the patients were recorded. Results 11 isolates were identified as N. animaloris and 2 as N. zoodegmatis due to the production of arginine dihydrolase. A majority of the patients had a history of dog bite. In 6 cases only grewth of N. animaloris or zoodegmatis was registered. When a patient received antibiotic treatment the most common drug of choice was penicillin V. Only 3 patients received treatment for which the isolated EF-4 bacterium was fully susceptible. Conclusion Human infections involving N. animaloris and N. zoodegmatis usually present themselves as local wound infection, but severe complications can occur. Despite their pathogenic potentia, l N. animaloris and N. zoodegmatis are often misidentified, dismissed as skin contaminants or not recognized at all. Due to the fact that N. animaloris and N. zoodegmatis are significant pathogens in animal bites, physicians should keep these bacteria in mind when choosing antibiotic therapy. PMID:23919115

  11. Analytical specificity and sensitivity of the APTIMA Combo 2 and APTIMA GC assays for detection of commensal Neisseria species and Neisseria gonorrhoeae on the Gen-Probe Panther instrument.

    PubMed

    Golparian, Daniel; Tabrizi, Sepehr N; Unemo, Magnus

    2013-02-01

    Genetic detection of Neisseria gonorrhoeae is replacing culture for increased diagnostic sensitivity. Specificity of several nucleic acid amplification tests is suboptimal. Herein, the Gen-Probe APTIMA Combo 2 and APTIMA GC assays had 100% specificity and 100% sensitivity after confirmatory testing, when testing 298 isolates of nongonococcal Neisseria and related species and 205 gonococcal isolates.

  12. Development of a test system for rapid differentiation of Neisseria and Haemophilus spp.

    PubMed Central

    Eriquez, L A; Hodinka, N E

    1983-01-01

    A qualitative micromethod (IDS Rapid NH system) employing conventional and single-substrate enzyme tests was developed for the biochemical characterization of Neisseria spp., Haemophilus spp., and other gram-negative species. A total of over 140 dehydrated, miniaturized biochemical tests were investigated for their ability to distinguish species. Computer-assisted test selection and pair separation analysis of the data allowed the selection of 11 4-h tests that would identify Haemophilus and Neisseria spp. implicated as etiological agents as well as differentiate them from other Neisseria spp., Moraxella spp., Branhamella catarrhalis, Centers for Disease Control M groups, and Kingella spp. The final test configuration included modified glucose, sucrose, galactosidase, nitrate, phosphatase, resazurin reduction, and two arylamidase tests. In addition, indole, urea, and ornithine decarboxylase tests were included to biochemically type strains of Haemophilus influenzae and Haemophilus parainfluenzae. PMID:6358247

  13. Phospholipid composition and phospholipase A activity of Neisseria gonorrhoeae.

    PubMed Central

    Senff, L M; Wegener, W S; Brooks, G F; Finnerty, W R; Makula, R A

    1976-01-01

    Exponential-phase cells of Neisseria gonorrhaeae 2686 were examined for phospholipid composition and for membrane-associated phospholipase A activity. When cells were harvested by centrifugation, washed, and lyophilized before extraction, approximately 74% of the total phospholipid was phosphatidylethanolamine, 18% was phosphatidylglycerol, 2% was cardiolipin, and 10% was lysophosphatidylethanolamine. However, when cells still suspended in growth medium were extracted, the amount of lysophosphatidylethanolamine decreased to approximately 1% of the phospholipid composition. This suggests that a gonococcal phospholipase A may be activated by conditions encountered during centrifugation and/or lyophilization of cells preceding extraction. Phospholipase A activity associated with cell membranes was assayed by measuring the conversion of tritiated phosphatidylethanolamine to lysophosphatidylethanolamine. Optimal activity was demonstrated in 10% methanol at pH 8.0 to 8.5, in the presence of calcium ions. The activity was both detergent sensitive and thermolabile. Comparisons of gonococcal colony types 1 and 4 showed no significant differences between the two types with respect to either phospholipid content or phospholipase A activity. Images PMID:821921

  14. Phospholipid composition and phospholipase A activity of Neisseria gonorrhoeae.

    PubMed

    Senff, L M; Wegener, W S; Brooks, G F; Finnerty, W R; Makula, R A

    1976-08-01

    Exponential-phase cells of Neisseria gonorrhaeae 2686 were examined for phospholipid composition and for membrane-associated phospholipase A activity. When cells were harvested by centrifugation, washed, and lyophilized before extraction, approximately 74% of the total phospholipid was phosphatidylethanolamine, 18% was phosphatidylglycerol, 2% was cardiolipin, and 10% was lysophosphatidylethanolamine. However, when cells still suspended in growth medium were extracted, the amount of lysophosphatidylethanolamine decreased to approximately 1% of the phospholipid composition. This suggests that a gonococcal phospholipase A may be activated by conditions encountered during centrifugation and/or lyophilization of cells preceding extraction. Phospholipase A activity associated with cell membranes was assayed by measuring the conversion of tritiated phosphatidylethanolamine to lysophosphatidylethanolamine. Optimal activity was demonstrated in 10% methanol at pH 8.0 to 8.5, in the presence of calcium ions. The activity was both detergent sensitive and thermolabile. Comparisons of gonococcal colony types 1 and 4 showed no significant differences between the two types with respect to either phospholipid content or phospholipase A activity.

  15. COMPARISON OF METHODS TO IDENTIFY Neisseria meningitidis IN ASYMPTOMATIC CARRIERS

    PubMed Central

    RIZEK, Camila F.; LUIZ, André Machado; de ASSIS, Gracilene Ramos; COSTA, Silvia Figueiredo; LEVIN, Anna Sara; LOPES, Marta Heloisa

    2016-01-01

    SUMMARY Neisseria meningitidis is a cause of several life-threatening diseases and can be a normal commensal in the upper respiratory tract of healthy carriers. The carrier rate is not well established especially because there is no standard method for the isolation of N. meningitidis. Therefore, the aim of this study was to compare identification methods for the carrier state. Two swabs were collected from 190 volunteers: one was cultured and the other had DNA extracted directly from the sample. The Polymerase Chain Reaction (PCR) was performed to determine species and serogroups and compared the results between the methods. PCR for species determination used two pairs of primers and when there was only one amplicon, it was sequenced. The culture technique was positive in 23 (12.1%) subjects while the direct extraction method was positive in 132 (69.5%), p < 0.001. Among the 135 subjects with positive N. meningitides tests, 88 (65.2%) were serogroup C; 3 (2.2%) serogroup B; 5 (3.7%) were positive for both serogroup B and C, and 39 (28.9%) did not belong to any of the tested serogroups. In this study, PCR from DNA extracted directly from swabs identified more N. meningitidis asymptomatic carriers than the culture technique. PMID:27680165

  16. [MOLECULAR MECHANISMS OF DRUG RESISTANCE NEISSERIA GONORRHOEAE HISTORY AND PROSPECTS].

    PubMed

    Bodoev, I N; Il'ina, E N

    2015-01-01

    Neisseria gonorrhoeae (gonococcus) is a strict human pathogen, which causes gonorrhea--an infectious disease, whose origin dates back to more than two thousand years. Due to the unique plasticity of the genetic material, these bacteria have acquired the capacity to adapt to the host immune system, cause repeated infections, as well as withstand antimicrobials. Since the introduction of antibiotics in 1930s, gonococcus has displayed its propensity to develop resistance to all clinically useful antibiotics. It is important to note that the known resistance determinants of N. gonorrhoeae were acquired through horizontal gene transfer, recombination and spontaneous mutagenesis, and may be located both in the chromosome and on the plasmid. After introduction of a new antimicrobial drug, gonococcus becomes resistant within two decades and replaces sensitive bacterial population. Currently Ceftriaxone is the last remaining antibiotic for first-line treatment of gonorrhea. However, the first gonococcus displaying high-level resistance to Ceftriaxone was isolated in Japan a few years ago. Therefore, in the near future, gonorrhea may become untreatable. In the present review, we discuss the chronology of the anti-gonorrhea drugs (antibiotics) replacement, the evolution of resistance mechanisms emergence and future perspectives of N. gonorrhoeae treatment.

  17. The obligate human pathogen, Neisseria gonorrhoeae, is polyploid.

    PubMed

    Tobiason, Deborah M; Seifert, H Steven

    2006-06-01

    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.

  18. Immunological basis of serum resistance of Neisseria gonorrhoeae.

    PubMed

    Schneider, H; Griffiss, J M; Williams, G D; Pier, G B

    1982-01-01

    The immunological basis for resistance of certain strains of Neisseria gonorrhoeae to the bactericidal action of normal human serum was studied by investigating the potential role of factors which are known to interfere with each of the sequential steps that result in immune lysis of Gram-negative bacteria. Strains of N. gonorrhoeae were characterized as serum-sensitive (sers) or serum-resistant (serr) on the basis of their sensitivity to lysis by the sera of six normal individuals. Neither intrinsic resistance to the lytic action of activated human complement nor inaccessibility of the cell membrane to C5b accounted for serr. Outer membrane lipopolysaccharide (LPS) was the target antigen for lytic antibody in normal human sera. The gross chemical composition and molecular size of the LPS of the strains were heterogeneous and no consistent patterns of differences between those extracted from serr and from sers strains were found. Neither IgA nor IgG 'blocking' antibody in normal human serum was responsible for serr. We conclude that serr results from the absence from the LPS of the strains of antigenic loci for the lytic antibody in most normal human sera, or, expressed as a function of the host, the absence from the sera of most normal humans of lytic antibody directed against LPS antigenic loci for immune lysis.

  19. Seroepidemiologic aspects of Neisseria meningitidis in homosexual men.

    PubMed Central

    Salit, I. E.; Frasch, C. E.

    1982-01-01

    Neisseria meningitidis has been isolated with increasing frequency from specimens obtained from patients attending venereal disease clinics and is an occasional cause of genital infection. Among 383 homosexual men attending either a venereal disease clinic or a community screening clinic meningococci were cultured from specimens obtained from 35.0% of all the subjects, and with similar frequency in the two groups. Of the positive specimens 93.5% were from the throat, 5.8% from the rectum and 0.72% from the urethra. The serogroups and serotypes of the isolates were characteristic of those commonly found in nasopharyngeal specimens from other asymptomatic carriers. Gonococci were isolated from 8.6% of all the subjects and were 1.4 times more common in those who also harboured meningococci. Of the cultures positive for gonococci, 14.7% were from the throat and 85.3% from the rectum. The two bacteria were rarely isolated from the same site in the same individual. Gonococci, but not meningococci, were significantly more common (P less than 0.05) in the group attending the venereal disease, clinic than in the group attending the screening clinic, the rates being 17.1% and 7.0%. PMID:6800626

  20. Neisseria meningitidis Lactate Permease Is Required for Nasopharyngeal Colonization

    PubMed Central

    Exley, Rachel M.; Goodwin, Linda; Mowe, Eva; Shaw, Jonathan; Smith, Harry; Read, Robert C.; Tang, Christoph M.

    2005-01-01

    Neisseria meningitidis is a human specific pathogen that is part of the normal nasopharyngeal flora. Little is known about the metabolic constraints on survival of the meningococcus during colonization of the upper airways. Here we show that glucose and lactate, both carbon energy sources for meningococcal growth, are present in millimolar concentrations within nasopharyngeal tissue. We used a mutant defective for the uptake of lactate (C311ΔlctP) to investigate the contribution of this energy source during colonization. Explants of nasopharyngeal tissue were inoculated with the wild-type strain (C311) and C311ΔlctP; the mutant was recovered at significantly lower levels (P = 0.01) than C311 18 h later. This defect was not due to changes in the expression of adhesins or initial adhesion in C311ΔlctP to epithelial cells. Instead, lactate appears to be important energy source for the bacterium during colonization and is necessary for growth of the bacterium in nasopharyngeal tissue. Studies with other strains defective for the uptake of specific nutrients should provide valuable information about the environment in which N. meningitidis persists during carriage. PMID:16113293

  1. Functional analysis of the Gonococcal Genetic Island of Neisseria gonorrhoeae.

    PubMed

    Pachulec, Emilia; Siewering, Katja; Bender, Tobias; Heller, Eva-Maria; Salgado-Pabon, Wilmara; Schmoller, Shelly K; Woodhams, Katelynn L; Dillard, Joseph P; van der Does, Chris

    2014-01-01

    Neisseria gonorrhoeae is an obligate human pathogen that is responsible for the sexually-transmitted disease gonorrhea. N. gonorrhoeae encodes a T4SS within the Gonococcal Genetic Island (GGI), which secretes ssDNA directly into the external milieu. Type IV secretion systems (T4SSs) play a role in horizontal gene transfer and delivery of effector molecules into target cells. We demonstrate that GGI-like T4SSs are present in other β-proteobacteria, as well as in α- and γ-proteobacteria. Sequence comparison of GGI-like T4SSs reveals that the GGI-like T4SSs form a highly conserved unit that can be found located both on chromosomes and on plasmids. To better understand the mechanism of DNA secretion by N. gonorrhoeae, we performed mutagenesis of all genes encoded within the GGI, and studied the effects of these mutations on DNA secretion. We show that genes required for DNA secretion are encoded within the yaa-atlA and parA-parB regions, while genes encoded in the yfeB-exp1 region could be deleted without any effect on DNA secretion. Genes essential for DNA secretion are encoded within at least four different operons. PMID:25340397

  2. The obligate human pathogen, Neisseria gonorrhoeae, is polyploid.

    PubMed

    Tobiason, Deborah M; Seifert, H Steven

    2006-06-01

    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. PMID:16719561

  3. NadA, a novel vaccine candidate of Neisseria meningitidis.

    PubMed

    Comanducci, Maurizio; Bambini, Stefania; Brunelli, Brunella; Adu-Bobie, Jeannette; Aricò, Beatrice; Capecchi, Barbara; Giuliani, Marzia Monica; Masignani, Vega; Santini, Laura; Savino, Silvana; Granoff, Dan M; Caugant, Dominique A; Pizza, Mariagrazia; Rappuoli, Rino; Mora, Marirosa

    2002-06-01

    Neisseria meningitidis is a human pathogen, which, in spite of antibiotic therapy, is still a major cause of mortality due to sepsis and meningitis. Here we describe NadA, a novel surface antigen of N. meningitidis that is present in 52 out of 53 strains of hypervirulent lineages electrophoretic types (ET) ET37, ET5, and cluster A4. The gene is absent in the hypervirulent lineage III, in N. gonorrhoeae and in the commensal species N. lactamica and N. cinerea. The guanine/cytosine content, lower than the chromosome, suggests acquisition by horizontal gene transfer and subsequent limited evolution to generate three well-conserved alleles. NadA has a predicted molecular structure strikingly similar to a novel class of adhesins (YadA and UspA2), forms high molecular weight oligomers, and binds to epithelial cells in vitro supporting the hypothesis that NadA is important for host cell interaction. NadA induces strong bactericidal antibodies and is protective in the infant rat model suggesting that this protein may represent a novel antigen for a vaccine able to control meningococcal disease caused by three hypervirulent lineages. PMID:12045242

  4. Ethylenediaminetetraacetic acid-sensitive antiphagocytic activity of Neisseria gonorrhoeae.

    PubMed Central

    Rosenthal, R S; Fulbright, R S; Eads, M E; Sawyer, W D

    1977-01-01

    Colonial types of Neisseria gonorrhoeae were examined for the presence of pilus-independent antiphagocytic activity. Type 3 and depiliated type 1 gonococci had a shearing- and protease-resistant antiphagocytic activity that was eliminated by treatment with ethylenediaminetetraacetic acid (EDTA) and that was not present on type 4 bacteria. Incubation of EDTA-treated bacteria 37 degrees C for 90 min resulted in fas prevented by antibiotics that block the final assembly of cell wall macromolecules that depend on the C55-isoprenoid carrier for export. These include both lipopolysaccharide and peptidoglycan. Restoration was, however, unaffected by drugs that interfere with the synthesis of peptidoglycan, but not that of lipopolysaccharide, and by inhibitors of protein synthesis. These data suggested that gonococci have an antiphagocytic mechanism in addition to the previously described determinant (presumably pili) that was removed by blending or by treatment with proteases. Of the two antiphagocytic activities, type 1 had both, type 3 had only the EDTA-sensitive component, and type 4 had neither. PMID:404246

  5. Molecular characterization of rifampin-resistant Neisseria meningitidis.

    PubMed Central

    Carter, P E; Abadi, F J; Yakubu, D E; Pennington, T H

    1994-01-01

    Primers were designed to amplify the rpoB gene of Neisseria meningitidis. The region of the gene amplified covered clusters I and II of the rifampin resistance (Rifr) mutation sites identified in Escherichia coli. DNAs from six Rifr isolates and 21 rifampin-susceptible isolates from the United Kingdom representing a number of serogroups were amplified and sequenced. All six Rifr isolates had identical DNA sequences and the same amino acid change, a His to an Asn change at position 35 (H35N). This His residue is equivalent to the His residue at position 526 in E. coli, one of the known Rifr mutation sites. DNAs from an additional six Rifr mutations generated in vitro were amplified and sequenced. Three had H35Y changes, one had an H35R change, one had an H35N change and one had an S40F change. The predominance of mutations at the His residue at position 35 in Rifr N. meningitidis isolates suggests that it plays a critical role in the selection of antibiotic-resistant variants. All six Rifr isolates belonged to the same clonal group when analyzed by restriction enzyme analysis and pulsed-field gel electrophoresis. These data suggest that a single clone of Rifr N. meningitidis is present and widespread throughout the United Kingdom. Images PMID:8092823

  6. Characterization of the Neisseria meningitidis Helicase RecG

    PubMed Central

    Beyene, Getachew Tesfaye; Balasingham, Seetha V.; Frye, Stephan A.; Namouchi, Amine; Homberset, Håvard; Kalayou, Shewit; Riaz, Tahira

    2016-01-01

    Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm. A transient reduction in transformation of DNA was observed in the Nm ΔrecG strain as compared to the wildtype. The gene encoding recGNm also contained an unusually high number of the DNA uptake sequence (DUS) that facilitate transformation in neisserial species. The differentially abundant protein profiles of the Nm wildtype and ΔrecG strains suggest that expression of RecGNm might be linked to expression of other proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity. This might explain the growth defect that was observed in the Nm ΔrecG null mutant. PMID:27736945

  7. Concomitant infection with Neisseria gonorrhoeae and Chlamydia trachomatis in pregnancy.

    PubMed

    Christmas, J T; Wendel, G D; Bawdon, R E; Farris, R; Cartwright, G; Little, B B

    1989-09-01

    Gonorrhea is an important marker for endocervical chlamydial infections in nonpregnant women. Concomitant infection rates as high as 50% have been reported. There are few data on concomitant infection rates in pregnant patients. The purpose of this study was to examine the prevalence of endocervical chlamydial infections in pregnant women with gonorrhea. Patients with cervical cultures positive for Neisseria gonorrhoeae at their initial prenatal visit had endocervical specimens for Chlamydia trachomatis culture obtained before anti-gonorrheal therapy. Control patients were selected at random from the same prenatal population. The prevalence of C trachomatis in patients with gonorrhea was significantly greater than that in the control population (46 versus 5%; P less than .001). Patients with gonorrhea were younger, less often married, and more often black than the control population, but these demographic differences did not account for the large difference in the chlamydial prevalence. Erythromycin 500 mg four times daily provided an excellent cure rate without intolerable side effects. Pregnant patients being evaluated or treated for gonorrhea should also be considered at high risk for concomitant cervical chlamydial infection.

  8. Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence

    PubMed Central

    Engman, Jakob; Negrea, Aurel; Sigurlásdóttir, Sara; Geörg, Miriam; Eriksson, Jens; Eriksson, Olaspers Sara; Kuwae, Asaomi; Sjölinder, Hong

    2016-01-01

    Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3′–5′ exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis. PMID:26930706

  9. 'Neisseria skkuensis' sp. nov., isolated from the blood of a diabetic patient with a foot ulcer.

    PubMed

    Lee, Mi Young; Park, Eu Gene; Choi, Ji Young; Cheong, Hae Suk; Chung, Doo Ryeon; Peck, Kyong Ran; Song, Jae-Hoon; Ko, Kwan Soo

    2010-07-01

    A Gram-negative bacterium was isolated from the blood of a patient with diabetes mellitus. However, it could not be identified by conventional microbiological methods, and so was characterized by phenotypic and genotypic analyses. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Neisseria. Based on the phenotypic and genotypic characteristics, we propose that strain SMC-A9199(T) (=KCTC 22696(T)=JCM 16127(T)) should be classified as a novel species, 'Neisseria skkuensis' sp. nov. The patient was further treated with amoxicillin-clavulanate and ciprofloxacin for 3 weeks.

  10. Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria.

    PubMed

    Yao, Jiangwei; Bruhn, David F; Frank, Matthew W; Lee, Richard E; Rock, Charles O

    2016-01-01

    Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.

  11. Distribution and diversity of the haemoglobin-haptoglobin iron-acquisition systems in pathogenic and non-pathogenic Neisseria.

    PubMed

    Harrison, Odile B; Bennett, Julia S; Derrick, Jeremy P; Maiden, Martin C J; Bayliss, Christopher D

    2013-09-01

    A new generation of vaccines containing multiple protein components that aim to provide broad protection against serogroup B meningococci has been developed. One candidate, 4CMenB (4 Component MenB), has been approved by the European Medicines Agency, but is predicted to provide at most 70-80 % strain coverage; hence there is a need for second-generation vaccines that achieve higher levels of coverage. Prior knowledge of the diversity of potential protein vaccine components is a key step in vaccine design. A number of iron import systems have been targeted in meningococcal vaccine development, including the HmbR and HpuAB outer-membrane proteins, which mediate the utilization of haemoglobin or haemoglobin-haptoglobin complexes as iron sources. While the genetic diversity of HmbR has been described, little is known of the diversity of HpuAB. Using whole genome sequences deposited in a Bacterial Isolate Genome Sequence Database (BIGSDB), the prevalence and diversity of HpuAB among Neisseria were investigated. HpuAB was widely present in a range of Neisseria species whereas HmbR was mainly limited to the pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae. Patterns of sequence variation in sequences from HpuAB proteins were suggestive of recombination and diversifying selection consistent with strong immune selection. HpuAB was subject to repeat-mediated phase variation in pathogenic Neisseria and the closely related non-pathogenic Neisseria species Neisseria lactamica and Neisseria polysaccharea but not in the majority of other commensal Neisseria species. These findings are consistent with HpuAB being subject to frequent genetic transfer potentially limiting the efficacy of this receptor as a vaccine candidate.

  12. Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis.

    PubMed

    Smyth, C J; Friedman-Kien, A E; Salton, M R

    1976-04-01

    Crossed immunoelectrophoresis was used to study two complex antigenic preparations from Neisseria gonorrhoeae, one of cytoplasmic origin and the other derived by Triton X-100 extraction of isolated washed gonococcal envelopes, with the aim of developing suitable reference antigen-antibody systems that could be subsequently used to investigate the immune response to gonococcal infection and to monitor envelope preparations for cytoplasmic contamination. A number of parameters were investigated to optimized and standardize antigen preparation, e.g., harvesting and washing of gonococci, methods of bacterial disruption, and washing of envelopes. The effects of Triton X-100 concentration, initial total envelope protein concentration, and the composition, pH, and concentration of buffer on cell envelope extractability were studied to obviate the need to concentrate material before use in crossed immunoelectrophoresis. The electroendoosmotic properties of agarose were a major determining factor in resolving envelope antigens. From 25 to 30 immunoprecipitates were revealed in the envelope antigen-antibody system; 75 to 80 were revealed in the cytoplasmic sytem. Envelope immunoprecipitates with reduced nicotinamide adenine dinucleotide and lactate dehydrogenase activities were identified. Crossed immunoelectrophoresis with intermediate gels revealed the presence of antibodies in a preimmune rabbit antiserum pool to a distinctive fact-moving component in both the envelope and cytoplasmic antigen preparations. The intermediate gel technique also demonstrated that extensive washing of envelope preparations with buffer did not remove cytoplasmic ontamination completely. The method provides a much more sensitive means of monitoring the purity of envelope fractions than the use of single enzy,e markers as indexes of such contamination. The use of rabbit antisera raised to formolized gonococci in intermediate gels indicated that both reference antigen-antibody systems were of

  13. Towards a synthetic glycoconjugate vaccine against Neisseria meningitidis A.

    PubMed

    Berkin, Ali; Coxon, Bruce; Pozsgay, Vince

    2002-10-01

    Albumin conjugates of synthetic fragments of the capsular polysaccharide of the Gram-negative bacterium Neisseria meningitidis serogroup A were prepared. The fragments include monosaccharides 1 [alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)] and 2 [6-O-P(O)(O(-))(2)-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)], disaccharide 3 [alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)], and trisaccharide 4 [alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)]. Two monosaccharide blocks were employed as key intermediates. The reducing-end mannose unit featured the NHAc group at C-2, and contained the aminoethyl spacer as the aglycon for the final bioconjugation. The interresidual phosphodiester linkages were fashioned from an anomerically positioned H-phosphonate group in a 2-azido-mannose building block. The spacer-linked saccharides 1-4 were N-acylated with hepta-4,6-dienoic acid and the resulting conjugated diene-equipped saccharides were subjected to Diels-Alder-type addition with maleimidobutyryl-group functionalized human serum albumin to form covalent conjugates containing up to 26 saccharide haptens per albumin molecule. Complete (1)H, (13)C, and (31)P NMR assignments for 1-4 are given. Antigenicity of the neoglycoconjugates containing 1-4 was demonstrated by a double immunodiffusion assay which indicated that a fragment as small as a monosaccharide is recognized by a polyclonal meningococcus group A antiserum and that the O-acetyl group(s) present in the natural capsular material is not essential for antigenicity. PMID:12355530

  14. Serological classification of Neisseria gonorrhoeae with monoclonal antibody coagglutination reagents.

    PubMed Central

    Coghill, D V; Young, H

    1987-01-01

    A total of 357 clinical isolates of Neisseria gonorrhoeae from 286 patients were classified serologically using two independently developed panels of monoclonal coagglutination reagents. The Pharmacia (Ph) Diagnostics panel comprised 14 reagents, five specific for serogroup WI strains and nine specific for serogroup WII/III strains, whereas the Genetic Systems (GS) panel comprised 14 reagents, seven specific for serogroup WI strains and seven specific for serogroup WII/III strains. Serogroup WI represented 45% and WII/III represented 55% of the patients. Using the monoclonal antibody reagents, the serogroups could be further subdivided into so-called serovars. The Ph reagents identified four WI serovars and 21 WII/III serovars, whereas the GS reagents identified 10 WI serovars and 18 WII/III serovars. By combining the results obtained with each panel, 15 Ph/GS WI serovars and 33 Ph/GS WII/III serovars were recognised. In the WI isolates, one predominating serovar was recognised, whereas in the WII/III isolates, no single serovar predominated and a much greater variety of serovars was identified. The serovar patterns for men and women patients were very similar, except for one WII/III serovar that was 10 times more common in isolates from men than from women. Most isolates from different anatomical sites in the same patient were of the same serogroup and serovar. Two double infections were found. One patient had a genital infection with serogroup WII/III and a rectal infection with serogroup WI. Another patient with genital, rectal, and throat infections with serogroup WI was found to have gonococci of different GS serovars at each site. It was concluded that the level of discrimination achieved with the monoclonal antibody reagents should prove to be valuable in studying the micro epidemiology of gonococcal infection. PMID:3115886

  15. History and epidemiology of antibiotic susceptibilities of Neisseria gonorrhoeae.

    PubMed

    Shigemura, Katsumi; Fujisawa, Masato

    2015-01-01

    Neisseria gonorrhoeae is a common causative microorganism of male urethritis. The most important problem with this infectious disease is antibiotic resistance. For instance, in the 1980's-1990's, most studies showed almost 100% susceptibility of N. gonorrhoeae to the representative cephalosporins, cefixime and cefpodoxime. By the late 1990s, the reported susceptibility decreased to 93.3-100% and further decreased to 82.9-100% in the early 2000's. However, reported susceptibility was revived to 95.8-100% in the late 2000's to 2010's. The susceptibility of N. gonorrhoeae to penicillins varied in different countries and regions. A 2002 Japanese study showed a resistance ratio of about 30% and while Laos, China and Korea showed 80-100% resistance. Fluoroquinolones have shown a dramatic change in their effect on N. gonorrhoeae. In the early 1990's, 0.3-1.3% of N. gonorrhoeae showed low susceptibility or resistance to ciprofloxacin in the US but this figure jumped to 9.5% by 1999. In Asia, N. gonorrhoeae ciprofloxacin resistance or lower susceptibility was about 80-90% in the early 2000's and this trend continues to the present day. Azithromycin is currently the possible last weapon for N. gonorrhoeae treatment per oral administration. The susceptibility of N. gonorrhoeae to azithromycin was 100% in Indonesia in 2004 and the latest study from Germany showed 6% resistance in strains from 2010-2011. This review summarizes the history and epidemiology of N. gonorrhoeae antibiotic susceptibilities, for which the most frequently used antibiotics vary between countries or regions.

  16. Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

    PubMed

    Petrov, A B; Semenov, B F; Vartanyan, Y P; Zakirov, M M; Torchilin, V P; Trubetskoy, V S; Koshkina, N V; L'Vov, V L; Verner, I K; Lopyrev, I V

    1992-09-01

    To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age.

  17. Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

    PubMed Central

    Petrov, A B; Semenov, B F; Vartanyan, Y P; Zakirov, M M; Torchilin, V P; Trubetskoy, V S; Koshkina, N V; L'Vov, V L; Verner, I K; Lopyrev, I V

    1992-01-01

    To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age. PMID:1500196

  18. Antimicrobial susceptibility and molecular epidemiology of Neisseria gonorrhoeae in Germany.

    PubMed

    Horn, Nicole Nari; Kresken, Michael; Körber-Irrgang, Barbara; Göttig, Stephan; Wichelhaus, Cornelia; Wichelhaus, Thomas A

    2014-07-01

    Antimicrobial drug resistance in Neisseria gonorrhoeae has become an increasing public health problem. Hence, surveillance of resistance development is of crucial importance to implement adequate treatment guidelines. Data on the spread of antibiotic resistance among gonococcal isolates in Germany, however, is scarce. In a resistance surveillance study conducted by the Paul Ehrlich Society for Chemotherapy between October 2010 and December 2011, 23 laboratories all over Germany were requested to send N. gonorrhoeae isolates to the study laboratory in Frankfurt am Main. Species verification was performed biochemically using ApiNH and with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed using the Etest method. For molecular epidemiological analysis, N. gonorrhoeae strains were genotyped by means of N. gonorrhoeae multi-antigen sequence typing. A total of 213 consecutive gonococcal isolates were analyzed in this nationwide study. Applying EUCAST breakpoints, high resistance rates were found for ciprofloxacin (74%) and tetracycline (41%). Penicillin non-susceptibility was detected in 80% of isolates. The rate of azithromycin resistance was 6%, while all strains were susceptible to spectinomycin, cefixime, and ceftriaxone. Molecular typing of gonococcal isolates revealed a great heterogeneity of 99 different sequence types (ST), but ST1407 predominated (n=39). This is the first comprehensive German multi-centre surveillance study on antibiotic susceptibility and molecular epidemiology of N. gonorrhoeae with implications for antibiotic choice for treatment of gonorrhoea. The World Health Organization supports the concept that an efficacious treatment of gonorrhoea results in at least 95% of infections being cured. Accordingly, as spectinomycin is not available on the German market, only the third generation cephalosporins cefixime and ceftriaxone are regarded as valuable drugs

  19. Optimization of intracellular product release from Neisseria denitrificans using microfluidizer.

    PubMed

    Stupak, Robert; Makauskas, Nerijus; Radzevičius, Kostas; Valančius, Zenonas

    2015-01-01

    Disruption of Neisseria denitrificans cells by microfluidizer was optimized using a factorial experiments design. The pH, pretreatment time, cell concentration, NaCl, ethylenediamine tetraacetic acid (EDTA) and Triton X-100 concentrations showed significant impact on disruption process and the process was optimized using central composite design and response surface methodology (RSM). Investigation revealed optimum conditions: 90 min pretreatment at pH 9.0 containing 110 g L(-1) cells (dry cell weight), 50 mM NaCl, 10 mM EDTA, and 0.2% Triton X-100. At optimized conditions, the disruption rate increased twofold, up to 5.62 ± 0.27 × 10(-3) MPa(-a); meanwhile, yield of intracellular content was increased by 26%, with 1 g of cells resulting in 113.2 ± 8.2 mg proteins, 12.1 ± 0.7 mg nucleic acids, 21.0 ± 1.2 mg polysaccharides, 0.99 ± 0.08 kU glucose-6-phosphate dehydrogenase (G6PD), and 10,100 ± 110 kU restriction endonuclease NdeI endonuclease. Particle size distribution analysis revealed nearly twofold larger cell lysate particles with diameter of 120 nm. For optimal release of intracellular content, 9200 J/g of energy was needed (95% confidence), yielding 6900 J/g energy savings. Model equations generated from RSM on cell disruption of N. denitrificans were found adequate to determine significant factors and its interaction. The results showed that optimized combination of known pretreatment and disruption methods could considerably improve cell disruption efficiency.

  20. Absence of mucosal immunity in the human upper respiratory tract to the commensal bacteria Neisseria lactamica but not pathogenic Neisseria meningitidis during the peak age of nasopharyngeal carriage.

    PubMed

    Vaughan, Andrew T; Gorringe, Andrew; Davenport, Victoria; Williams, Neil A; Heyderman, Robert S

    2009-02-15

    The normal flora that colonizes the mucosal epithelia has evolved diverse strategies to evade, modulate, or suppress the immune system and avoid clearance. Neisseria lactamica and Neisseria meningitidis are closely related obligate inhabitants of the human upper respiratory tract. N. lactamica is a commensal but N. meningitidis is an opportunistic pathogen that occasionally causes invasive disease such as meningitis and septicemia. We demonstrate that unlike N. meningitidis, N. lactamica does not prime the development of mucosal T or B cell memory during the peak period of colonization. This cannot be explained by the induction of peripheral tolerance or regulatory CD4(+)CD25(+) T cell activity. Instead, N. lactamica mediates a B cell-dependent mitogenic proliferative response that is absent to N. meningitidis. This mitogenic response is associated with the production of T cell-independent polyclonal IgM that we propose functions by shielding colonizing N. lactamica from the adaptive immune system, maintaining immunological ignorance in the host. We conclude that, in contrast to N. meningitidis, N. lactamica maintains a commensal relationship with the host in the absence of an adaptive immune response. This may prolong the period of susceptibility to colonization by both pathogenic and nonpathogenic Neisseria species.

  1. Description of an unusual Neisseria meningitidis isolate containing and expressing Neisseria gonorrhoeae-Specific 16S rRNA gene sequences.

    PubMed

    Walcher, Marion; Skvoretz, Rhonda; Montgomery-Fullerton, Megan; Jonas, Vivian; Brentano, Steve

    2013-10-01

    An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination.

  2. Positive Selection Pressure Drives Variation on the Surface-Exposed Variable Proteins of the Pathogenic Neisseria

    PubMed Central

    Hill, Stuart

    2016-01-01

    Pathogenic species of Neisseria utilize variable outer membrane proteins to facilitate infection and proliferation within the human host. However, the mechanisms behind the evolution of these variable alleles remain largely unknown due to analysis of previously limited datasets. In this study, we have expanded upon the previous analyses to substantially increase the number of analyzed sequences by including multiple diverse strains, from various geographic locations, to determine whether positive selective pressure is exerted on the evolution of these variable genes. Although Neisseria are naturally competent, this analysis indicates that only intrastrain horizontal gene transfer among the pathogenic Neisseria principally account for these genes exhibiting linkage equilibrium which drives the polymorphisms evidenced within these alleles. As the majority of polymorphisms occur across species, the divergence of these variable genes is dependent upon the species and is independent of geographical location, disease severity, or serogroup. Tests of neutrality were able to detect strong selection pressures acting upon both the opa and pil gene families, and were able to locate the majority of these sites within the exposed variable regions of the encoded proteins. Evidence of positive selection acting upon the hypervariable domains of Opa contradicts previous beliefs and provides evidence for selection of receptor binding. As the pathogenic Neisseria reside exclusively within the human host, the strong selection pressures acting upon both the opa and pil gene families provide support for host immune system pressure driving sequence polymorphisms within these variable genes. PMID:27532335

  3. Correia Repeat Enclosed Elements and Non-Coding RNAs in the Neisseria Species

    PubMed Central

    Roberts, Sabrina B.; Spencer-Smith, Russell; Shah, Mahwish; Nebel, Jean-Christophe; Cook, Richard T.; Snyder, Lori A. S.

    2016-01-01

    Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Neisseria meningitidis is a closely related pathogen that shares many of the same genomic features and virulence factors, but causes the life threatening diseases meningococcal meningitis and septicaemia. The importance of non-coding RNAs in gene regulation has become increasingly evident having been demonstrated to be involved in regulons responsible for iron acquisition, antigenic variation, and virulence. Neisseria spp. contain an IS-like element, the Correia Repeat Enclosed Element, which has been predicted to be mobile within the genomes or to have been in the past. This repeat, present in over 100 copies in the genome, has the ability to alter gene expression and regulation in several ways. We reveal here that Correia Repeat Enclosed Elements tend to be near non-coding RNAs in the Neisseria spp., especially N. gonorrhoeae. These results suggest that Correia Repeat Enclosed Elements may have disrupted ancestral regulatory networks not just through their influence on regulatory proteins but also for non-coding RNAs. PMID:27681925

  4. Correia Repeat Enclosed Elements and Non-Coding RNAs in the Neisseria Species.

    PubMed

    Roberts, Sabrina B; Spencer-Smith, Russell; Shah, Mahwish; Nebel, Jean-Christophe; Cook, Richard T; Snyder, Lori A S

    2016-01-01

    Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Neisseria meningitidis is a closely related pathogen that shares many of the same genomic features and virulence factors, but causes the life threatening diseases meningococcal meningitis and septicaemia. The importance of non-coding RNAs in gene regulation has become increasingly evident having been demonstrated to be involved in regulons responsible for iron acquisition, antigenic variation, and virulence. Neisseria spp. contain an IS-like element, the Correia Repeat Enclosed Element, which has been predicted to be mobile within the genomes or to have been in the past. This repeat, present in over 100 copies in the genome, has the ability to alter gene expression and regulation in several ways. We reveal here that Correia Repeat Enclosed Elements tend to be near non-coding RNAs in the Neisseria spp., especially N. gonorrhoeae. These results suggest that Correia Repeat Enclosed Elements may have disrupted ancestral regulatory networks not just through their influence on regulatory proteins but also for non-coding RNAs. PMID:27681925

  5. Draft Genome Sequence of Neisseria gonorrhoeae Sequence Type 1407, a Multidrug-Resistant Clinical Isolate

    PubMed Central

    Anselmo, A.; Ciammaruconi, A.; Carannante, A.; Neri, A.; Fazio, C.; Fortunato, A.; Palozzi, A. M.; Vacca, P.; Fillo, S.; Lista, F.

    2015-01-01

    Gonorrhea may become untreatable due to the spread of resistant or multidrug-resistant strains. Cefixime-resistant gonococci belonging to sequence type 1407 have been described worldwide. We report the genome sequence of Neisseria gonorrhoeae strain G2891, a multidrug-resistant isolate of sequence type 1407, collected in Italy in 2013. PMID:26272575

  6. Conjunctivitis caused by Neisseria gonorrhoeae isolates with reduced cephalosporin susceptibility and multidrug resistance.

    PubMed

    Suzuki, Takashi; Kitagawa, Yutaka; Maruyama, Yosuke; Yamaguchi, Satoshi; Sakane, Yuri; Miyamoto, Hitoshi; Ohashi, Yuichi

    2013-12-01

    We report two cases of conjunctivitis caused by Neisseria gonorrhoeae with reduced cephalosporin susceptibility. Patients showed no response to cefmenoxime eye drops and intravenous ceftriaxone administration. The patients' condition improved after the addition of oral minocycline. The isolates contained the mosaic penA for reduction of β-lactam susceptibility.

  7. Acute urethritis due to Neisseria meningitidis group A acquired by orogenital contact: case report.

    PubMed Central

    Wilson, A P; Wolff, J; Atia, W

    1989-01-01

    A case of heterosexual transmission of Neisseria meningitidis group A by fellatio, which resulted in acute purulent urethritis in the male partner, is presented. The emotional impact of a mistaken diagnosis of gonococcal infection and the need to treat asymptomatic throat carriage in sexual contacts makes early recognition of meningococcal infection important in this unusual circumstance. PMID:2502493

  8. Correia Repeat Enclosed Elements and Non-Coding RNAs in the Neisseria Species.

    PubMed

    Roberts, Sabrina B; Spencer-Smith, Russell; Shah, Mahwish; Nebel, Jean-Christophe; Cook, Richard T; Snyder, Lori A S

    2016-08-25

    Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Neisseria meningitidis is a closely related pathogen that shares many of the same genomic features and virulence factors, but causes the life threatening diseases meningococcal meningitis and septicaemia. The importance of non-coding RNAs in gene regulation has become increasingly evident having been demonstrated to be involved in regulons responsible for iron acquisition, antigenic variation, and virulence. Neisseria spp. contain an IS-like element, the Correia Repeat Enclosed Element, which has been predicted to be mobile within the genomes or to have been in the past. This repeat, present in over 100 copies in the genome, has the ability to alter gene expression and regulation in several ways. We reveal here that Correia Repeat Enclosed Elements tend to be near non-coding RNAs in the Neisseria spp., especially N. gonorrhoeae. These results suggest that Correia Repeat Enclosed Elements may have disrupted ancestral regulatory networks not just through their influence on regulatory proteins but also for non-coding RNAs.

  9. Characterization of Neisseria gonorrhoeae reference strains used in development of serologic classification systems.

    PubMed Central

    Evins, G M; Knapp, J S

    1988-01-01

    Certain strains of Neisseria gonorrhoeae have been used by numerous investigators to develop serologic classification systems. Some of these strains have been used by investigators to study gonococcal virulence. A reference consisting of strain classification by auxotype and serovar, a strain history, and a selected bibliography are provided cohesively. PMID:3125223

  10. Gonococcal aneurysm of the ascending aorta: case report and review of Neisseria gonorrhoeae endovascular infections.

    PubMed

    Markowicz, Samuel; Anstey, James Richards; Hites, Maya; Montesinos, Isabel; Roisin, Sandrine; Keyzer, Caroline; Jacobs, Frederique

    2014-02-01

    We present the case of a man with a bicuspid aortic valve who presented with persistent fever. Blood cultures yielded Neisseria gonorrhoeae, and the diagnosis of infected mycotic aneurysm was confirmed by detection of the bacterial genome in the aortic wall. The patient was cured with surgery and intravenous ceftriaxone.

  11. Decline in Decreased Cephalosporin Susceptibility and Increase in Azithromycin Resistance in Neisseria gonorrhoeae, Canada

    PubMed Central

    Sawatzky, P.; Liu, G.; Allen, V; Lefebvre, B.; Hoang, L.; Drews, S.; Horsman, G.; Wylie, J.; Haldane, D.; Garceau, R.; Ratnam, S.; Wong, T.; Archibald, C.; Mulvey, M.R.

    2016-01-01

    Antimicrobial resistance profiles were determined for Neisseria gonorrhoeae strains isolated in Canada during 2010–2014. The proportion of isolates with decreased susceptibility to cephalosporins declined significantly between 2011 and 2014, whereas azithromycin resistance increased significantly during that period. Continued surveillance of antimicrobial drug susceptibilities is imperative to inform treatment guidelines. PMID:26689114

  12. Neisseria gonorrhoeae strain with reduced susceptibilities to extended-spectrum cephalosporins.

    PubMed

    Nguyen, Duylinh; Gose, Severin; Castro, Lina; Chung, Kathleen; Bernstein, Kyle; Samuel, Micheal; Bauer, Heidi; Pandori, Mark

    2014-07-01

    The spread of Neisseria gonorrhoeae strains with reduced susceptibility to extended-spectrum cephalosporins is an increasing public health threat. Using Etest and multiantigen sequence typing, we detected sequence type 1407, which is associated with reduced susceptibilities to extended-spectrum cephalosporins, in 4 major populated regions in California, USA, in 2012.

  13. Decline in Decreased Cephalosporin Susceptibility and Increase in Azithromycin Resistance in Neisseria gonorrhoeae, Canada.

    PubMed

    Martin, I; Sawatzky, P; Liu, G; Allen, V; Lefebvre, B; Hoang, L; Drews, S; Horsman, G; Wylie, J; Haldane, D; Garceau, R; Ratnam, S; Wong, T; Archibald, C; Mulvey, M R

    2016-01-01

    Antimicrobial resistance profiles were determined for Neisseria gonorrhoeae strains isolated in Canada during 2010-2014. The proportion of isolates with decreased susceptibility to cephalosporins declined significantly between 2011 and 2014, whereas azithromycin resistance increased significantly during that period. Continued surveillance of antimicrobial drug susceptibilities is imperative to inform treatment guidelines.

  14. Case-Fatality Rates and Sequelae Resulting from Neisseria meningitidis Serogroup C Epidemic, Niger, 2015

    PubMed Central

    Salou, Halidou; Sidikou, Fati; Goumbi, Kadadé; Djibo, Ali; Lechevalier, Pauline; Compaoré, Idrissa; Grais, Rebecca F.

    2016-01-01

    We describe clinical symptoms, case-fatality rates, and prevalence of sequelae during an outbreak of Neisseria meningitidis serogroup C infection in a rural district of Niger. During home visits, we established that household contacts of reported case-patients were at higher risk for developing meningitis than the general population. PMID:27649257

  15. 21 CFR 866.3390 - Neisseria spp. direct serological test reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Neisseria spp. direct serological test reagents. 866.3390 Section 866.3390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  16. 21 CFR 866.3390 - Neisseria spp. direct serological test reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Neisseria spp. direct serological test reagents. 866.3390 Section 866.3390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  17. 21 CFR 866.3390 - Neisseria spp. direct serological test reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Neisseria spp. direct serological test reagents. 866.3390 Section 866.3390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  18. 21 CFR 866.3390 - Neisseria spp. direct serological test reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Neisseria spp. direct serological test reagents. 866.3390 Section 866.3390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  19. 21 CFR 866.3390 - Neisseria spp. direct serological test reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Neisseria spp. direct serological test reagents. 866.3390 Section 866.3390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  20. Effectiveness of Meningococcal B Vaccine against Endemic Hypervirulent Neisseria meningitidis W Strain, England

    PubMed Central

    Giuliani, Marzia Monica; Biolchi, Alessia; Pizza, Mariagrazia; Beebeejaun, Kazim; Lucidarme, Jay; Findlow, Jamie; Ramsay, Mary E.; Borrow, Ray

    2016-01-01

    Serum samples from children immunized with a meningococcal serogroup B vaccine demonstrated potent serum bactericidal antibody activity against the hypervirulent Neisseria meningitidis serogroup W strain circulating in England. The recent introduction of this vaccine into the United Kingdom national immunization program should also help protect infants against this endemic strain. PMID:26811872

  1. Correia Repeat Enclosed Elements and Non-Coding RNAs in the Neisseria Species

    PubMed Central

    Roberts, Sabrina B.; Spencer-Smith, Russell; Shah, Mahwish; Nebel, Jean-Christophe; Cook, Richard T.; Snyder, Lori A. S.

    2016-01-01

    Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Neisseria meningitidis is a closely related pathogen that shares many of the same genomic features and virulence factors, but causes the life threatening diseases meningococcal meningitis and septicaemia. The importance of non-coding RNAs in gene regulation has become increasingly evident having been demonstrated to be involved in regulons responsible for iron acquisition, antigenic variation, and virulence. Neisseria spp. contain an IS-like element, the Correia Repeat Enclosed Element, which has been predicted to be mobile within the genomes or to have been in the past. This repeat, present in over 100 copies in the genome, has the ability to alter gene expression and regulation in several ways. We reveal here that Correia Repeat Enclosed Elements tend to be near non-coding RNAs in the Neisseria spp., especially N. gonorrhoeae. These results suggest that Correia Repeat Enclosed Elements may have disrupted ancestral regulatory networks not just through their influence on regulatory proteins but also for non-coding RNAs.

  2. Case-Fatality Rates and Sequelae Resulting from Neisseria meningitidis Serogroup C Epidemic, Niger, 2015.

    PubMed

    Coldiron, Matthew E; Salou, Halidou; Sidikou, Fati; Goumbi, Kadadé; Djibo, Ali; Lechevalier, Pauline; Compaoré, Idrissa; Grais, Rebecca F

    2016-10-01

    We describe clinical symptoms, case-fatality rates, and prevalence of sequelae during an outbreak of Neisseria meningitidis serogroup C infection in a rural district of Niger. During home visits, we established that household contacts of reported case-patients were at higher risk for developing meningitis than the general population. PMID:27649257

  3. Complete Genome Sequences of Neisseria gonorrhoeae with Coresistance to First-Line Antimicrobials.

    PubMed

    Bharat, Amrita; Martin, Irene; Demczuk, Walter; Allen, Vanessa; Haldane, David; Hoang, Linda; Mulvey, Michael R

    2016-01-01

    Neisseria gonorrhoeae strains with coresistance to the first-line antimicrobial treatments azithromycin and ceftriaxone are an emerging public health threat. Here, we present the complete genome sequences of three strains of N. gonorrhoeae, including one susceptible strain and two strains with coresistance to ceftriaxone and azithromycin. PMID:27609929

  4. Complete Genome Sequences of Neisseria gonorrhoeae with Coresistance to First-Line Antimicrobials

    PubMed Central

    Bharat, Amrita; Martin, Irene; Demczuk, Walter; Allen, Vanessa; Haldane, David; Hoang, Linda

    2016-01-01

    Neisseria gonorrhoeae strains with coresistance to the first-line antimicrobial treatments azithromycin and ceftriaxone are an emerging public health threat. Here, we present the complete genome sequences of three strains of N. gonorrhoeae, including one susceptible strain and two strains with coresistance to ceftriaxone and azithromycin. PMID:27609929

  5. Emergence of high level azithromycin-resistant Neisseria gonorrhoeae strain isolated in Argentina.

    PubMed

    Galarza, Patricia G; Alcalá, Belén; Salcedo, Celia; Canigia, Liliana Fernández; Buscemi, Luis; Pagano, Irene; Oviedo, Claudia; Vázquez, Julio A

    2009-12-01

    One Neisseria gonorrhoeae strains highly resistant to azithromycin AzHLR (MIC >2048 mg/L) was isolated in Argentina in 2001 and it has been characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST) as ST696, suggesting a different event to other isolates in Europe. Neither, mtrR mutations or presence of mef gene were detected.

  6. Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Like most microorganisms, Neisseria gonorrhoeae alters gene expression in response to iron availability. The ferric uptake regulator Fur has been shown to be involved in controlling this response, but the extent of this involvement remains unknown. It is known that in addition to working directly to...

  7. Neisseria oralis sp. nov., isolated from healthy gingival plaque and clinical samples.

    PubMed

    Wolfgang, William J; Passaretti, Teresa V; Jose, Reashma; Cole, Jocelyn; Coorevits, An; Carpenter, Andrea N; Jose, Sherly; Van Landschoot, Anita; Izard, Jacques; Kohlerschmidt, Donna J; Vandamme, Peter; Dewhirst, Floyd E; Fisher, Mark A; Musser, Kimberlee A

    2013-04-01

    A polyphasic analysis was undertaken of seven independent isolates of gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7-100 %, and the closest species with a validly published name was Neisseria lactamica (96.9 % similarity to the type strain). DNA-DNA hybridization confirmed that these isolates are of the same species and are distinct from their nearest phylogenetic neighbour, N. lactamica. Phylogenetic analysis of 16S and 23S rRNA gene sequences indicated that the novel species belongs in the genus Neisseria. The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The cellular fatty acid profile, together with other phenotypic characters, further supports the inclusion of the novel species in the genus Neisseria. The name Neisseria oralis sp. nov. (type strain 6332(T)  = DSM 25276(T)  = LMG 26725(T)) is proposed.

  8. A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

    PubMed

    Jen, Freda E-C; Djoko, Karrera Y; Bent, Stephen J; Day, Christopher J; McEwan, Alastair G; Jennings, Michael P

    2015-09-01

    Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species.

  9. Effectiveness of Meningococcal B Vaccine against Endemic Hypervirulent Neisseria meningitidis W Strain, England.

    PubMed

    Ladhani, Shamez N; Giuliani, Marzia Monica; Biolchi, Alessia; Pizza, Mariagrazia; Beebeejaun, Kazim; Lucidarme, Jay; Findlow, Jamie; Ramsay, Mary E; Borrow, Ray

    2016-02-01

    Serum samples from children immunized with a meningococcal serogroup B vaccine demonstrated potent serum bactericidal antibody activity against the hypervirulent Neisseria meningitidis serogroup W strain circulating in England. The recent introduction of this vaccine into the United Kingdom national immunization program should also help protect infants against this endemic strain.

  10. Tied down: tethering redox proteins to the outer membrane in Neisseria and other genera.

    PubMed

    Li, Xi; Parker, Steven; Deeudom, Manu; Moir, James W

    2011-12-01

    Typically, the redox proteins of respiratory chains in Gram-negative bacteria are localized in the cytoplasmic membrane or in the periplasm. An alternative arrangement appears to be widespread within the betaproteobacterial genus Neisseria, wherein several redox proteins are covalently associated with the outer membrane. In the present paper, we discuss the structural properties of these outer membrane redox proteins and the functional consequences of this attachment. Several tethered outer membrane redox proteins of Neisseria contain a weakly conserved repeated structure between the covalent tether and the redox protein globular domain that should enable the redox cofactor-containing domain to extend from the outer membrane, across the periplasm and towards the inner membrane. It is argued that the constraints imposed on the movement and orientation of the globular domains by these tethers favours the formation of electron-transfer complexes for entropic reasons. The attachment to the outer membrane may also affect the exposure of the host to redox proteins with a moonlighting function in the host-microbe interaction, thus affecting the host response to Neisseria infection. We identify putative outer membrane redox proteins from a number of other bacterial genera outside Neisseria, and suggest that this organizational arrangement may be more common than previously recognized.

  11. A genome-wide identification of genes undergoing recombination and positive selection in Neisseria.

    PubMed

    Yu, Dong; Jin, Yuan; Yin, Zhiqiu; Ren, Hongguang; Zhou, Wei; Liang, Long; Yue, Junjie

    2014-01-01

    Currently, there is particular interest in the molecular mechanisms of adaptive evolution in bacteria. Neisseria is a genus of gram negative bacteria, and there has recently been considerable focus on its two human pathogenic species N. meningitidis and N. gonorrhoeae. Until now, no genome-wide studies have attempted to scan for the genes related to adaptive evolution. For this reason, we selected 18 Neisseria genomes (14 N. meningitidis, 3 N. gonorrhoeae and 1 commensal N. lactamics) to conduct a comparative genome analysis to obtain a comprehensive understanding of the roles of natural selection and homologous recombination throughout the history of adaptive evolution. Among the 1012 core orthologous genes, we identified 635 genes with recombination signals and 10 genes that showed significant evidence of positive selection. Further functional analyses revealed that no functional bias was found in the recombined genes. Positively selected genes are prone to DNA processing and iron uptake, which are essential for the fundamental life cycle. Overall, the results indicate that both recombination and positive selection play crucial roles in the adaptive evolution of Neisseria genomes. The positively selected genes and the corresponding amino acid sites provide us with valuable targets for further research into the detailed mechanisms of adaptive evolution in Neisseria.

  12. Lack of lipid A pyrophosphorylation and functional lptA reduces inflammation by Neisseria commensals.

    PubMed

    John, Constance M; Liu, Mingfeng; Phillips, Nancy J; Yang, Zhijie; Funk, Courtney R; Zimmerman, Lindsey I; Griffiss, J McLeod; Stein, Daniel C; Jarvis, Gary A

    2012-11-01

    The interaction of the immune system with Neisseria commensals remains poorly understood. We have previously shown that phosphoethanolamine on the lipid A portion of lipooligosaccharide (LOS) plays an important role in Toll-like receptor 4 (TLR4) signaling. For pathogenic Neisseria, phosphoethanolamine is added to lipid A by the phosphoethanolamine transferase specific for lipid A, which is encoded by lptA. Here, we report that Southern hybridizations and bioinformatics analyses of genomic sequences from all eight commensal Neisseria species confirmed that lptA was absent in 15 of 17 strains examined but was present in N. lactamica. Mass spectrometry of lipid A and intact LOS revealed the lack of both pyrophosphorylation and phosphoethanolaminylation in lipid A of commensal species lacking lptA. Inflammatory signaling in human THP-1 monocytic cells was much greater with pathogenic than with commensal Neisseria strains that lacked lptA, and greater sensitivity to polymyxin B was consistent with the absence of phosphoethanolamine. Unlike the other commensals, whole bacteria of two N. lactamica commensal strains had low inflammatory potential, whereas their lipid A had high-level pyrophosphorylation and phosphoethanolaminylation and induced high-level inflammatory signaling, supporting previous studies indicating that this species uses mechanisms other than altering lipid A to support commensalism. A meningococcal lptA deletion mutant had reduced inflammatory potential, further illustrating the importance of lipid A pyrophosphorylation and phosphoethanolaminylation in the bioactivity of LOS. Overall, our results indicate that lack of pyrophosphorylation and phosphoethanolaminylation of lipid A contributes to the immune privilege of most commensal Neisseria strains by reducing the inflammatory potential of LOS.

  13. Recognition of Neisseria meningitidis by the long pentraxin PTX3 and its role as an endogenous adjuvant.

    PubMed

    Bottazzi, Barbara; Santini, Laura; Savino, Silvana; Giuliani, Marzia M; Dueñas Díez, Ana I; Mancuso, Giuseppe; Beninati, Concetta; Sironi, Marina; Valentino, Sonia; Deban, Livija; Garlanda, Cecilia; Teti, Giuseppe; Pizza, Mariagrazia; Rappuoli, Rino; Mantovani, Alberto

    2015-01-01

    Long pentraxin 3 (PTX3) is a non-redundant component of the humoral arm of innate immunity. The present study was designed to investigate the interaction of PTX3 with Neisseria meningitidis. PTX3 bound acapsular meningococcus, Neisseria-derived outer membrane vesicles (OMV) and 3 selected meningococcal antigens (GNA0667, GNA1030 and GNA2091). PTX3-recognized microbial moieties are conserved structures which fulfil essential microbial functions. Ptx3-deficient mice had a lower antibody response in vaccination protocols with OMV and co-administration of PTX3 increased the antibody response, particularly in Ptx3-deficient mice. Administration of PTX3 reduced the bacterial load in infant rats challenged with Neisseria meningitidis. These results suggest that PTX3 recognizes a set of conserved structures from Neisseria meningitidis and acts as an amplifier/endogenous adjuvant of responses to this bacterium.

  14. Structural, functional and immunogenic insights on Cu,Zn Superoxide Dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and he...

  15. [Healthcare-associated Neisseria meningitidis W135 conjunctivitis].

    PubMed

    Unal Yılmaz, Gülizar; Alkan, Metin; Vatansever Özbek, Ulfet; Tuğrul, H Murat

    2013-10-01

    Neisseria meningitidis is an unusual pathogen among the causes of acute bacterial conjunctivitis. Meningococcal conjunctivitis may present as primary or secondary infection, while primary meningococcal conjunctivitis may emerge as invasive or non-invasive forms. N.meningitidis W135 strain is not common in Turkey, and is rarely reported as the cause of meningitis. Moreover, no cases of conjunctivitis due to N.meningitidis W135 were reported from Turkey. In this report a case of N.meningitidis W135 conjunctivitis has been presented who acquired the infection from another patient with meningococcal meningitis by close contact in the hospital environment. A 2-month-old male infant was admitted to our hospital with poor health condition, feeding difficulty and weight loss. He was hospitalized in intensive care unit and fluid replacement started due to severe dehydration. The infant had stigmata of Down's Syndrome, and since conjunctivitis were detected on physical examination, swab samples were obtained from both eyes for direct microscopic examination and cultivation. Abundant lekocytes and gram-negative diplococci were observed in Gram-stained smears, and bacterial growth were detected in the culture from left eye samples. The isolate have been identified as N.meningitidis by conventional microbiological methods, and serotyping of the isolate yielded W135 strain. The infant was treated with systemic cefotaxime and ampicillin-sulbactam, together with topical tobramycin and gentamycin. Since no symptoms of meningitis appeared during the follow-ups, the case was diagnosed as non-invasive primary meningococcal conjunctivitis. Investigation for a probable source revealed that the infant had close contact with a six-year-old boy with high fever, unconsciousness and vomiting a week ago in the outpatient clinic of Tekirdag State Hospital. N.meningitidis was also isolated from the cerebrospinal fluid culture of probable index case with meningitis and identified as W135 strain

  16. Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB.

    PubMed Central

    Lee, F K; Stephens, D S; Gibson, B W; Engstrom, J J; Zhou, D; Apicella, M A

    1995-01-01

    Neisseria meningitidis is the etiologic agent of epidemic bacterial meningitis. Lipooligosaccharide (LOS) is a principal virulence factor associated with the organism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LOS has demonstrated that there is considerable microheterogeneity in the molecule. To begin our understanding of the nature of this heterogeneity, we identified a Tn916-generated LOS mutant of N. meningitidis NMB (serotype L3, monoclonal antibodies 3F11+, 6B4+, and 4C4-) that was designated NMB-SS3 (monoclonal antibodies 3F11-, 6B4-, and 4C4+). The transposon insertion was localized to the amino terminus of the functional copy of the UDP-Glc 4-epimerase gene (galE). UDP-Glc 4-epimerase (EC 5.1.3.2) activity was present in N. meningitidis NMB but not in NMB-SS3, indicating that the Tn916 insertion had abolished this activity. Mass spectrometric analysis of the LOS from strain NMB revealed multiple species of LOS, which is consistent with extensive microheterogeneity. While the most predominant structure was consistent with a terminal lacto-N-neotetrose structure found in other strains of N. meningitidis, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-->(GlcNAc)-->Hep2PEA-->KDO2 (where Hep is heptose, PEA is phosphoethanolamine, and KDO is 2-keto-3-deoxymannooctulosonic acid), structures containing repetitive hexoses which are not precursors of this structure were also identified. Compositional analysis of LOS from strain NMB-SS3 revealed that there were no galactoses present in the structure. Mass spectrometric analysis of O-deacylated LOS revealed the presence of multiple species, with the predominant LOS species in this mutant strain formed by the Hex-->(HexNAc)-->Hep2PEA-->KDO2 (where Hex is hexose and HexNAc is N-acetylhexosamine) structure. However, LOS structures with repetitive hexoses, e.g., Hexn-->(HexNAc)-->Hep2PEA-->KDO2 (n = 2, 3, or 4), emanating from one or both heptoses were also identified. Since this mutant

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

    PubMed Central

    Müller, A; Günther, D; Düx, F; Naumann, M; Meyer, T F; Rudel, T

    1999-01-01

    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. PMID:9889191

  18. Genetically modified L3,7 and L2 lipooligosaccharides from Neisseria meningitidis serogroup B confer a broad cross-bactericidal response.

    PubMed

    Weynants, V; Denoël, P; Devos, N; Janssens, D; Feron, C; Goraj, K; Momin, P; Monnom, D; Tans, C; Vandercammen, A; Wauters, F; Poolman, Jan T

    2009-05-01

    Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

  19. Genetically Modified L3,7 and L2 Lipooligosaccharides from Neisseria meningitidis Serogroup B Confer a Broad Cross-Bactericidal Response▿

    PubMed Central

    Weynants, V.; Denoël, P.; Devos, N.; Janssens, D.; Feron, C.; Goraj, K.; Momin, P.; Monnom, D.; Tans, C.; Vandercammen, A.; Wauters, F.; Poolman, Jan T.

    2009-01-01

    Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease. PMID:19289516

  20. Molecular and Serological Diversity of Neisseria meningitidis Carrier Strains Isolated from Italian Students Aged 14 to 22 Years

    PubMed Central

    Comanducci, Maurizio; Amicizia, Daniela; Ansaldi, Filippo; Canepa, Paola; Orsi, Andrea; Icardi, Giancarlo; Rizzitelli, Emanuela; De Angelis, Gabriella; Bambini, Stefania; Moschioni, Monica; Comandi, Sara; Simmini, Isabella; Boccadifuoco, Giueseppe; Brunelli, Brunella; Giuliani, Marzia Monica; Pizza, Mariagrazia

    2014-01-01

    Neisseria meningitidis is an obligate human commensal that commonly colonizes the oropharyngeal mucosa. Carriage is age dependent and very common in young adults. The relationships between carriage and invasive disease are not completely understood. In this work, we performed a longitudinal carrier study in adolescents and young adults (173 subjects). Overall, 32 subjects (18.5%) had results that were positive for meningococcal carriage in at least one visit (average monthly carriage rate, 12.1%). Only five subjects tested positive at all four visits. All meningococcal isolates were characterized by molecular and serological techniques. Multilocus sequence typing, PorA typing, and sequencing of the 4CMenB vaccine antigens were used to assess strain diversity. The majority of positive subjects were colonized by capsule null (34.4%) and capsular group B strains (28.1%), accounting for 23.5% and 29.4% of the total number of isolates, respectively. The fHbp and nhba genes were present in all isolates, while the nadA gene was present in 5% of the isolates. The genetic variability of the 4CMenB vaccine antigens in this collection was relatively high compared with that of other disease-causing strain panels. Indications about the persistence of the carriage state were limited to the time span of the study. All strains isolated from the same subject were identical or cumulated minor changes over time. The expression levels and antigenicities of the 4CMenB vaccine antigens in each strain were analyzed by the meningococcal antigen typing system (MATS), which revealed that expression can change over time in the same individual. Future analysis of antigen variability and expression in carrier strains after the introduction of the MenB vaccine will allow for a definition of its impact on nasopharyngeal/oropharyngeal carriage. PMID:24648565

  1. Molecular and serological diversity of Neisseria meningitidis carrier strains isolated from Italian students aged 14 to 22 years.

    PubMed

    Gasparini, Roberto; Comanducci, Maurizio; Amicizia, Daniela; Ansaldi, Filippo; Canepa, Paola; Orsi, Andrea; Icardi, Giancarlo; Rizzitelli, Emanuela; De Angelis, Gabriella; Bambini, Stefania; Moschioni, Monica; Comandi, Sara; Simmini, Isabella; Boccadifuoco, Giueseppe; Brunelli, Brunella; Giuliani, Marzia Monica; Pizza, Mariagrazia; Panatto, Donatella

    2014-06-01

    Neisseria meningitidis is an obligate human commensal that commonly colonizes the oropharyngeal mucosa. Carriage is age dependent and very common in young adults. The relationships between carriage and invasive disease are not completely understood. In this work, we performed a longitudinal carrier study in adolescents and young adults (173 subjects). Overall, 32 subjects (18.5%) had results that were positive for meningococcal carriage in at least one visit (average monthly carriage rate, 12.1%). Only five subjects tested positive at all four visits. All meningococcal isolates were characterized by molecular and serological techniques. Multilocus sequence typing, PorA typing, and sequencing of the 4CMenB vaccine antigens were used to assess strain diversity. The majority of positive subjects were colonized by capsule null (34.4%) and capsular group B strains (28.1%), accounting for 23.5% and 29.4% of the total number of isolates, respectively. The fHbp and nhba genes were present in all isolates, while the nadA gene was present in 5% of the isolates. The genetic variability of the 4CMenB vaccine antigens in this collection was relatively high compared with that of other disease-causing strain panels. Indications about the persistence of the carriage state were limited to the time span of the study. All strains isolated from the same subject were identical or cumulated minor changes over time. The expression levels and antigenicities of the 4CMenB vaccine antigens in each strain were analyzed by the meningococcal antigen typing system (MATS), which revealed that expression can change over time in the same individual. Future analysis of antigen variability and expression in carrier strains after the introduction of the MenB vaccine will allow for a definition of its impact on nasopharyngeal/oropharyngeal carriage.

  2. Molecular and serological diversity of Neisseria meningitidis carrier strains isolated from Italian students aged 14 to 22 years.

    PubMed

    Gasparini, Roberto; Comanducci, Maurizio; Amicizia, Daniela; Ansaldi, Filippo; Canepa, Paola; Orsi, Andrea; Icardi, Giancarlo; Rizzitelli, Emanuela; De Angelis, Gabriella; Bambini, Stefania; Moschioni, Monica; Comandi, Sara; Simmini, Isabella; Boccadifuoco, Giueseppe; Brunelli, Brunella; Giuliani, Marzia Monica; Pizza, Mariagrazia; Panatto, Donatella

    2014-06-01

    Neisseria meningitidis is an obligate human commensal that commonly colonizes the oropharyngeal mucosa. Carriage is age dependent and very common in young adults. The relationships between carriage and invasive disease are not completely understood. In this work, we performed a longitudinal carrier study in adolescents and young adults (173 subjects). Overall, 32 subjects (18.5%) had results that were positive for meningococcal carriage in at least one visit (average monthly carriage rate, 12.1%). Only five subjects tested positive at all four visits. All meningococcal isolates were characterized by molecular and serological techniques. Multilocus sequence typing, PorA typing, and sequencing of the 4CMenB vaccine antigens were used to assess strain diversity. The majority of positive subjects were colonized by capsule null (34.4%) and capsular group B strains (28.1%), accounting for 23.5% and 29.4% of the total number of isolates, respectively. The fHbp and nhba genes were present in all isolates, while the nadA gene was present in 5% of the isolates. The genetic variability of the 4CMenB vaccine antigens in this collection was relatively high compared with that of other disease-causing strain panels. Indications about the persistence of the carriage state were limited to the time span of the study. All strains isolated from the same subject were identical or cumulated minor changes over time. The expression levels and antigenicities of the 4CMenB vaccine antigens in each strain were analyzed by the meningococcal antigen typing system (MATS), which revealed that expression can change over time in the same individual. Future analysis of antigen variability and expression in carrier strains after the introduction of the MenB vaccine will allow for a definition of its impact on nasopharyngeal/oropharyngeal carriage. PMID:24648565

  3. Lipopolysaccharide structure and serum sensitivity of non-serogroupable Neisseria meningitidis.

    PubMed

    Blackwell, C C; Winstanley, F P; Weir, D M; Kinane, D F

    1987-09-01

    Bactericidal activities of normal human serum for non-serogroupable strains of Neisseria meningitidis were determined. In similar experiments with isolates of Neisseria gonorrhoeae from localized infections, strains with group I lipopolysaccharide (LPS) were uniformly serum resistant and those with group II were serum sensitive. We found no similar association between serum sensitivity of the meningococcal strains and their lipopolysaccharide groups determined by the same pyocin typing system used to classify the gonococcal isolates. Immune mouse sera raised against non-serogroupable meningococci of either LPS group I or II were bactericidal for non-serogroupable strains of the same LPS group and also cross-reactive for strains of the opposite group. They were not bactericidal for the majority (13/17) of the serogroupable strains tested. These findings suggest there are antigens, in addition to the LPS and capsules, that elicit some of the "natural" bactericidal antibodies to pathogenic meningococci.

  4. Pharyngeal Neisseria gonorrhoeae detection in oral-throat wash specimens of male patients with urethritis.

    PubMed

    Takahashi, Satoshi; Kurimura, Yuichiro; Hashimoto, Jiro; Takeyama, Koh; Koroku, Mikio; Tanda, Hitoshi; Nishimura, Masahiro; Tsukamoto, Taiji

    2008-12-01

    Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in the pharynx has been highlighted in the prevention of the unexpected spread of sexually transmitted diseases. We tried to clarify the detection rate of Neisseria gonorrhoeae in the pharynx and the clinical relevance of oral-throat wash specimens to detect the organism in heterosexual men with gonococcal and nongonococcal urethritis. In our cohort of 79 male patients with urethritis, oral throat wash specimens were collected after they had gargled with normal saline for approximately 30 to 60 s. Positive pharyngeal N. gonorrhoeae was defined as a positive result on the strand displacement amplification test for the specimen from the oral-throat wash. N. gonorrhoeae was detected in the oral-throat wash specimens of 13 (31.7%) of the 41 male patients with gonococcal urethritis. Oral-throat wash with a nucleic acid amplification test can detect pharyngeal N. gonorrhoeae easily and efficiently.

  5. Efficacy of a Novel Tricyclic Topoisomerase Inhibitor in a Murine Model of Neisseria gonorrhoeae Infection.

    PubMed

    Savage, Victoria J; Charrier, Cédric; Salisbury, Anne-Marie; Box, Helen; Chaffer-Malam, Nathan; Huxley, Anthony; Kirk, Ralph; Noonan, Gary M; Mohmed, Sarfraz; Craighead, Mark W; Ratcliffe, Andrew J; Best, Stuart A; Stokes, Neil R

    2016-09-01

    There is an urgent need for new antibiotics to treat multidrug-resistant Neisseria gonorrhoeae In this report, the microbiology, in vivo pharmacokinetics, and efficacy of REDX05931, a representative novel tricyclic topoisomerase inhibitor, were evaluated. REDX05931 demonstrated high oral bioavailability in mice and reduced N. gonorrhoeae infection after a single dose in a mouse model of gonorrhea. These data support the potential of this series of small molecules as a new treatment for drug-resistant gonorrheal infections. PMID:27324777

  6. Penicillinase-producing plasmid types in Neisseria gonorrhoeae clinical isolates from Australia.

    PubMed

    Whiley, David; Trembizki, Ella; Buckley, Cameron; Freeman, Kevin; Lawrence, Andrew; Limnios, Athena; Pearson, Julie; Smith, Helen; Stevens, Kerrie; Lahra, Monica M

    2014-12-01

    Penicillinase-producing Neisseria gonorrhoeae (PPNG) carrying the blaTEM-135 gene is of particular concern, as it is considered a stepping stone toward resistance to extended-spectrum cephalosporins. Here, we sought to characterize plasmid types and the occurrence of the blaTEM-135 gene for N. gonorrhoeae clinical isolates from Australia. We found that blaTEM-135 was prevalent in Australian PPNG and was detected on all three major plasmid types.

  7. Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence.

    PubMed Central

    Seifert, H S; Ajioka, R S; Paruchuri, D; Heffron, F; So, M

    1990-01-01

    The method of shuttle mutagenesis has been extended to Neisseria gonorrhoeae. We have constructed a defective mini-Tn3 derivative that encodes chloramphenicol resistance in both N. gonorrhoeae and Escherichia coli and selected for mutations in the chloramphenicol resistance gene that express higher levels of antibiotic resistance in N. gonorrhoeae. Isogenic N. gonorrhoeae strains that differ only in pilin expression were constructed and used to test the effect of pilin null mutations on DNA transformation competence. PMID:2152910

  8. In Vitro selection of Neisseria gonorrhoeae mutants with elevated MIC values and increased resistance to cephalosporins.

    PubMed

    Johnson, Steven R; Grad, Yonatan; Ganakammal, Satishkumar Ranganathan; Burroughs, Mark; Frace, Mike; Lipsitch, Marc; Weil, Ryan; Trees, David

    2014-11-01

    Strains of Neisseria gonorrhoeae with mosaic penA genes bearing novel point mutations in penA have been isolated from ceftriaxone treatment failures. Such isolates exhibit significantly higher MIC values to third-generation cephalosporins. Here we report the in vitro isolation of two mutants with elevated MICs to cephalosporins. The first possesses a point mutation in the transpeptidase region of the mosaic penA gene, and the second contains an insertion mutation in pilQ.

  9. Zabofloxacin (DW-224a) activity against Neisseria gonorrhoeae including quinolone-resistant strains.

    PubMed

    Jones, Ronald N; Biedenbach, Douglas J; Ambrose, Paul G; Wikler, Matthew A

    2008-09-01

    Zabofloxacin, a new fluoroquinolone compound (DW-224a), was tested by reference agar dilution methods against 35 strains of multiresistant Neisseria gonorrhoeae. The potency of zabofloxacin (MIC(50), 0.016 microg/mL) was generally comparable with azithromycin but 8-fold superior to ciprofloxacin. This novel naphthyridine should be explored as an alternative therapy for quinolone-nonsusceptible gonorrhea and Chlamydia trachomatis infections. PMID:18620833

  10. In Vitro selection of Neisseria gonorrhoeae mutants with elevated MIC values and increased resistance to cephalosporins.

    PubMed

    Johnson, Steven R; Grad, Yonatan; Ganakammal, Satishkumar Ranganathan; Burroughs, Mark; Frace, Mike; Lipsitch, Marc; Weil, Ryan; Trees, David

    2014-11-01

    Strains of Neisseria gonorrhoeae with mosaic penA genes bearing novel point mutations in penA have been isolated from ceftriaxone treatment failures. Such isolates exhibit significantly higher MIC values to third-generation cephalosporins. Here we report the in vitro isolation of two mutants with elevated MICs to cephalosporins. The first possesses a point mutation in the transpeptidase region of the mosaic penA gene, and the second contains an insertion mutation in pilQ. PMID:25199775

  11. In-house growth-promoting transport system for Neisseria gonorrhoeae.

    PubMed

    Sharma, Dharmendra; Sethi, Sunil; Mehta, Swami Das; Sharma, Meera

    2007-08-01

    Eno powder (GlaxoSmithKline), an antacid preparation readily available over the counter, was used instead of a CO(2) generator for the growth of 15 strains of Neisseria gonorrhoeae obtained from men with urethritis. Due to its easy accessibility and low cost, Eno powder can be useful in developing countries for transporting clinical specimens from resource-poor peripheral labs to reference laboratories. PMID:17537947

  12. First case of Neisseria meningitidis capsule null locus infection in China.

    PubMed

    Xu, Zheng; Zhu, Bingqing; Xu, Li; Gao, Yuan; Shao, Zhujun

    2015-08-01

    Invasive meningococcal diseases are rarely caused by capsule null locus (cnl) strains, which are unencapsulated owing to an absence of capsule synthesis genes. Only a few cases of cnl meningococcemia in severely immunocompromised patients have been reported. Here, we describe and discuss the first case of invasive disease in an immunocompetent patient due to a cnl strain of Neisseria meningitidis in China. We characterize this strain and discuss the special ST-198 complex of cnl.

  13. Population structure in the Neisseria, and the biological significance of fuzzy species.

    PubMed

    Corander, Jukka; Connor, Thomas R; O'Dwyer, Clíona A; Kroll, J Simon; Hanage, William P

    2012-06-01

    Phenotypic and genetic variation in bacteria can take bewilderingly complex forms even within a single genus. One of the most intriguing examples of this is the genus Neisseria, which comprises both pathogens and commensals colonizing a variety of body sites and host species, and causing a range of disease. Complex relatedness among both named species and previously identified lineages of Neisseria makes it challenging to study their evolution. Using the largest publicly available collection of bacterial sequence data in combination with a population genetic analysis and experiment, we probe the contribution of inter-species recombination to neisserial population structure, and specifically whether it is more common in some strains than others. We identify hybrid groups of strains containing sequences typical of more than one species. These groups of strains, typical of a fuzzy species, appear to have experienced elevated rates of inter-species recombination estimated by population genetic analysis and further supported by transformation experiments. In particular, strains of the pathogen Neisseria meningitidis in the fuzzy species boundary appear to follow a different lifestyle, which may have considerable biological implications concerning distribution of novel resistance elements and meningococcal vaccine development. Despite the strong evidence for negligible geographical barriers to gene flow within the population, exchange of genetic material still shows directionality among named species in a non-uniform manner.

  14. Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis.

    PubMed Central

    Ropp, P A; Nicholas, R A

    1997-01-01

    The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences. PMID:9098083

  15. Direct bacterial profiling by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry for identification of pathogenic Neisseria.

    PubMed

    Ilina, Elena N; Borovskaya, Alexandra D; Malakhova, Maja M; Vereshchagin, Vladimir A; Kubanova, Anna A; Kruglov, Alexander N; Svistunova, Tatyana S; Gazarian, Anaida O; Maier, Thomas; Kostrzewa, Markus; Govorun, Vadim M

    2009-01-01

    The present study investigates the suitability of direct bacterial profiling as a tool for the identification and subtyping of pathogenic Neisseria. The genus Neisseria includes two human pathogens, Neisseria meningitidis and Neisseria gonorrhoeae, as well as several nonpathogenic Neisseria species. Here, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling protocol was optimized using a laboratory strain of E. coli DH5alpha to guarantee high quality and reproducible results. Subsequently, mass spectra for both laboratory and clinical strains of N. gonorrhoeae, N. meningitidis, and several nonpathogenic Neisseria species were collected. Significant interspecies differences but little intraspecies diversity were revealed by means of a visual inspection and bioinformatics examination using the MALDI BioTyper software. Cluster analysis successfully separated mass spectra collected from three groups that corresponded to N. gonorrhoeae, N. meningitidis, and nonpathogenic Neisseria isolates. Requiring only one bacterial colony for testing and using a fast and easy measuring protocol, this approach represents a powerful tool for the rapid identification of pathogenic Neisseria and can be adopted for other microorganisms.

  16. A misleading false-negative result using Neisseria gonorrhoeae opa MGB multiplex PCR assay in patient's rectal sample due to partial mutations of the opa gene.

    PubMed

    Vahidnia, Ali; van Empel, Pieter Jan; Costa, Sandra; Oud, Rob T N; van der Straaten, Tahar; Bliekendaal, Harry; Spaargaren, Joke

    2015-07-01

    A 53-year-old homosexual man presented at his general practitioner (GP) practice with a suspicion of sexually transmitted infection. Initial NAAT screening was performed for Chlamydia trachomatis and Neisseria gonorrhoeae. The patient was positive for Neisseria gonorrhoeae both for his urine and rectal sample. The subsequent confirmation test for Neisseria gonorrhoeae by a second laboratory was only confirmed for the urine sample and the rectal sample was negative. We report a case of a potential false-negative diagnosis of Neisseria gonorrhoeae due to mutations of DNA sequence in the probe region of opa-MGB assay of the rectal sample. The patient did not suffer any discomfort as diagnosis of Neisseria gonorrhoeae in his urine sample had already led to treatment by prescribing the patient with Ceftriaxone 500 mg IV dissolved in 1 ml lidocaine 2% and 4 mL saline. The patient also received a prescription for Azithromycin (2x500 mg).

  17. Determination of serotyping antigens, clonal analysis and genetic characterization of the 4CMenB vaccine antigen genes in invasive Neisseria meningitidis from Western Canada, 2009 to 2013.

    PubMed

    Law, Dennis K S; Zhou, Jianwei; Deng, Saul; Hoang, Linda; Tyrrell, Gregory; Horsman, Greg; Wylie, John; Tsang, Raymond S W

    2014-11-01

    This study examined invasive Neisseria meningitidis recovered from invasive meningococcal disease (IMD) cases in Western Canada between 2009 and 2013. A total of 161 isolates from individual IMD cases were analysed for serogroup, serotype, serosubtype, PorA genotype, multi-locus sequence type and nucleotide sequence of their 4CMenB vaccine antigen genes. Sixty-nine isolates were serogroup B (MenB), 47 were serogroup Y (MenY), 22 were serogroup C (MenC), 19 were serogroup W (MenW), three were serogroup E and one was non-encapsulated. MenC, MenY and MenW were mainly clonal, represented primarily by clonal complex (cc) 11, cc23 or cc167, and cc22, respectively. In contrast, MenB were composed of eight different ccs together with 11 isolates not assigned to any known cc. Antigenic analysis and PorA genotyping confirmed the heterogeneity of MenB isolates, while such results supported the clonal nature of most MenC, MenY and MenW isolates. Thirty-four (21.1%) isolates had at least one gene that encoded one matching vaccine protein component of the 4CMenB vaccine (i.e. PorA P1.4; fHbp variant 1.1; NHBA peptide 2; and NadA-1, -2, or -3). An additional 18 isolates had genes that encoded variant 1 or subfamily B factor H binding proteins of this same vaccine.

  18. Porin-mediated antibiotic resistance in Neisseria gonorrhoeae: ion, solute, and antibiotic permeation through PIB proteins with penB mutations.

    PubMed

    Olesky, Melanie; Zhao, Shuqing; Rosenberg, Robert L; Nicholas, Robert A

    2006-04-01

    Neisseria gonorrhoeae has two porins, PIA and PIB, whose genes (porA and porB, respectively) are alleles of a single por locus. We recently demonstrated that penB mutations at positions 120 and 121 in PIB, which are presumed to reside in loop 3 that forms the pore constriction zone, confer intermediate-level resistance to penicillin and tetracycline (M. Olesky, M. Hobbs, and R. A. Nicholas, Antimicrob. Agents Chemother. 46:2811-2820, 2002). In the present study, we investigated the electrophysiological properties as well as solute and antibiotic permeation rates of recombinant PIB proteins containing penB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H). In planar lipid bilayers, the predominant conducting state of each porin variant was 30 to 40% of the wild type, even though the anion selectivity and maximum channel conductance of each PIB variant was similar to that of the wild type. Liposome-swelling experiments revealed no significant differences in the permeation of sugars or beta-lactam antibiotics through the wild type or PIB variants. Although these results are seemingly contradictory with the ability of these variants to increase antibiotic resistance, they are consistent with MIC data showing that these porin mutations confer resistance only in strains containing an mtrR mutation, which increases expression of the MtrC-MtrD-MtrE efflux pump. Moreover, both the mtrR and penB mutations were required to decrease in vivo permeation rates below those observed in the parental strain containing either mtrR or porin mutations alone. Thus, these data demonstrate a novel mechanism of porin-mediated resistance in which mutations in PIB have no affect on antibiotic permeation alone but instead act synergistically with the MtrC-MtrD-MtrE efflux pump in the development of antibiotic resistance in gonococci. PMID:16547016

  19. Application of atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry for rapid identification of Neisseria species.

    PubMed

    Gudlavalleti, Seshu K; Sundaram, Appavu K; Razumovski, Jane; Doroshenko, Vladimir

    2008-07-01

    Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI MS) was applied to develop a proteomics-based method to detect and identify Neisseria species. Heat-inactivated clinical isolate cell suspensions of Neisseria gonorrhoeae and strains belonging to five serogroups (A, B, C, W135, and Y) of Neisseria meningitidis were subjected to on-probe protein/peptide extraction and tryptic digestion followed by AP-MALDI tandem MS (MS/MS)-based proteomic analysis. Amino acid sequences derived from three protonated peptides with m/z values of 1743.8, 1894.8, and 1946.8 were identified by AP-MALDI MS/MS and MASCOT proteome database search analysis as belonging to neisserial acyl carrier protein, neisserial-conserved hypothetical protein, and neisserial putative DNA binding protein, respectively. These three peptide masses can thus be potential biomarkers for neisserial species identification by AP-MALDI MS.

  20. Potential impact of vaccination against Neisseria meningitidis on Neisseria gonorrhoeae in the United States: results from a decision-analysis model.

    PubMed

    Régnier, Stéphane A; Huels, Jasper

    2014-01-01

    Components in 4CMenB vaccine against Neisseria meningitidis serogroup B have shown to potentially cross-react with Neisseria gonorrhoeae. We modeled the theoretical impact of a US 4CMenB vaccination program on gonorrhea outcomes. A decision-analysis model was populated using published healthcare utilization and cost data. A two-dose adolescent vaccination campaign was assumed, with protective immunity starting at age 15 years and a base-case efficacy against gonorrhea of 20%. The 20%-efficacy level is an assumption since no clinical data have yet quantified the efficacy of 4CMenB against Neisseria gonorrhoea. Key outcome measures were reductions in gonorrhea and HIV infections, reduction in quality-adjusted life-years (QALYs) lost, and the economically justifiable price assuming a willingness-to-pay threshold of $75,000 per QALY gained. Adolescent vaccination with 4CMenB would prevent 83,167 (95% credible interval [CrI], 44,600-134,600) gonorrhea infections and decrease the number of HIV infections by 55 (95% CrI, 2-129) per vaccinated birth cohort in the USA. Excluding vaccination costs, direct medical costs for gonorrhea would reduce by $28.7 million (95% CrI, $6.8-$70.0 million), and income and productivity losses would reduce by $40.0 million (95% CrI, $8.2-$91.7 million). Approximately 83% of the reduction in lost productivity is generated by avoiding HIV infections. At a cost of $75,000 per QALY gained, and incremental to the vaccine's effect on meningococcal disease, a price of $26.10 (95% CrI, $9.10-$57.20) per dose, incremental to the price of the meningococcal vaccine, would be justified from the societal perspective. At this price, the net cost per infection averted would be $1,677 (95% CrI, $404-$2,564). Even if the cross-immunity of 4CMenB vaccine and gonorrhea is only 20%, the reduction in gonorrhea infections and associated costs would be substantial.

  1. Review and International Recommendation of Methods for Typing Neisseria gonorrhoeae Isolates and Their Implications for Improved Knowledge of Gonococcal Epidemiology, Treatment, and Biology

    PubMed Central

    Unemo, Magnus; Dillon, Jo-Anne R.

    2011-01-01

    Summary: Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae, together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding. PMID:21734242

  2. Amino acid substitutions in mosaic penicillin-binding protein 2 associated with reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae.

    PubMed

    Takahata, Sho; Senju, Nami; Osaki, Yumi; Yoshida, Takuji; Ida, Takashi

    2006-11-01

    The molecular mechanisms of reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae, particularly amino acid substitutions in mosaic penicillin-binding protein 2 (PBP2), were examined. The complete sequence of ponA, penA, and por genes, encoding, respectively, PBP1, PBP2, and porin, were determined for 58 strains isolated in 2002 from Japan. Replacement of leucine 421 by proline in PBP1 and the mosaic-like structure of PBP2 were detected in 48 strains (82.8%) and 28 strains (48.3%), respectively. The presence of mosaic PBP2 was the main cause of the elevated cefixime MIC (4- to 64-fold). In order to identify the mutations responsible for the reduced susceptibility to cefixime in isolates with mosaic PBP2, penA genes with various mutations were transferred to a susceptible strain by genetic transformation. The susceptibility of partial recombinants and site-directed mutants revealed that the replacement of glycine 545 by serine (G545S) was the primary mutation, which led to a two- to fourfold increase in resistance to cephems. Replacement of isoleucine 312 by methionine (I312M) and valine 316 by threonine (V316T), in the presence of the G545S mutation, reduced susceptibility to cefixime, ceftibuten, and cefpodoxime by an additional fourfold. Therefore, three mutations (G545S, I312M, and V316T) in mosaic PBP2 were identified as the amino acid substitutions responsible for reduced susceptibility to cefixime in N. gonorrhoeae.

  3. Characterization of a class II pilin expression locus from Neisseria meningitidis: evidence for increased diversity among pilin genes in pathogenic Neisseria species.

    PubMed Central

    Aho, E L; Botten, J W; Hall, R J; Larson, M K; Ness, J K

    1997-01-01

    Strains of Neisseria meningitidis elaborate one of two classes of pili. Meningococcal class I pili have many features in common with pili produced by N. gonorrhoeae, including the ability to bind monoclonal antibody SM1 and a common gene and protein structure consisting of conserved, semivariable, and hypervariable regions. Class II pili are SM1 nonreactive and display smaller subunit molecular weights than do gonococcal or meningococcal class I pili. In this study, we have determined the N-terminal amino acid sequence for class II pilin and isolated the expression locus encoding class II pilin from N. meningitidis FAM18. Meningococcal class II pilin displays features typical of type IV pili and shares extensive amino acid identity with the N-terminal conserved regions of other neisserial pilin proteins. However, the deduced class II pilin sequence displays several unique features compared with previously reported meningococcal class I and gonococcal pilin sequences. Class II pilin lacks several conserved peptide regions found within the semivariable and hypervariable regions of other neisserial pilins and displays a large deletion in a hypervariable region of the protein believed to be exposed on the pilus face in gonococcal pili. DNA sequence comparisons within all three regions of the coding sequence also suggest that the meningococcal class II pilin gene is the most dissimilar of the three types of neisserial pilE loci. Additionally, the class II locus fails to display flanking-sequence homology to class I and gonococcal genes and lacks a downstream Sma/Cla repeat sequence, a feature present in all other neisserial pilin genes examined to date. These data indicate meningococcal class II pili represent a structurally distinct class of pili and suggest that relationships among pilin genes in pathogenic Neisseria do not necessarily follow species boundaries. PMID:9199428

  4. Divergence and transcriptional analysis of the division cell wall (dcw) gene cluster in Neisseria spp.

    PubMed

    Snyder, Lori A S; Shafer, William M; Saunders, Nigel J

    2003-01-01

    Three of the 18 open reading frames in the division and cell wall synthesis cluster of the pathogenic Neisseria spp. are not present in the clusters of other bacterial species. The region containing two of these, dcaB and dcaC, displays interstrain and interspecies variability uncharacteristic of such clusters. 3' of dcaB is a Correia repeat enclosed element (CREE), which is only present in some strains. It has been suggested that this CREE is a transcriptional terminator, although we demonstrate otherwise. A gearbox-like promoter within this CREE is active in Escherichia coli but not in Neisseria meningitidis. There is an active promoter 5' of dcaC, although its sequence is not conserved. The presence of similarly located promoters has not been demonstrated in other species. In Neisseria lactamica, this promoter involves another dcw-associated CREE, the first demonstration of active promoter generation at the 5' end of this common intergenic, apparently mobile, element. Upstream of this promoter is an inverted pair of neisserial uptake signal sequences, which are commonly considered to be transcriptional terminators. It has been proposed to terminate transcription in this location, although we have demonstrated transcript extending through this uptake signal sequence. dcaC contains a 108 bp tandem repeat, which is present in different copy numbers in the neisserial strains examined. This investigation reveals extensive sequence variation, disputes the presence of transcriptional terminators and identifies active internal promoters in this normally highly conserved cluster of essential genes, and addresses the transcriptional activity of two common neisserial intergenic components.

  5. Expression of epithelial cell iron-related genes upon infection by Neisseria meningitidis.

    PubMed

    Bonnah, Robert A; Muckenthaler, Martina U; Carlson, Hanqian; Minana, Belen; Enns, Caroline A; Hentze, Matthias W; So, Magdalene

    2004-05-01

    Infection by the obligate human pathogens Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) reduces the expression of host epithelial cell transferrin receptor 1 (TfR-1) (Bonnah et al., 2000, Cellular Microbiology 2: 207-218). In addition, the rate and pattern of TfR-1 cycling is altered, leading to diminished uptake of Tf-iron by infected host cells. As Tf-iron is important for maintaining iron homeostasis in the eukaryotic cell, these findings raised the possibility that Neisseria infection might affect further pathways of epithelial cell iron metabolism. We used a specialized cDNA microarray platform, the 'IronChip', to investigate the expression of genes involved in iron transport, storage and regulation. We show that mRNA expression of several host genes involved in iron homeostasis is altered. Surprisingly, the general mRNA expression profile of infected cells closely resembled that of uninfected cells grown in an iron-limited environment. An important exception to this profile is TfR-1, the mRNA level of which is strongly reduced. Low TfR-1 expression may be explained in part by decreased activity of the iron-regulatory proteins (IRPs) in MC-infected cells, which may result in the destabilization of TfR-1 mRNA. Intriguingly, low IRP activity contrasts with the decrease in H-ferritin protein levels in infected cells. This finding suggests that low IRP activity may be responsible in part for the decrease in TfR-1 mRNA levels. A discussion of these novel findings in relation to MC infection and virulence is provided. PMID:15056217

  6. Use of Restriction Fragment Length Polymorphisms to Investigate Strain Variation Within Neisseria Meningitidis.

    NASA Astrophysics Data System (ADS)

    Williams, Shelley Diane

    Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty -six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P ^{32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analysed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population. This analysis demonstrates the lack of structure within Neisseria meningitidis due primarily to a heterogenous population and the lack of geographic segregation. The potential utility of this technique as a

  7. Analysis of penicillinase-producing Neisseria gonorrhoeae isolates in Madrid (Spain) from 1983-85.

    PubMed Central

    Fenoll, A.; Berrón, S.; Vázquez, J. A.

    1987-01-01

    Between April 1983 and December 1985, 576 strains of Neisseria gonorrhoeae were isolated in our laboratory from patients attending Sexually Transmitted Diseases (STD) clinics. Of these, 61 (10.6%) were penicillinase-producing. Studies on these strains by plasmid analysis, auxotyping and serogrouping showed that the predominant type strains harboured the Asian resistance plasmid, were prototrophic, and were of serogroup W II/W III. About half of the strains, both of the African and Asian type, harboured the transfer plasmid. Strains of serogroup W II/W III were less sensitive to tetracycline and cefoxitin than serogroup W I strains. Images Fig. 2 PMID:2960555

  8. First Neisseria gonorrhoeae strain with resistance to cefixime causing gonorrhoea treatment failure in Austria, 2011.

    PubMed

    Unemo, M; Golparian, D; Stary, A; Eigentler, A

    2011-10-27

    We describe the first cefixime-resistant Neisseria gonorrhoeae strain in Austria that caused treatment failure.It follows the first five cases in Europe of cefixime treatment failure, reported in Norway in 2010 and the United Kingdom in 2011. Effective treatment of gonorrhoea is crucial for public health control and, at present, requires substantially enhanced awareness, more frequent test-of-cure, interaction with experts after therapeutic failure, tracing and therapy of contacts, and surveillance of gonococcal antimicrobial resistance and treatment failures worldwide.

  9. Control of Neisseria gonorrhoeae in the era of evolving antimicrobial resistance.

    PubMed

    Barbee, Lindley A; Dombrowski, Julia C

    2013-12-01

    Neisseria gonorrhoeae has developed resistance to all previous first-line antimicrobial therapies over the past 75 years. Today the cephalosporins, the last available antibiotic class that is sufficiently effective, are also threatened by evolving resistance. Screening for asymptomatic gonorrhea in women and men who have sex with men, treating with a dual antibiotic regimen, ensuring effective partner therapy, and remaining vigilant for treatment failures constitute critical activities for clinicians in responding to evolving antimicrobial resistance. This article reviews the epidemiology, history of antimicrobial resistance, current screening and treatment guidelines, and future treatment options for gonorrhea.

  10. High-level azithromycin-resistant Neisseria gonorrhoeae clinical isolate in France, March 2014.

    PubMed

    Bercot, B; Belkacem, A; Goubard, A; Mougari, F; Sednaoui, P; La Ruche, G; Cambau, E

    2014-11-06

    We report the first case in France of a high-level azithromycin-resistant Neisseria gonorrhoeae (minimum inhibitory concentration (MIC) = 96 mg/L) assigned to MLST7363 (NG-MAST ST6360), also resistant to ciprofloxacin and tetracycline but susceptible to ceftriaxone. The patient was a 51 year-old heterosexual man who returned following 1g azithromycin monotherapy. Mechanisms of azithromycin resistance were a C2599T mutation in the four copies of the rrl gene and a novel mutation in the promoter of the mtrR gene.

  11. [In vitro activities of 9 aminosides against of 250 strains of Neisseria gonorrhoeae].

    PubMed

    Casin, I; Sanson-Le Pors, M J; Barateau, V; Morel, P; Perol, Y

    1987-05-01

    We compared the in vitro activities of 9 aminoglycosides including trospectomycin sulfate against 250 selected strains of Neisseria gonorrhoeae according to their penicillin resistance (penicillinase production), streptomycin resistance, spectinomycin resistance. Trospectomycin sulfate inhibited all spectinomycin-sensible strain at a concentration of 4 mg/l but the spectinomycin-resistant strain needed 32 mg/l for its inhibition. Among the other aminoglycosides tested netilmicin had the most in vitro effectiveness. Its minimal inhibitory concentrations were comparable to that of trospectomycin sulfate: furthermore this compound demonstrated in vitro activity against the spectino-resistant strain.

  12. Multicenter Investigation of Gepotidacin (GSK2140944) Agar Dilution Quality Control Determinations for Neisseria gonorrhoeae ATCC 49226.

    PubMed

    Jones, Ronald N; Fedler, Kelley A; Scangarella-Oman, Nicole E; Ross, James E; Flamm, Robert K

    2016-07-01

    Gepotidacin, a novel triazaacenaphthylene antibacterial agent, is the first in a new class of type IIA topoisomerase inhibitors with activity against many biothreat and conventional pathogens, including Neisseria gonorrhoeae To assist ongoing clinical studies of gepotidacin to treat gonorrhea, a multilaboratory quality assurance investigation determined the reference organism (N. gonorrhoeae ATCC 49226) quality control MIC range to be 0.25 to 1 μg/ml (88.8% of gepotidacin MIC results at the 0.5 μg/ml mode). PMID:27161642

  13. Induction of HIV-1 long terminal repeat-mediated transcription by Neisseria gonorrhoeae.

    PubMed

    Chen, Adrienne; Boulton, Ian C; Pongoski, Jodi; Cochrane, Alan; Gray-Owen, Scott D

    2003-03-01

    Gonorrhoea enhances the transmission of HIV through increased viral shedding and the increased probability of seroconversion among previously HIV-negative individuals. However, the mechanism(s) underlying these influences remain poorly understood. We demonstrated that exposure to Neisseria gonorrhoeae induces the nuclear factor kappa B-dependent transcription from the HIV-1 long terminal repeat in derivatives of the Jurkat CD4 T cell line. These data suggest that gonococcal infection directly impacts HIV-1 transmission through the localized stimulation of viral expression. PMID:12598784

  14. High-level azithromycin-resistant Neisseria gonorrhoeae clinical isolate in France, March 2014.

    PubMed

    Bercot, B; Belkacem, A; Goubard, A; Mougari, F; Sednaoui, P; La Ruche, G; Cambau, E

    2014-01-01

    We report the first case in France of a high-level azithromycin-resistant Neisseria gonorrhoeae (minimum inhibitory concentration (MIC) = 96 mg/L) assigned to MLST7363 (NG-MAST ST6360), also resistant to ciprofloxacin and tetracycline but susceptible to ceftriaxone. The patient was a 51 year-old heterosexual man who returned following 1g azithromycin monotherapy. Mechanisms of azithromycin resistance were a C2599T mutation in the four copies of the rrl gene and a novel mutation in the promoter of the mtrR gene. PMID:25394255

  15. Alterations in peptidoglycan of Neisseria gonorrhoeae induced by sub-MICs of beta-lactam antibiotics.

    PubMed Central

    Garcia-Bustos, J F; Dougherty, T J

    1987-01-01

    Exposure of Neisseria gonorrhoeae to sub-MICs of selected beta-lactam antibiotics caused distortion of normal cell morphology. Analysis of the peptidoglycan indicated that the cells were accumulating increased quantities of disaccharide pentapeptide in their cell walls. The O-acetylated form of the disaccharide pentapeptide was not detected among the major peaks. The correlation of antibiotic binding to gonococcal penicillin-binding protein 2 and accumulation of non-O-acetylated disaccharide pentapeptide suggested an explanation for the previously observed relationship of penicillin-binding protein 2 and O-acetylation of peptidoglycan. PMID:3105447

  16. Draft Genome Sequence of Neisseria gonorrhoeae Strain NG_869 with Penicillin, Tetracycline and Ciprofloxacin Resistance Determinants Isolated from Malaysia.

    PubMed

    Ang, Geik Yong; Yu, Choo Yee; Yong, Delicia Ann; Cheong, Yuet Meng; Yin, Wai-Fong; Chan, Kok-Gan

    2016-06-01

    Gonorrhea is a sexually transmitted infection caused by Neisseria gonorrhoeae and the increasing reports of multidrug-resistant gonococcal isolates are a global public health care concern. Herein, we report the genome sequence of N. gonorrhoeae strain NG_869 isolated from Malaysia which may provide insights into the drug resistance determinants in gonococcal bacteria. PMID:27570316

  17. Neisseria gonorrhoeae with high-level resistance to azithromycin: case report of the first isolate identified in the United States.

    PubMed

    Katz, Alan R; Komeya, Alan Y; Soge, Olusegun O; Kiaha, Mandy I; Lee, Maria Veneranda C; Wasserman, Glenn M; Maningas, Eloisa V; Whelen, A Christian; Kirkcaldy, Robert D; Shapiro, Steven J; Bolan, Gail A; Holmes, King K

    2012-03-01

    We report on the first Neisseria gonorrhoeae isolate in the United States identified with high-level resistance to azithromycin. This report discusses the epidemiologic case investigation, the molecular studies of resistance-associated mutations and N. gonorrhoeae multiantigen sequence typing, and challenges posed by emerging gonococcal antimicrobial resistance. PMID:22184617

  18. Comparison of Neisseria gonorrhoeae MICs obtained by Etest and agar dilution for ceftriaxone, cefpodoxime, cefixime and azithromycin.

    PubMed

    Gose, Severin; Kong, Carol J; Lee, Yer; Samuel, Michael C; Bauer, Heidi M; Dixon, Paula; Soge, Olusegun O; Lei, John; Pandori, Mark

    2013-12-01

    We evaluated Neisseria gonorrhoeae Etest minimum inhibitory concentrations (MICs) relative to agar dilution MICs for 664 urethral isolates for ceftriaxone (CRO) and azithromycin (AZM), 351 isolates for cefpodoxime (CPD) and 315 isolates for cefixime (CFM). Etest accurately determined CPD, CFM and AZM MICs, but resulted in higher CRO MICs.

  19. Comparison of Neisseria gonorrhoeae MICs Obtained by Etest and Agar Dilution for Ceftriaxone, Cefpodoxime, Cefixime and Azithromycin.

    PubMed

    Gose, Severin; Kong, Carol J; Lee, Yer; Samuel, Michael C; Bauer, Heidi M; Dixon, Paula; Soge, Olusegun O; Lei, John; Pandori, Mark

    2013-10-24

    We evaluated Neisseria gonorrhoeae Etest minimum inhibitory concentrations (MICs) relative to agar dilution MICs for 664 urethral isolates for ceftriaxone (CRO) and azithromycin (AZM), 351 isolates for cefpodoxime (CPD) and 315 isolates for cefixime (CFM). Etest accurately determined CPD, CFM and AZM MICs, but resulted in higher CRO MICs.

  20. Genetic, structural, and antigenic analyses of glycan diversity in the O-linked protein glycosylation systems of human Neisseria species.

    PubMed

    Børud, Bente; Aas, Finn Erik; Vik, Ashild; Winther-Larsen, Hanne C; Egge-Jacobsen, Wolfgang; Koomey, Michael

    2010-06-01

    Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.

  1. Changing Antimicrobial Resistance Profiles among Neisseria gonorrhoeae Isolates in Italy, 2003 to 2012

    PubMed Central

    Carannante, Anna; Renna, Giovanna; Dal Conte, Ivano; Ghisetti, Valeria; Matteelli, Alberto; Prignano, Grazia; Impara, Giampaolo; Cusini, Marco; D'Antuono, Antonietta; Vocale, Caterina; Antonetti, Raffaele; Gaino, Marina; Busetti, Marina; Latino, Maria Agnese; Mencacci, Antonella; Bonanno, Carmen; Cava, Maria Carmela; Giraldi, Cristina

    2014-01-01

    The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobial agents is a major public health concern and a serious issue related to the occurrence of further untreatable gonorrhea infections. A retrospective analysis on 1,430 N. gonorrhoeae isolates, collected from 2003 through 2012, for antimicrobial susceptibility by Etest and molecular characterization by Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) was carried out in Italy. Azithromycin-resistant gonococci decreased from 14% in 2007 to 2.2% in 2012. Similarly, isolates with high MICs to cefixime (>0.125 mg/liter) decreased from 11% in 2008 to 3.3% in 2012. The ciprofloxacin resistance rate remains quite stable, following an increasing trend up to 64% in 2012. The percentage of penicillinase-producing N. gonorrhoeae (PPNG) significantly declined from 77% in 2003 to 7% in 2012. A total of 81 multidrug-resistant (MDR) gonococci were identified, showing 11 different antimicrobial resistance patterns. These were isolated from men who have sex with men (MSM) and from heterosexual patients. Two sequence types (STs), ST661 and ST1407, were the most common. Genogroup 1407, which included cefixime-, ciprofloxacin-, and azithromycin-resistant isolates, was found. In conclusion, a change in the antimicrobial resistance profiles among gonococci was identified in Italy together with a percentage of MDR isolates. PMID:25070110

  2. Opa proteins and CEACAMs: pathways of immune engagement for pathogenic Neisseria.

    PubMed

    Sadarangani, Manish; Pollard, Andrew J; Gray-Owen, Scott D

    2011-05-01

    Neisseria meningitidis and Neisseria gonorrhoeae are globally important pathogens, which in part owe their success to their ability to successfully evade human immune responses over long periods. The phase-variable opacity-associated (Opa) adhesin proteins are a major surface component of these organisms, and are responsible for bacterial adherence and entry into host cells and interactions with the immune system. Most immune interactions are mediated via binding to members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family. These Opa variants are able to bind to different receptors of the CEACAM family on epithelial cells, neutrophils, and T and B lymphocytes, influencing the innate and adaptive immune responses. Increased epithelial cell adhesion creates the potential for prolonged infection, invasion and dissemination. Furthermore, Opa proteins may inhibit T-lymphocyte activation and proliferation, B-cell antibody production, and innate inflammatory responses by infected epithelia, in addition to conferring increased resistance to antibody-dependent, complement-mediated killing. While vaccines containing Opa proteins could induce adhesion-blocking and bactericidal antibodies, the consequence of CEACAM binding by a candidate Opa-containing vaccine requires further investigation. This review summarizes current knowledge of the immunological consequences of the interaction between meningococcal and gonococcal Opa proteins and human CEACAMs, considering the implications for pathogenesis and vaccine development.

  3. Will targeting oropharyngeal gonorrhoea delay the further emergence of drug-resistant Neisseria gonorrhoeae strains?

    PubMed

    Lewis, D A

    2015-06-01

    Gonorrhoea is an important sexually transmitted infection associated with serious complications and enhanced HIV transmission. Oropharyngeal infections are often asymptomatic and will only be detected by screening. Gonococcal culture has low sensitivity (<50%) for detecting oropharyngeal gonorrhoea, and, although not yet approved commercially, nucleic acid amplification tests (NAAT) are the assay of choice. Screening for oropharyngeal gonorrhoea should be performed in high-risk populations, such as men-who-have-sex-with-men(MSM). NAATs have a poor positive predictive value when used in low-prevalence populations. Gonococci have repeatedly thwarted gonorrhoea control efforts since the first antimicrobial agents were introduced. The oropharyngeal niche provides an enabling environment for horizontal transfer of genetic material from commensal Neisseria and other bacterial species to Neisseria gonorrhoeae. This has been the mechanism responsible for the generation of mosaic penA genes, which are responsible for most of the observed cases of resistance to extended-spectrum cephalosporins (ESC). As antimicrobial-resistant gonorrhoea is now an urgent public health threat, requiring improved antibiotic stewardship, laboratory-guided recycling of older antibiotics may help reduce ESC use. Future trials of antimicrobial agents for gonorrhoea should be powered to test their efficacy at the oropharynx as this is the anatomical site where treatment failure is most likely to occur. It remains to be determined whether a combination of frequent screening of high-risk individuals and/or laboratory-directed fluoroquinolone therapy of oropharyngeal gonorrhoea will delay the further emergence of drug-resistant N. gonorrhoeae strains.

  4. Analysis of damage to human ciliated nasopharyngeal epithelium by Neisseria meningitidis.

    PubMed Central

    Stephens, D S; Whitney, A M; Melly, M A; Hoffman, L H; Farley, M M; Frasch, C E

    1986-01-01

    We used an in vitro model of human nasopharyngeal tissue in organ culture to evaluate the effects of Neisseria meningitidis on human cilia and ciliary function. Encapsulated, viable meningococci damaged ciliated epithelium of nasopharyngeal organ cultures, whereas Neisseria subflava, a commensal species, did not. Meningococcus-induced ciliary damage was due to loss of ciliated cells to which meningococci were not attached. Damage was seen with piliated and nonpiliated meningococci and did not appear to require the presence of other specific meningococcal surface proteins. Meningococcal viability was a requirement for both ciliary damage and interactions of meningococci with microvilli of nonciliated epithelial cells. That is, filter-sterilized supernatants from meningococcus-infected organ cultures, heat-killed meningococci at high inoculum, and purified meningococcal or gonococcal lipopolysaccharide at concentrations of 100 micrograms/ml did not damage ciliary activity of nasopharyngeal organ cultures. In contrast, meningococcal lipopolysaccharide at 10 micrograms/ml markedly damaged ciliary activity of human fallopian tube organ cultures, suggesting a selective toxicity of lipopolysaccharide for specific human ciliated cells. Damage to nasopharyngeal ciliated epithelium by N. meningitidis may be an important first step in meningococcal colonization of the human nasopharynx, but meningococcal lipopolysaccharide does not appear to be directly responsible for this toxicity. Images PMID:2867973

  5. Pili-mediated Interactions between Neisseria Gonorrhoeae Bacteria are the Driving Mechanism of Microcolony Merging

    NASA Astrophysics Data System (ADS)

    Poenisch, Wolfram; Weber, Christoph; Alzurqa, Khaled; Nasrollahi, Hadi; Biais, Nicolas; Zaburdaev, Vasily; Collective Dynamics of Cells Team; Mechano-Micro-Biology Lab Team

    2015-03-01

    During the early infection with Neisseria gonorrhoeae the bacteria form microcolonies consisting of a few hundreds to a few thousands of cells. The formation of colonies is mediated by type IV pili, thin and long filaments that are also involved in the motion of single cells over a substrate. A related process causes attractive cell-cell-interactions. While the motion of single cells has been extensively studied during the past years, the physical principles driving the growth of these colonies are poorly understood. One key mechanism of colony growth is coalescence of smaller colonies. Therefore we experimentally examine the process of merging of two Neisseria gonorrhoeae colonies. We develop a theoretical microscopic model of single cells interacting solely by their pili. The experimental data and the results obtained from our model are in excellent quantitative agreement. We observe a fast initial approach of the two merging colonies within a few minutes, that is followed by a slow relaxation of the colony shape with a characteristic time of several hours. These findings suggest that pili-mediated interactions are the primary driving mechanism of the microcolony merging process.

  6. Activation of NOD receptors by Neisseria gonorrhoeae modulates the innate immune response.

    PubMed

    Mavrogiorgos, Nikolaos; Mekasha, Samrawit; Yang, Yibin; Kelliher, Michelle A; Ingalls, Robin R

    2014-05-01

    NOD1 and NOD2 are members of the NOD-like receptor family of cytosolic pattern recognition receptors that recognize specific fragments of the bacterial cell wall component peptidoglycan. Neisseria species are unique amongst Gram-negative bacteria in that they turn over large amounts of peptidoglycan during growth. We examined the ability of NOD1 and NOD2 to recognize Neisseria gonorrhoeae, and determined the role of NOD-dependent signaling in regulating the immune response to gonococcal infection. Gonococci, as well as conditioned medium from mid-logarithmic phase grown bacteria, were capable of activating both human NOD1 and NOD2, as well as mouse NOD2, leading to the activation of the transcription factor NF-κB and polyubiquitination of the adaptor receptor-interacting serine-threonine kinase 2. We identified a number of cytokines and chemokines that were differentially expressed in wild type versus NOD2-deficient macrophages in response to gonococcal infection. Moreover, NOD2 signaling up-regulated complement pathway components and cytosolic nucleic acid sensors, suggesting a broad impact of NOD activation on innate immunity. Thus, NOD1 and NOD2 are important intracellular regulators of the immune response to infection with N. gonorrhoeae. Given the intracellular lifestyle of this pathogen, we believe these cytosolic receptors may provide a key innate immune defense mechanism for the host during gonococcal infection. PMID:23884094

  7. Presenilin/gamma-secretase cleaves CD46 in response to Neisseria infection.

    PubMed

    Weyand, Nathan J; Calton, Christine M; Higashi, Dustin L; Kanack, Kristen J; So, Magdalene

    2010-01-15

    CD46 is a type I transmembrane protein with complement and T cell regulatory functions in human cells. CD46 has signaling and receptor properties in immune and nonimmune cells, many of which are dependent on the expression of cytoplasmic tail (cyt) isoforms cyt1 or cyt2. Little is known about how cyt1 and cyt2 mediate cellular responses. We show that CD46-cyt1 and CD46-cyt2 are substrates for presenilin/gamma-secretase (PS/gammaS), an endogenous protease complex that regulates many important signaling proteins through proteolytic processing. PS/gammaS processing of CD46 releases immunoprecipitable cyt1 and cyt2 tail peptides into the cell, is blocked by chemical inhibitors, and is prevented in dominant negative presenilin mutant cell lines. Two human pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, stimulate PS/gammaS processing of CD46-cyt1 and CD46-cyt2. This stimulation requires type IV pili and PilT, the type IV pilus retraction motor, implying that mechanotransduction plays a role in this event. We present a model for PS/gammaS processing of CD46 that provides a mechanism by which signals are transduced via the cyt1 and cyt2 tails to regulate CD46-dependent cellular responses. Our findings have broad implications for understanding the full range of CD46 functions in infection and noninfection situations. PMID:20018629

  8. Mechanism of action of Neisseria gonorrhoeae O-acetylpeptidoglycan esterase, an SGNH serine esterase.

    PubMed

    Pfeffer, John M; Weadge, Joel T; Clarke, Anthony J

    2013-01-25

    O-Acetylpeptidoglycan esterase from Neisseria gonorrhoeae functions to release O-acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan, thereby permitting the continued metabolism of this essential cell wall heteropolymer. It has been demonstrated to be a serine esterase with sequence similarity to the family CE-3 carbohydrate esterases of the CAZy classification system. In the absence of a three-dimensional structure for any Ape, further knowledge of its structure and function relationship is dependent on modeling and kinetic studies. In this study, we predicted Neisseria gonorrhoeae Ape1a to be an SGNH hydrolase with an adopted α/β-hydrolase fold containing a central twisted four-stranded parallel β-sheet flanked by six α-helices with the putative catalytic triad, Asp-366, His-369, and Ser-80 appropriately aligned within a pocket. The role of eight invariant and highly conserved residues localized to the active site was investigated by site-directed replacements coupled with kinetic characterization and binding studies of the resultant engineered enzymes. Based on these data and theoretical considerations, Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism, whereas Gly-78, Asp-79, His-81, Asn-235, Thr-267, and Val-368 are proposed to position appropriately the catalytic residues and participate in substrate binding. PMID:23209280

  9. Incidence, epidemiology and evolution of reduced susceptibility to ciprofloxacin in Neisseria gonorrhoeae in Korea.

    PubMed

    Lee, Kyungwon; Chong, Yunsop; Erdenechemeg, L.; Soon Song, Kyung; Hun Shin, Kwang

    1998-01-01

    OBJECTIVE: To verify the decrease of susceptibility to ciprofloxacin in Neisseria gonorrhoeae, determine the size of the recently reported new beta-lactamase plasmid and explain the high prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG). METHODS: Gonococci were isolated from prostitutes in Korea. Antimicrobial susceptibility was tested by NCCLS disk diffusion and agar dilution methods. Plasmid was isolated by an alkaline lysis method. Patterns of Nhel-digested genomic DNA were compared after pulsed-field gel electrophoresis (PFGE). RESULTS: The minimum inhibitory concentration of ciprofloxacin for 50% of the isolates rose from 0.015 mg/L in 1993 to 0.12 mg/L in 1996. The proportion of PPNG remained at 70% or over during the 5-year period. The size of a novel beta-lactamase plasmid, first reported in 1994, was determined to be approximately 3.2 MDa, and 48% of the PPNG isolates contained it. Twelve of 50 isolates had the same PFGE pattern and nine others another pattern. CONCLUSION: The rapid decrease of fluoroquinolone-susceptible gonococci suggests that in the near future the drug may become less useful for gonorrhea treatment. The new 3.2-MDa plasmid may have been introduced as a result of the recent increase in overseas travel. The PFGE pattern suggests that high prevalence of PPNG may be due to dissemination of a few resistant clones among the high-risk groups. PMID:11864261

  10. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

    PubMed Central

    Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

    1990-01-01

    We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis. Images PMID:2106493

  11. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

    PubMed

    Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

    1990-03-01

    We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis.

  12. Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction.

    PubMed

    John, Constance M; Liu, Mingfeng; Jarvis, Gary A

    2009-03-01

    Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically. PMID:18832773

  13. Structural characterization of outer membrane components of the type IV pili system in pathogenic Neisseria.

    PubMed

    Jain, Samta; Mościcka, Katarzyna B; Bos, Martine P; Pachulec, Emilia; Stuart, Marc C A; Keegstra, Wilko; Boekema, Egbert J; van der Does, Chris

    2011-01-31

    Structures of the type IV pili secretin complexes from Neisseria gonorrhoeae and Neisseria meningitidis, embedded in outer membranes were investigated by transmission electron microscopy. Single particle averaging revealed additional domains not observed previously. Secretin complexes of N. gonorrhoeae showed a double ring structure with a 14-15-fold symmetry in the central ring, and a 14-fold symmetry of the peripheral ring with 7 spikes protruding. In secretin complexes of N. meningitidis, the spikes were absent and the peripheral ring was partly or completely lacking. When present, it had a 19-fold symmetry. The structures of the complexes in several pil mutants were determined. Structures obtained from the pilC1/C2 adhesin and the pilW minor pilin deletion strains were similar to wild-type, whereas deletion of the homologue of N. meningitidis PilW resulted in the absence of secretin structures. Remarkably, the pilE pilin subunit and pilP lipoprotein deletion mutants showed a change in the symmetry of the peripheral ring from 14 to 19 and loss of spikes. The pilF ATPase mutant also lost the spikes, but maintained 14-fold symmetry. These results show that secretin complexes contain previously unidentified large and flexible extra domains with a probable role in stabilization or assembly of type IV pili.

  14. Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction.

    PubMed

    John, Constance M; Liu, Mingfeng; Jarvis, Gary A

    2009-03-01

    Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.

  15. Identifying Neisseria species by use of the 50S ribosomal protein L6 (rplF) gene.

    PubMed

    Bennett, Julia S; Watkins, Eleanor R; Jolley, Keith A; Harrison, Odile B; Maiden, Martin C J

    2014-05-01

    The comparison of 16S rRNA gene sequences is widely used to differentiate bacteria; however, this gene can lack resolution among closely related but distinct members of the same genus. This is a problem in clinical situations in those genera, such as Neisseria, where some species are associated with disease while others are not. Here, we identified and validated an alternative genetic target common to all Neisseria species which can be readily sequenced to provide an assay that rapidly and accurately discriminates among members of the genus. Ribosomal multilocus sequence typing (rMLST) using ribosomal protein genes has been shown to unambiguously identify these bacteria. The PubMLST Neisseria database (http://pubmlst.org/neisseria/) was queried to extract the 53 ribosomal protein gene sequences from 44 genomes from diverse species. Phylogenies reconstructed from these genes were examined, and a single 413-bp fragment of the 50S ribosomal protein L6 (rplF) gene was identified which produced a phylogeny that was congruent with the phylogeny reconstructed from concatenated ribosomal protein genes. Primers that enabled the amplification and direct sequencing of the rplF gene fragment were designed to validate the assay in vitro and in silico. Allele sequences were defined for the gene fragment, associated with particular species names, and stored on the PubMLST Neisseria database, providing a curated electronic resource. This approach provides an alternative to 16S rRNA gene sequencing, which can be readily replicated for other organisms for which more resolution is required, and it has potential applications in high-resolution metagenomic studies.

  16. Structural alterations in a component of cytochrome c oxidase and molecular evolution of pathogenic Neisseria in humans.

    PubMed

    Aspholm, Marina; Aas, Finn Erik; Harrison, Odile B; Quinn, Diana; Vik, Ashild; Viburiene, Raimonda; Tønjum, Tone; Moir, James; Maiden, Martin C J; Koomey, Michael

    2010-08-19

    Three closely related bacterial species within the genus Neisseria are of importance to human disease and health. Neisseria meningitidis is a major cause of meningitis, while Neisseria gonorrhoeae is the agent of the sexually transmitted disease gonorrhea and Neisseria lactamica is a common, harmless commensal of children. Comparative genomics have yet to yield clear insights into which factors dictate the unique host-parasite relationships exhibited by each since, as a group, they display remarkable conservation at the levels of nucleotide sequence, gene content and synteny. Here, we discovered two rare alterations in the gene encoding the CcoP protein component of cytochrome cbb(3) oxidase that are phylogenetically informative. One is a single nucleotide polymorphism resulting in CcoP truncation that acts as a molecular signature for the species N. meningitidis. We go on to show that the ancestral ccoP gene arose by a unique gene duplication and fusion event and is specifically and completely distributed within species of the genus Neisseria. Surprisingly, we found that strains engineered to express either of the two CcoP forms conditionally differed in their capacity to support nitrite-dependent, microaerobic growth mediated by NirK, a nitrite reductase. Thus, we propose that changes in CcoP domain architecture and ensuing alterations in function are key traits in successive, adaptive radiations within these metapopulations. These findings provide a dramatic example of how rare changes in core metabolic proteins can be connected to significant macroevolutionary shifts. They also show how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host.

  17. Portable exhausters POR-004 SKID B, POR-005 SKID C, POR-006 SKID D storage plan

    SciTech Connect

    Nelson, O.D.

    1997-09-04

    This document provides a storage plan for portable exhausters POR-004 SKID B, POR-005 SKID C, AND POR-006 SKID D. The exhausters will be stored until they are needed by the TWRS (Tank Waste Remediation Systems) Saltwell Pumping Program. The storage plan provides criteria for portable exhauster storage, periodic inspections during storage, and retrieval from storage.

  18. Identification of Neisseria gonorrhoeae by the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry System Is Improved by a Database Extension.

    PubMed

    Schweitzer, Valentijn A; van Dam, Alje P; Hananta, I Putu Yuda; Schuurman, Rob; Kusters, Johannes G; Rentenaar, Rob J

    2016-04-01

    Identification ofNeisseria gonorrhoeaeby the Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system may be affected by "B consistency categorization." A supplementary database of 17N. gonorrhoeaemain spectra was constructed. Twelve of 64N. gonorrhoeaeidentifications were categorized with B consistency, which disappeared using the supplementary database. Database extension did not result in misidentification ofNeisseria meningitidis. PMID:26763972

  19. Identification of Neisseria gonorrhoeae by the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System Is Improved by a Database Extension

    PubMed Central

    Schweitzer, Valentijn A.; van Dam, Alje P.; Hananta, I Putu Yuda; Schuurman, Rob; Kusters, Johannes G.

    2016-01-01

    Identification of Neisseria gonorrhoeae by the Bruker matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) system may be affected by “B consistency categorization.” A supplementary database of 17 N. gonorrhoeae main spectra was constructed. Twelve of 64 N. gonorrhoeae identifications were categorized with B consistency, which disappeared using the supplementary database. Database extension did not result in misidentification of Neisseria meningitidis. PMID:26763972

  20. HexR Controls Glucose-Responsive Genes and Central Carbon Metabolism in Neisseria meningitidis

    PubMed Central

    Antunes, Ana; Golfieri, Giacomo; Ferlicca, Francesca; Giuliani, Marzia M.; Scarlato, Vincenzo

    2015-01-01

    ABSTRACT Neisseria meningitidis, an exclusively human pathogen and the leading cause of bacterial meningitis, must adapt to different host niches during human infection. N. meningitidis can utilize a restricted range of carbon sources, including lactate, glucose, and pyruvate, whose concentrations vary in host niches. Microarray analysis of N. meningitidis grown in a chemically defined medium in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. Most such genes are implicated in energy metabolism and transport, and some are implicated in virulence. In particular, genes involved in glucose catabolism were upregulated, whereas genes involved in the tricarboxylic acid cycle were downregulated. Several genes encoding surface-exposed proteins, including the MafA adhesins and Neisseria surface protein A, were upregulated in the presence of glucose. Our microarray analysis led to the identification of a glucose-responsive hexR-like transcriptional regulator that controls genes of the central carbon metabolism of N. meningitidis in response to glucose. We characterized the HexR regulon and showed that the hexR gene is accountable for some of the glucose-responsive regulation; in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of the bacterium. Based on DNA sequence alignment of the target sites, we propose a 17-bp pseudopalindromic consensus HexR binding motif. Furthermore, N. meningitidis strains lacking hexR expression were deficient in establishing successful bacteremia in an infant rat model of infection, indicating the importance of this regulator for the survival of this pathogen in vivo. IMPORTANCE Neisseria meningitidis grows on a limited range of nutrients during infection. We analyzed the gene expression of N. meningitidis in response to glucose, the main energy source available in human blood, and we found that glucose regulates many genes

  1. Erythrocyte gangliosides act as receptors for Neisseria subflava: identification of the Sia-1 adhesin.

    PubMed Central

    Nyberg, G; Strömberg, N; Jonsson, A; Karlsson, K A; Normark, S

    1990-01-01

    Neisseria gonorrhoeae was recently shown to bind to a subset of lactose-containing glycolipids (N. Strömberg, C. Deal, G. Nyberg, S. Normark, M. So, and K.-A. Karlsson, Proc. Natl. Acad. Sci. USA 85:4902-4906, 1988). A number of commensal Neisseria strains were also shown to be lactose binders. In addition, Neisseria subflava bound to immobilized gangliosides, such as hematoside and sialosyl paragloboside, carrying the NeuAc alpha 2-3Gal beta 1-4Glc sequence. To a lesser extent, N. gonorrhoeae also bound to this receptor in vitro. In N. subflava GN01, this binding property mediated agglutination of human erythrocytes in a neuraminidase-sensitive fashion. Nitrosoguanidine-induced nonhemagglutinative mutants of N. subflava GN01 had lost the ability to bind hematoside and sialosylparagloboside but remained able to bind lactosylceramide and gangliotetraosylceramide. These mutants fell into three classes with respect to their outer membrane protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Class 1 mutants were identical to the parent strain save for the loss of a 27-kilodalton (kDa) protein. Class 2 mutants showed an outer membrane protein profile identical to that of the wild type, whereas mutants belonging to class 3 showed a number of changes, including the apparent absence of the 27-kDa protein. The 27-kDa protein from N. subflava GN01 was purified from the supernatant. A polyclonal antiserum to the purified Sia-1 protein as well as a Sia-1-specific monoclonal antibody inhibited hemagglutination by strain GN01. The purified Sia-1 protein in the presence of diluted anti-Sia-1 antiserum mediated a neuraminidase-sensitive hemagglutination. The purified Sia protein from a class 2 mutant was not able to hemagglutinate when cross-linked with antibodies, suggesting that it is a mutant form of Sia-1 affected in the receptor-binding site. Immunoelectron microscopy with a Sia-1-specific monoclonal antibody revealed that the adhesin was

  2. [Primary Neisseria meningitidis conjunctivitis in a 14-month-old child].

    PubMed

    Bayramoğlu, Gülçin; Çakır, Murat; Kola, Mehmet; Buruk, Kurtuluş; Kılıç, Selçuk

    2015-07-01

    Meningococcal conjunctivitis is a rare but important infection since it can lead to severe complications and can threaten public health. It may emerge in two forms, either primary or secondary type which is developed after a systemic infection. Accurate diagnosis of primary meningococcal conjunctivitis is very important in addition to ocular complications which can result in loss of vision, the condition can also lead to severe complications like systemic meningococcal disease. However, the lack of specific symptoms which can distinguish meningococcal conjunctivitis from other forms of bacterial conjunctivitis, initiation of empiric antibiotic therapy without performing culture and nonaccurate differentiation of Neisseria gonorrhoeae and Neisseria meningitidis with commercial kits/systems used in laboratories cause problematic situations. This report describes a case of primary unilateral conjunctivitis in a 14-month-old girl caused by non-groupable N.meningitidis that was resolved without sequelae following treatment. A pre-healthy 14-month-old girl was brought to the pediatric emergency department with redness, crusts and discharge in the left eye that had begun two days earlier. Ocular examination revealed hyperemia and purulent discharge in the left conjunctiva. Purulent conjunctivitis was diagnosed. A conjunctival swab specimen was taken for culture, and the patient was started on topical netilmicin (4x1), topical fusidic acid (2x1) and artificial tears. Microscopic examination of the conjunctival swab revealed polymorphonuclear leukocytes and no visible bacteria. Catalase and oxidase positive, gram-negative diplococci grew purely in culture. The first Gram stain preparation was evaluated again after the growth and small numbers of gram-negative diplococci were observed. The cultivated bacteria were identified as N.meningitidis using MALDI-TOF MS (Bruker Daltonics, Germany), but as N.gonorrhoeae with BBL Crystal N/H (Neisseria/Haemophilus) (BD Diagnostic

  3. Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone.

    PubMed

    Osaka, Kazuyoshi; Takakura, Tadakazu; Narukawa, Kayo; Takahata, Masahiro; Endo, Katsuhisa; Kiyota, Hiroshi; Onodera, Shoichi

    2008-06-01

    Neisseria gonorrhoeae strains with reduced susceptibility to cefixime and ceftriaxone, with minimum inhibitory concentrations (MICs) of cefixime of 0.125-0.25 microg/ml and ceftriaxone of 0.031-0.125 microg/ml, were isolated from male urethritis patients in Tokyo, Japan, in 2006. The amino acid sequences of PenA, penicillin-binding protein 2, in these strains were of two types: PenA mosaic and nonmosaic strains. In the PenA mosaic strain, some regions in the transpeptidase-encoding domain in PenA were similar to those of Neisseria perflava/sicca, Neisseria cinerea, Neisseria flavescens, Neisseria polysaccharea, and Neisseria meningitidis. In the PenA nonmosaic strain, there was a mutation of Ala-501 to Val in PenA. In addition, we performed homology modeling of PenA wild-type and mosaic strains and compared them. The results of the modeling studies suggested that reduced susceptibility to cephems such as cefixime and ceftriaxone is due to a conformational alteration of the beta-lactam-binding pocket. These results also indicated that the mosaic structures and the above point mutation in PenA make a major contribution to the reduced susceptibility to cephem antibiotics.

  4. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display

    PubMed Central

    Connor, Daniel O.; Zantow, Jonas; Hust, Michael; Bier, Frank F.; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae. PMID:26859666

  5. Crystal structure of the open state of the Neisseria gonorrhoeae MtrE outer membrane channel.

    PubMed

    Lei, Hsiang-Ting; Chou, Tsung-Han; Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Do, Sylvia V; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2014-01-01

    Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel.

  6. Antimicrobial susceptibility testing of Neisseria gonorrhoeae and implications for epidemiology and therapy.

    PubMed Central

    Fekete, T

    1993-01-01

    Antimicrobial susceptibility testing (AST) of Neisseria gonorrhoeae has been under development since the early days of antimicrobial agents. However, it is rarely applied to clinical isolates today. The history of the various in vitro tests to determine the susceptibility of N. gonorrhoeae to antibiotics is rich with evidence that these results predict response to therapy for almost all agents tested. Further, AST is a useful and important aspect of strain characterization and disease epidemiology in conjunction with the more specific but laborious techniques of auxotyping, serotyping, and plasmid analysis. Current technology has overcome many of the objections to AST for N. gonorrhoeae with standardization of test media and the development of an accurate disk diffusion AST method that is suited to most clinical laboratories regardless of volume or level of technical expertise. Ironically, the very low level of resistance to the current primary treatment strategy in the United States, ceftriaxone or another potent cephalosporin, makes the use of AST somewhat superfluous. PMID:8457978

  7. Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme

    PubMed Central

    Potocki De Montalk, G.; Remaud-Simeon, M.; Willemot, R. M.; Planchot, V.; Monsan, P.

    1999-01-01

    The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)8-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione S-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75. PMID:9882648

  8. Detection of Neisseria meningitidis using surface plasmon resonance based DNA biosensor.

    PubMed

    Kaur, Gurpreet; Paliwal, Ayushi; Tomar, Monika; Gupta, Vinay

    2016-04-15

    Herein, we report the development of a surface plasmon resonance (SPR) based biosensor for the detection of Neisseria meningitidis DNA employing Kretschmann configuration. Highly c-axis oriented ZnO thin film of thickness 200nm was deposited on gold coated glass prisms by RF sputtering technique. Single stranded probe DNA was immobilized on the surface of ZnO thin film by physical adsorption method. SPR reflectance curves were recorded as a function of incident angle of He-Ne laser beam using a laboratory assembled SPR setup. The prepared biosensor exhibits a linear response towards target meningitidis DNA over the concentration range from 10 to 180 ng/μl with a high sensitivity of about 0.03°/(ng/μl) and a low limit of detection of 5 ng/μl. The SPR biosensor demonstrated high specificity and long shelf life thus, pointing towards a promising application in the field of meningitidis diagnosis. PMID:26599479

  9. Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis.

    PubMed

    Power, P M; Roddam, L F; Rutter, K; Fitzpatrick, S Z; Srikhanta, Y N; Jennings, M P

    2003-08-01

    Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pglB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pglB2 polymorphisms were not found in strain C311 musical sharp 3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311 musical sharp 3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating

  10. Characteristics of antisera against periodate-resistant membrane antigens from Neisseria gonorrhoeae.

    PubMed

    Røe, S F; Eggset, G; Iversen, O J; Maeland, J A

    1980-12-01

    Crude outer membrane (OM) was prepared by extraction of bacteria of the Neisseria gonorrhoeae strains 8551. V, and VII, with an EDTA-containing buffer. The preparations contained the lipopolysaccharide (LPS) and at least 10 proteins as shown by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with untreated OM resulted in production of antibodies against several antigens, including LPS. Antisera raised against periodate-treated OM did not contain antibodies against LPS. These latter antisera agglutinated heat-treated (100 degrees C, 60 min) gonoccal cells by means of antibodies to one or more common agglutinogens and against a strain-specific agglutinogen that was susceptible to digestion with proteolytic enzymes. Both side agglutination and a plate agglutination test could be used to detect antibodies against these agglutinogens. PMID:6261525

  11. Detection of Neisseria meningitidis using surface plasmon resonance based DNA biosensor.

    PubMed

    Kaur, Gurpreet; Paliwal, Ayushi; Tomar, Monika; Gupta, Vinay

    2016-04-15

    Herein, we report the development of a surface plasmon resonance (SPR) based biosensor for the detection of Neisseria meningitidis DNA employing Kretschmann configuration. Highly c-axis oriented ZnO thin film of thickness 200nm was deposited on gold coated glass prisms by RF sputtering technique. Single stranded probe DNA was immobilized on the surface of ZnO thin film by physical adsorption method. SPR reflectance curves were recorded as a function of incident angle of He-Ne laser beam using a laboratory assembled SPR setup. The prepared biosensor exhibits a linear response towards target meningitidis DNA over the concentration range from 10 to 180 ng/μl with a high sensitivity of about 0.03°/(ng/μl) and a low limit of detection of 5 ng/μl. The SPR biosensor demonstrated high specificity and long shelf life thus, pointing towards a promising application in the field of meningitidis diagnosis.

  12. A thermonuclease of Neisseria gonorrhoeae enhances bacterial escape from killing by neutrophil extracellular traps.

    PubMed

    Juneau, Richard A; Stevens, Jacqueline S; Apicella, Michael A; Criss, Alison K

    2015-07-15

    Acute gonorrhea is characterized by neutrophilic inflammation that is insufficient to clear Neisseria gonorrhoeae. Activated neutrophils release extracellular traps (NETs), which are composed of chromatin and decorated with antimicrobial proteins. The N. gonorrhoeae NG0969 open reading frame contains a gene (nuc) that encodes a putatively secreted thermonuclease (Nuc) that contributes to biofilm remodeling. Here, we report that Nuc degrades NETs to help N. gonorrhoeae resist killing by neutrophils. Primary human neutrophils released NETs after exposure to N. gonorrhoeae, but NET integrity declined over time with Nuc-containing bacteria. Recombinant Nuc and conditioned medium from Nuc-containing N. gonorrhoeae degraded human neutrophil DNA and NETs. NETs were found to have antimicrobial activity against N. gonorrhoeae, and Nuc expression enhanced N. gonorrhoeae survival in the presence of neutrophils that released NETs. We propose that Nuc enables N. gonorrhoeae to escape trapping and killing by NETs during symptomatic infection, highlighting Nuc as a multifunctional virulence factor for N. gonorrhoeae.

  13. Modulation of HIV transmission by Neisseria gonorrhoeae: molecular and immunological aspects.

    PubMed

    Jarvis, Gary A; Chang, Theresa L

    2012-04-01

    Neisseria gonorrhoeae (GC), a major cause of pelvic inflammatory disease, can facilitate HIV transmission. In response to GC infection, genital epithelial cells can produce cytokines, chemokines and defensins to modulate HIV infection and infectivity. GC can also induce the production of cytokines and chemokines in monocytes and modulate T cell activation. In vivo, an increase in the number of endocervical CD4+ T cells has been found in GC-infected women. Additionally, GC appears to modulate HIV-specific immune responses in HIV-exposed sex workers. Interestingly, in vitro, GC exhibits HIV enhancing or inhibitory effects depending on the HIV target cells. This review summarizes molecular and immunological aspects of the modulation of HIV infection and transmission by GC. Future studies using a multi-cellular system or in animal models will offer insight into the mechanisms by which GC increases HIV transmission.

  14. The chick embryo in studies of virulence and immunity with Neisseria Gonorrhoeae.

    PubMed

    Diena, B B; Lavergne, G; Ryan, A; Ashton, F E; Wallace, R; Perry, M; Daoust, V

    1975-12-01

    Intravenous inoculation of 11-day old chick embryos with Neisseria gonorrhoeae has confirmed the original observation of Bumgarner and Finkelstein that T1 and T2 gonocci are significantly more virulent than T3 and T4. Pili do not seem to be solely responsible for this virulence, since elimination of pili did not effect either the viability or the virulence of N. gonorrhoeae. In neutralization studies, there was only one log difference between the ability of normal and hyperimmune rabbit serum to protect the embryo against gonoccocal infection. However, when mouse sera were used in the same chick embryo system a clear difference was noted between the protective activity of normal serum and that antisera elicited in mice by purified gonococcal antigens. It is suggested therefore that mouse antisera be used in this chick embryo model for the assay for gonococcal antigens.

  15. Contrasting within- and between-host immune selection shapes Neisseria Opa repertoires.

    PubMed

    Watkins, Eleanor R; Grad, Yonatan H; Gupta, Sunetra; Buckee, Caroline O

    2014-10-09

    Pathogen evolution is influenced strongly by the host immune response. Previous studies of the effects of herd immunity on the population structure of directly transmitted, short-lived pathogens have primarily focused on the impact of competition for hosts. In contrast, for long-lived infections like HIV, theoretical work has focused on the mechanisms promoting antigenic variation within the host. In reality, successful transmission requires that pathogens balance both within- and between-host immune selection. The Opa adhesins in the bacterial Neisseria genus provide a unique system to study the evolution of the same antigens across two major pathogens: while N. meningitidis is an airborne, respiratory pathogen colonising the nasopharynx relatively transiently, N. gonorrhoeae can cause sexually transmitted, long-lived infections. We use a simple mathematical model and genomic data to show that trade-offs between immune selection pressures within- and between-hosts can explain the contrasting Opa repertoires observed in meningococci and gonococci.

  16. An evaluation of Neisseria gonorrhoeae antimicrobial susceptibility testing in the UK.

    PubMed

    Jain, Anu; Cole, Michelle J; Planche, Tim; Ison, Catherine A

    2014-11-01

    The only method currently available to perform Neisseria gonorrhoeae antimicrobial susceptibility testing (Ng-AST) requires a viable organism obtained by culture. Reports of in vitro resistance to extended-spectrum cephalosporins, the treatment of choice for gonorrhoea, coupled with increasing gonorrhoea diagnoses is worrying. The aim of this study was to identify various methodologies employed by the UK microbiology laboratories to perform Ng-AST. Of the 118 laboratories that responded, 114 offered Ng-AST; the majority (82.5%, 94/114) of the laboratories used British Society for Antimicrobial Chemotherapy methodology for Ng-AST. The other main findings were infrequent use of quality control procedures and inconsistent susceptibility testing of the antibiotics used routinely for treatment.

  17. Population structure of Neisseria gonorrhoeae based on whole genome data and its relationship with antibiotic resistance

    PubMed Central

    Ezewudo, Matthew N.; Joseph, Sandeep J.; Castillo-Ramirez, Santiago; Dean, Deborah; del Rio, Carlos; Didelot, Xavier; Dillon, Jo-Anne; Selden, Richard F.; Shafer, William M.; Turingan, Rosemary S.; Unemo, Magnus

    2015-01-01

    Neisseria gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted infection (STI) of major importance. As a result of antibiotic resistance, there are now limited options for treating patients. We collected draft genome sequence data and associated metadata data on 76 N. gonorrhoeae strains from around the globe and searched for known determinants of antibiotics resistance within the strains. The population structure and evolutionary forces within the pathogen population were analyzed. Our results indicated a cosmopolitan gonoccocal population mainly made up of five subgroups. The estimated ratio of recombination to mutation (r/m = 2.2) from our data set indicates an appreciable level of recombination occurring in the population. Strains with resistance phenotypes to more recent antibiotics (azithromycin and cefixime) were mostly found in two of the five population subgroups. PMID:25780762

  18. Antigenic potential of a highly conserved Neisseria meningitidis lipopolysaccharide inner core structure defined by chemical synthesis.

    PubMed

    Reinhardt, Anika; Yang, You; Claus, Heike; Pereira, Claney L; Cox, Andrew D; Vogel, Ulrich; Anish, Chakkumkal; Seeberger, Peter H

    2015-01-22

    Neisseria meningitidis is a leading cause of bacterial meningitis worldwide. We studied the potential of synthetic lipopolysaccharide (LPS) inner core structures as broadly protective antigens against N. meningitidis. Based on the specific reactivity of human serum antibodies to synthetic LPS cores, we selected a highly conserved LPS core tetrasaccharide as a promising antigen. This LPS inner core tetrasaccharide induced a robust IgG response in mice when formulated as an immunogenic glycoconjugate. Binding of raised mouse serum to a broad collection of N. meningitidis strains demonstrated the accessibility of the LPS core on viable bacteria. The distal trisaccharide was identified as the crucial epitope, whereas the proximal Kdo moiety was immunodominant and induced mainly nonprotective antibodies that are responsible for lack of functional protection in polyclonal serum. Our results identified key antigenic determinants of LPS core glycan and, hence, may aid the design of a broadly protective immunization against N. meningitidis. PMID:25601073

  19. Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins

    PubMed Central

    Morand, Philippe C; Bille, Emmanuelle; Morelle, Sandrine; Eugène, Emmanuel; Beretti, Jean-Luc; Wolfgang, Matthew; Meyer, Thomas F; Koomey, Michael; Nassif, Xavier

    2004-01-01

    Pathogenic Neisseria express type IV pili (tfp), which have been shown to play a central role in the interactions of bacteria with their environment. The regulation of piliation thus constitutes a central element in bacterial life cycle. The PilC proteins are outer membrane-associated proteins that have a key role in tfp biogenesis since PilC-null mutants appear defective for fibre expression. Moreover, tfp are also subjected to retraction, which is under the control of the PilT nucleotide-binding protein. In this work, we bring evidence that fibre retraction involves the translocation of pilin subunits to the cytoplasmic membrane. Furthermore, by engineering meningococcal strains that harbour inducible pilC genes, and with the use of meningococcus–cell interaction as a model for the sequential observation of fibre expression and retraction, we show that the PilC proteins regulate PilT-mediated fibre retraction. PMID:15103324

  20. Proteins that appear to be associated with pili in Neisseria gonorrhoeae.

    PubMed Central

    Muir, L L; Strugnell, R A; Davies, J K

    1988-01-01

    Pili of Neisseria gonorrhoeae are thought to be composed entirely of identical subunits, called pilin, that self-assemble in vitro. Previous pilus purification methods have relied on this latter point, and dissociation and reassociation of pilin subunits has yielded pilin preparations of high purity. Such a procedure could result in the loss of any pilus-associated proteins. We have developed a procedure for the isolation of intact native pili in a deoxycholate-urea buffer in which the pili are fractionated on the basis of size and hydrophobicity. Electron microscopy indicates that the pili are largely free from outer membrane vesicles and other cellular material. Electrophoretic analysis has shown that a number of proteins copurify with pilin. Antibodies to these proteins could be removed from an antiserum against whole piliated cells by absorption with piliated cells but not by absorption with nonpiliated cells. Hence, our results indicate that these proteins could be pilus associated. Images PMID:2898429

  1. Comparison of antigenic heterogeneity of Neisseria gonorrhoeae strains by micro-immunofluorescence and serum bactericidal tests.

    PubMed Central

    Mark, J A; Wang, S P

    1978-01-01

    The antigenic heterogeneity of Neisseria gonorrhoeae strains was assessed by the micro-immunofluorescence (micro-IF) and the serum bactericidal tests. The micro-IF test verified the antigenic heterogeneity of nine strains received from the Center for Disease Control and placed them into immunotypes A and B. The serum bactericidal system also detected different antigenic determinants among the strains. Although the micro-IF and bactericidal assays did not correspond in each instance, the overall pattern of similarities and differences among these gonococcal strains was similar. The micro-IF pattern obtained with mouse antisera was identical to the pattern revealed with guinea pig antisera. Different colony type organisms showed similar sensitivity in the bactericidal test. The micro-IF test is a rapid technique for the immunotyping of N. gonorrhoeae and has the additional advantages of reproducibility and simplicity. PMID:83299

  2. Evaluation of the Microcult system for isolating and identifying Neisseria gonorrhoeae.

    PubMed Central

    Williams, R J; Ratnatunga, C S; Hamilton-Miller, J M; Brumfitt, W

    1978-01-01

    Specimens from 95 patients attending a venereal diseases clinic were examined for gonococci by three methods--a conventional culture technique using modified Thayer-Martin medium, microscopy of a Gram-stained direct smear, and the Microcult system. For 56% of the specimens the results by all three methods agreed. Assuming the results obtained by culture on Thayer-Martin medium to be correct, the largest source of error was due to false-positive results: microscopy gave 26 and Microcult gave 15 such results. False-negative results were less common: Microcult gave 14, microscopy six. Microcult gave positive results more quickly than the conventional Thayer-Martin cultural method, but the gonococci were difficult to isolate by subculture from the Microcult culture pads. The Microcult medium was not absolutely specific for Neisseria gonorrhoeae. Nevertheless, the Microcult test may well prove to be a useful adjunct to the diagnosis of gonorrhoea, especially when laboratory facilities are not readily available. PMID:417090

  3. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    PubMed

    Connor, Daniel O; Zantow, Jonas; Hust, Michael; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.

  4. The class III ribonucleotide reductase from Neisseria bacilliformis can utilize thioredoxin as a reductant

    PubMed Central

    Wei, Yifeng; Funk, Michael A.; Rosado, Leonardo A.; Baek, Jiyeon; Drennan, Catherine L.; Stubbe, JoAnne

    2014-01-01

    The class III anaerobic ribonucleotide reductases (RNRs) studied to date couple the reduction of ribonucleotides to deoxynucleotides with the oxidation of formate to CO2. Here we report the cloning and heterologous expression of the Neisseria bacilliformis class III RNR and show that it can catalyze nucleotide reduction using the ubiquitous thioredoxin/thioredoxin reductase/NADPH system. We present a structural model based on a crystal structure of the homologous Thermotoga maritima class III RNR, showing its architecture and the position of conserved residues in the active site. Phylogenetic studies suggest that this form of class III RNR is present in bacteria and archaea that carry out diverse types of anaerobic metabolism. PMID:25157154

  5. Differences in the adhesive properties of Neisseria meningitidis for human buccal epithelial cells and erythrocytes.

    PubMed Central

    Trust, T J; Gillespie, R M; Bhatti, A R; White, L A

    1983-01-01

    The ability of clinical and carrier isolates of Neisseria meningitidis to adhere to human buccal epithelial cells and erythrocytes was investigated. Four of the 10 fimbriated strains were able to hemagglutinate. Serial subculture of three of these strains resulted in a loss of ability to hemagglutinate and was coincident with a loss of fimbriation. Other fimbriated strains were unable to hemagglutinate but did adhere to buccal epithelial cells. Subculture of one of these strains for as many as 42 passages did not result in loss of fimbriation or ability to adhere to buccal epithelial cells. The attachment of this strain to buccal epithelial cells was inhibited by glycoconjugates. Further, pH exerted different influences on the attachment of hemagglutinating and non-hemagglutinating fimbriated strains to buccal epithelial cells and erythrocytes. The results suggest that different fimbrial mechanisms are involved in the attachment of N. meningitidis to different cell types and that hemagglutination is not an absolute test for fimbriae. PMID:6134676

  6. Immunogenicity of recombinant class 1 protein from Neisseria meningitidis refolded into phospholipid vesicles and detergent.

    PubMed

    Niebla, O; Alvarez, A; Martín, A; Rodríguez, A; Delgado, M; Falcón, V; Guillén, G

    2001-05-14

    The possibility of eliciting bactericidal antibodies against a recombinant class 1 protein (P1) from Neisseria meningitidis, joined to the first 45 amino acids of the neisserial LpdA protein (PM82), was examined. P1 was produced in Escherichia coli as intracellular inclusion bodies, from which it was purified and reconstituted by (a) inclusion into phospholipid vesicles and detergent and (b) refolding in 0.1% SDS. When Balb/c mice were immunised, high titres of subtype-specific bactericidal antibodies against P1 were obtained in both cases. These results suggest that in spite of being a denaturing agent, it is possible to use SDS to reconstitute the P1 protein in a conformation that exposes the immunodominat regions.

  7. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    PubMed

    Connor, Daniel O; Zantow, Jonas; Hust, Michael; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae. PMID:26859666

  8. Evidence for a reserpine-affected mechanism of resistance to tetracycline in Neisseria gonorrhoeae.

    PubMed

    Ruiz, Joaquim; Ribera, Anna; Jurado, Angels; Marco, Francesc; Vila, Jordi

    2005-10-01

    The presence of a reserpine-affected mechanism of tetracycline resistance was investigated in 17 Neisseria gonorrhoeae clinical isolates. To establish this fact the MIC of tetracycline in the presence and absence of reserpine was determined, and, in addition, mechanisms of tetracycline resistance were analyzed by PCR. The results showed that reserpine affects the MIC of tetracycline at least 4-fold in all isolates, including those containing the tetM gene. An inhibitory effect of reserpine against the MtrCDE efflux system was ruled out by using strains either with an inactive or with an unrepressed MtrCDE system. The results suggest the presence of a constitutive system of resistance to tetracycline, by a possible efflux pump, which may be inhibited by reserpine. Further studies are required to determine the exact nature of the action of reserpine on the MIC of tetracycline. PMID:16309425

  9. Neisseria gonorrhoeae enhances infection of dendritic cells by HIV type 1.

    PubMed

    Zhang, Jizhong; Li, Geling; Bafica, Andre; Pantelic, Milica; Zhang, Pei; Broxmeyer, Hal; Liu, Ying; Wetzler, Lee; He, Johnny J; Chen, Tie

    2005-06-15

    Clinical studies indicate that Neisseria gonorrhoeae (gonococci (GC)) has the capacity to enhance HIV type 1 (HIV-1) infection. We studied whether GC enhances HIV infection of activated dendritic cells (DCs). The results show that GC can dramatically enhance HIV replication in human DCs during coinfection. The GC component responsible for HIV infection enhancement may be peptidoglycan, which activates TLR2. TLR2 involvement is suggested by bacterial lipoprotein, a TLR2-specific inducer, which stimulates a strong enhancement of HIV infection by human DCs. Moreover, participation of TLR2 is further implicated because GC is unable to stimulate expression of HIV in DCs of TLR2-deficient HIV-1-transgenic mice. These results provide one potential mechanism through which GC infection increases HIV replication in patients infected with both GC and HIV. PMID:15944306

  10. Crystal structure of the open state of the Neisseria gonorrhoeae MtrE outer membrane channel.

    PubMed

    Lei, Hsiang-Ting; Chou, Tsung-Han; Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Do, Sylvia V; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2014-01-01

    Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel. PMID:24901251

  11. The U.S. military's Neisseria gonorrhoeae resistance surveillance initiatives in selected populations of five countries.

    PubMed

    Tsai, Alice Y; Dueger, Erica; Macalino, Grace E; Montano, Silvia M; Tilley, Drake H; Mbuchi, Margaret; Wurapa, Eyako K; Saylors, Karen; Duplessis, Christopher C; Puplampu, Naiki; Garges, Eric C; McClelland, R Scott; Sanchez, Jose L

    2013-02-01

    Multi-drug resistant Neisseria gonorrhoeae (GC) threatens the successful treatment of gonorrhea. This report presents preliminary findings with regard to the prevalence of laboratory-confirmed GC and the extent of drug-resistance among sample populations in five countries. Between October 2010 and January 2013, 1,694 subjects (54% male; 45% female; 1% unknown) were enrolled and screened for the presence of laboratory-confirmed GC in the United States, Djibouti, Ghana, Kenya, and Peru. Overall, 108 (6%) of enrolled subjects tested positive for GC. Antimicrobial susceptibility testing results were available for 66 GC isolates. Resistance to at least three antibiotics was observed at each overseas site. All isolates tested in Ghana (n=6) were resistant to ciprofloxacin, penicillin, and tetracycline. In Djibouti, preliminary results suggested resistance to penicillin, tetracycline, ciprofloxacin, cefepime, and ceftriaxone. The small sample size and missing data prevent comparative analysis and limit the generalizability of these preliminary findings. PMID:23461308

  12. Multilocus variable-number tandem-repeat analysis of Neisseria meningitidis serogroup C in China.

    PubMed

    Shan, X Y; Zhou, H J; Zhang, J; Zhu, B Q; Xu, L; Xu, Z; Hu, G C; Bai, A Y; Shi, Y W; Jiang, B F; Shao, Z J

    2015-10-01

    This study characterized Neisseria meningitidis serogroup C strains in China in order to establish their genetic relatedness and describe the use of multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) to provide useful epidemiological information. A total of 215 N. meningitidis serogroup C strains, obtained from 2003 to 2012 in China, were characterized by MLVA with different published schemes as well as multilocus sequence typing. (i) Based on the MLVA scheme with a combination of five highly variable loci, 203 genotypes were identified; this level of discrimination supports its use for resolving closely related isolates. (ii) Based on a combination of ten low variable loci, clear phylogenetic relationships were established within sequence type complexes. In addition, there was evidence of microevolution of VNTR loci over the decade as strain lineages spread from Anhui to other provinces, the more distant the provinces from Anhui, the higher the genetic variation.

  13. A Case of Diabetic Mellitus Foot Infection by a Newly Reported Neisseria Skkuensis: Case Report.

    PubMed

    Cho, Chi Hyun; Lee, Chang Kyu; Nam, Myung-Hyun; Yoon, Soo-Young; Lim, Chae Seung; Cho, Yunjung; Kim, Young Kee

    2015-01-01

    In pus and wound samples collected from the right second toe of a 61-year-old woman with diabetes mellitus (DM), gram-negative diplococci bacterium was observed. However, the bacterium could not be identified by conventional microbiological methods and mass spectrometry. In the partial 16S rRNA gene sequence analysis, the bacterium showed a 100% identity match with GenBank sequence FJ0763637.1 (Neisseria skkuensis). N. skkuensis, SMC-A9199 strain, was reported as a novel species in 2010 based on its phenotypic characteristics and the 16S rRNA gene sequence, which was isolated from the blood and wound pus of a DM patient with a foot ulcer. The second reported N. skkuensis was identified from the blood cultures of a patient with endocarditis. To the best of our knowledge, this is only the third report of N. skkuensis.

  14. Utilizing complement evasion strategies to design complement-based antibacterial immunotherapeutics: Lessons from the pathogenic Neisseriae.

    PubMed

    Ram, Sanjay; Shaughnessy, Jutamas; DeOliveira, Rosane B; Lewis, Lisa A; Gulati, Sunita; Rice, Peter A

    2016-10-01

    Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac and resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms. PMID:27297292

  15. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    PubMed

    Obergfell, Kyle P; Seifert, H Steven

    2016-05-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants

  16. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    PubMed

    Obergfell, Kyle P; Seifert, H Steven

    2016-05-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants

  17. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation

    PubMed Central

    2016-01-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic

  18. Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae.

    PubMed

    Zhao, Shuqing; Duncan, Margaret; Tomberg, Joshua; Davies, Christopher; Unemo, Magnus; Nicholas, Robert A

    2009-09-01

    All strains of Neisseria gonorrhoeae with reduced susceptibility to ceftriaxone and cefixime (cephalosporin-intermediate-resistant [Ceph(i)] strains) contain a mosaic penA allele encoding penicillin-binding protein 2 (PBP 2) with nearly 60 amino acid differences compared to the sequence of wild-type PBP 2, together with a set of resistance determinants (i.e., mtrR, penB, and/or ponA1) that are required for high-level penicillin resistance. To define the individual contributions of these determinants to reduced susceptibility to ceftriaxone and cefixime, we created isogenic strains containing the mosaic penA allele from the Ceph(i) strain 35/02 (penA35) together with one or more of the other resistance determinants and determined the MICs of penicillin G, ceftriaxone, and cefixime. The majority of cefixime resistance is conferred by the penA35 allele, with only a small contribution coming from mtrR and penB, whereas ceftriaxone resistance is nearly equally dependent upon mtrR and penB. Unlike high-level penicillin resistance, the ponA1 allele does not appear to be important for Ceph(i). A strain containing all four determinants has increased resistance to ceftriaxone and cefixime but not to the levels that the donor Ceph(i) strain does, suggesting that Ceph(i) strains, similar to high-level-penicillin-resistant strains, contain an additional unknown determinant that is required to reach donor levels of resistance. Our data also suggest that the original Ceph(i) strains arose from the transformation of penA genes from commensal Neisseria species into a penicillin-resistant strain already harboring mtrR, penB, ponA1, and the unknown gene(s) involved in high-level penicillin resistance.

  19. Will targeting oropharyngeal gonorrhoea delay the further emergence of drug-resistant Neisseria gonorrhoeae strains?

    PubMed

    Lewis, D A

    2015-06-01

    Gonorrhoea is an important sexually transmitted infection associated with serious complications and enhanced HIV transmission. Oropharyngeal infections are often asymptomatic and will only be detected by screening. Gonococcal culture has low sensitivity (<50%) for detecting oropharyngeal gonorrhoea, and, although not yet approved commercially, nucleic acid amplification tests (NAAT) are the assay of choice. Screening for oropharyngeal gonorrhoea should be performed in high-risk populations, such as men-who-have-sex-with-men(MSM). NAATs have a poor positive predictive value when used in low-prevalence populations. Gonococci have repeatedly thwarted gonorrhoea control efforts since the first antimicrobial agents were introduced. The oropharyngeal niche provides an enabling environment for horizontal transfer of genetic material from commensal Neisseria and other bacterial species to Neisseria gonorrhoeae. This has been the mechanism responsible for the generation of mosaic penA genes, which are responsible for most of the observed cases of resistance to extended-spectrum cephalosporins (ESC). As antimicrobial-resistant gonorrhoea is now an urgent public health threat, requiring improved antibiotic stewardship, laboratory-guided recycling of older antibiotics may help reduce ESC use. Future trials of antimicrobial agents for gonorrhoea should be powered to test their efficacy at the oropharynx as this is the anatomical site where treatment failure is most likely to occur. It remains to be determined whether a combination of frequent screening of high-risk individuals and/or laboratory-directed fluoroquinolone therapy of oropharyngeal gonorrhoea will delay the further emergence of drug-resistant N. gonorrhoeae strains. PMID:25911525

  20. Genetic Studies of Sulfadiazine-resistant and Methionine-requiring Neisseria Isolated From Clinical Material

    PubMed Central

    Catlin, B. Wesley

    1967-01-01

    Deoxyribonucleate (DNA) preparations were extracted from Neisseria meningitidis (four isolates from spinal fluid and blood) and N. gonorrhoeae strains, all of which were resistant to sulfadiazine upon primary isolation. These DNA preparations, together with others from in vitro mutants of N. meningitidis and N. perflava, were examined in transformation tests by using as recipient a drug-susceptible strain of N. meningitidis (Ne 15 Sul-s Met+) which was able to grow in a methionine-free defined medium. The sulfadiazine resistance typical of each donor was introduced into the uniform constitution of this recipient. Production of p-aminobenzoic acid was not significantly altered thereby. Transformants elicited by DNA from the N. meningitidis clinical isolates were resistant to at least 200 μg of sulfadiazine/ml, and did not show a requirement for methionine (Sul-r Met+). DNA from six strains of N. gonorrhoeae, which were isolated during the period of therapeutic use of sulfonamides, conveyed lower degrees of resistance and, invariably, a concurrent methionine requirement (Sul-r/Met−). The requirement of these transformants, and that of in vitro mutants selected on sulfadiazine-agar, was satisfied by methionine, but not by vitamin B12, homocysteine, cystathionine, homoserine, or cysteine. Sul-r Met+ and Sul-r/Met− loci could coexist in the same genome, but were segregated during transformation. On the other hand, the dual Sul-r/Met− properties were not separated by recombination, but were eliminated together. DNA from various Sul-r/Met− clones tested against recipients having nonidentical Sul-r/Met− mutant sites yielded Sul-s Met+ transformants. The met locus involved is genetically complex, and will be a valuable tool for studies of genetic fine structure of members of Neisseria, and of genetic homology between species. Images PMID:4962305

  1. Use of restriction fragment length polymorphisms to investigate strain variation within Neisseria meningitidis

    SciTech Connect

    Williams, S.D.

    1989-01-01

    Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty-six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P{sup 32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analyzed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population.

  2. Utilizing complement evasion strategies to design complement-based antibacterial immunotherapeutics: Lessons from the pathogenic Neisseriae.

    PubMed

    Ram, Sanjay; Shaughnessy, Jutamas; DeOliveira, Rosane B; Lewis, Lisa A; Gulati, Sunita; Rice, Peter A

    2016-10-01

    Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac and resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms.

  3. Genotypic comparison of invasive Neisseria meningitidis serogroup Y isolates from the United States, South Africa, and Israel, isolated from 1999 through 2002.

    PubMed

    Whitney, Anne M; Coulson, Garry B; von Gottberg, Anne; Block, Colin; Keller, Nathan; Mayer, Leonard W; Messonnier, Nancy E; Klugman, Keith P

    2009-09-01

    The proportion of meningococcal disease in the United States, South Africa, and Israel caused by Neisseria meningitidis serogroup Y (NmY) was greater than the worldwide average during the period 1999-2002. Genotypic characterization of 300 NmY isolates by multilocus sequence typing, 16S rRNA gene sequencing, and PorA variable region typing was conducted to determine the relationships of the isolates from these three countries. Seventy different genotypes were found. Two groups of ST-23 clonal complex isolates accounted for 88% of the U.S. isolates, 12% of the South African isolates, and 96% of the isolates from Israel. The single common clone (ST-23/16S-19/P1.5-2,10-1) represented 57, 5, and 35% of the NmY isolates from the United States, South Africa, and Israel. The predominant clone in South Africa (ST-175/16S-21/P1.5-1,2-2), and 11 other closely related clones made up 77% of the South African study isolates and were not found among the isolates from the United States or Israel. ST-175 was the predicted founder of the ST-175 clonal complex, and isolates of ST-175 and related sequence types have been described previously in other African countries. Continued active surveillance and genetic characterization of NmY isolates causing disease in the United States, South Africa, and Israel will provide valuable data for local and global epidemiology and allow monitoring for any expansion of existing clonal complexes and detection of the emergence of new virulent clones in the population. PMID:19571028

  4. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure.

    PubMed

    Unemo, Magnus; Golparian, Daniel; Nicholas, Robert; Ohnishi, Makoto; Gallay, Anne; Sednaoui, Patrice

    2012-03-01

    Recently, the first Neisseria gonorrhoeae strain (H041) highly resistant to the expanded-spectrum cephalosporins (ESCs) ceftriaxone and cefixime, which are the last remaining options for first-line gonorrhea treatment, was isolated in Japan. Here, we confirm and characterize a second strain (F89) with high-level cefixime and ceftriaxone resistance which was isolated in France and most likely caused a treatment failure with cefixime. F89 was examined using six species-confirmatory tests, antibiograms (33 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of known gonococcal resistance determinants (penA, mtrR, penB, ponA, and pilQ). F89 was assigned to MLST sequence type 1901 (ST1901) and NG-MAST ST1407, which is a successful gonococcal clone that has spread globally. F89 has high-level resistance to cefixime (MIC = 4 μg/ml) and ceftriaxone (MIC = 1 to 2 μg/ml) and resistance to most other antimicrobials examined. A novel penA mosaic allele (penA-CI), which was penA-XXXIV with an additional A501P alteration in penicillin-binding protein 2, was the primary determinant for high-level ESC resistance, as determined by transformation into a set of recipient strains. N. gonorrhoeae appears to be emerging as a superbug, and in certain circumstances and settings, gonorrhea may become untreatable. Investigations of the biological fitness and enhanced understanding and monitoring of the ESC-resistant clones and their international transmission are required. Enhanced disease control activities, antimicrobial resistance control and surveillance worldwide, and public health response plans for global (and national) perspectives are also crucial. Nevertheless, new treatment strategies and/or drugs and, ideally, a vaccine are essential to develop for efficacious gonorrhea management.

  5. Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhea?: detailed characterization of the first strain with high-level resistance to ceftriaxone.

    PubMed

    Ohnishi, Makoto; Golparian, Daniel; Shimuta, Ken; Saika, Takeshi; Hoshina, Shinji; Iwasaku, Kazuhiro; Nakayama, Shu-ichi; Kitawaki, Jo; Unemo, Magnus

    2011-07-01

    Recently, the first Neisseria gonorrhoeae strain (H041) that is highly resistant to the extended-spectrum cephalosporin (ESC) ceftriaxone, the last remaining option for empirical first-line treatment, was isolated. We performed a detailed characterization of H041, phenotypically and genetically, to confirm the finding, examine its antimicrobial resistance (AMR), and elucidate the resistance mechanisms. H041 was examined using seven species-confirmatory tests, antibiograms (30 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of ESC resistance determinants (penA, mtrR, penB, ponA, and pilQ). Transformation, using appropriate recipient strains, was performed to confirm the ESC resistance determinants. H041 was assigned to serovar Bpyust, MLST sequence type (ST) ST7363, and the new NG-MAST ST4220. H041 proved highly resistant to ceftriaxone (2 to 4 μg/ml, which is 4- to 8-fold higher than any previously described isolate) and all other cephalosporins, as well as most other antimicrobials tested. A new penA mosaic allele caused the ceftriaxone resistance. In conclusion, N. gonorrhoeae has now shown its ability to also develop ceftriaxone resistance. Although the biological fitness of ceftriaxone resistance in N. gonorrhoeae remains unknown, N. gonorrhoeae may soon become a true superbug, causing untreatable gonorrhea. A reduction in the global gonorrhea burden by enhanced disease control activities, combined with wider strategies for general AMR control and enhanced understanding of the mechanisms of emergence and spread of AMR, which need to be monitored globally, and public health response plans for global (and national) perspectives are important. Ultimately, the development of new drugs for efficacious gonorrhea treatment is necessary.

  6. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure.

    PubMed

    Unemo, Magnus; Golparian, Daniel; Nicholas, Robert; Ohnishi, Makoto; Gallay, Anne; Sednaoui, Patrice

    2012-03-01

    Recently, the first Neisseria gonorrhoeae strain (H041) highly resistant to the expanded-spectrum cephalosporins (ESCs) ceftriaxone and cefixime, which are the last remaining options for first-line gonorrhea treatment, was isolated in Japan. Here, we confirm and characterize a second strain (F89) with high-level cefixime and ceftriaxone resistance which was isolated in France and most likely caused a treatment failure with cefixime. F89 was examined using six species-confirmatory tests, antibiograms (33 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of known gonococcal resistance determinants (penA, mtrR, penB, ponA, and pilQ). F89 was assigned to MLST sequence type 1901 (ST1901) and NG-MAST ST1407, which is a successful gonococcal clone that has spread globally. F89 has high-level resistance to cefixime (MIC = 4 μg/ml) and ceftriaxone (MIC = 1 to 2 μg/ml) and resistance to most other antimicrobials examined. A novel penA mosaic allele (penA-CI), which was penA-XXXIV with an additional A501P alteration in penicillin-binding protein 2, was the primary determinant for high-level ESC resistance, as determined by transformation into a set of recipient strains. N. gonorrhoeae appears to be emerging as a superbug, and in certain circumstances and settings, gonorrhea may become untreatable. Investigations of the biological fitness and enhanced understanding and monitoring of the ESC-resistant clones and their international transmission are required. Enhanced disease control activities, antimicrobial resistance control and surveillance worldwide, and public health response plans for global (and national) perspectives are also crucial. Nevertheless, new treatment strategies and/or drugs and, ideally, a vaccine are essential to develop for efficacious gonorrhea management. PMID:22155830

  7. The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity.

    PubMed

    Valeri, Maria; Zurli, Vanessa; Ayala, Inmaculada; Colanzi, Antonino; Lapazio, Lucia; Corda, Daniela; Soriani, Marco; Pizza, Mariagrazia; Rossi Paccani, Silvia

    2015-01-01

    Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis.

  8. Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.

    PubMed Central

    Makino, S; van Putten, J P; Meyer, T F

    1991-01-01

    Neisseria gonorrhoeae is a facultative intracellular bacterium capable of penetrating into certain human epithelial cell types. In order to identify gonococcal factors essential for invading Chang human conjunctiva cells, a gentamicin selection assay for the quantification of viable intracellular bacteria was used in conjunction with microscopy. The results demonstrate a correlation between the invasive behaviour of gonococci and the expression of Opa proteins, a family of variable outer membrane proteins present in all pathogenic Neisseria species. However, only particular Opa proteins supported invasion into Chang cells as indicated by the use of two unrelated gonococcal strains. Invasion was sensitive to cytochalasin D, and strong adherence mediated by the Opa proteins appeared to be essential for the internalization of gonococci. In contrast pili, which also conferred binding to Chang conjunctiva cells, did not support cellular invasion but rather were inhibitory. Images PMID:1673923

  9. recA and catalase in H sub 2 O sub 2 -mediated toxicity in Neisseria gonorrhoeae

    SciTech Connect

    Hassett, D.J.; Charniga, L.; Cohen, M.S. )

    1990-12-01

    Neisseria gonorrhoeae cells defective in the biosynthesis of the recA gene product are no more sensitive to hydrogen peroxide than wild-type cells. Although gonococci possess nearly 100-fold-greater catalase levels than Escherichia coli, they are more susceptible to hydrogen peroxide than this organism. The natural niche of gonococci undoubtedly results in exposure to oxidant stress; however, they do not demonstrate particularly efficient antioxidant defense systems.

  10. Rapid carbohydrate fermentation test for confirmation of the pathogenic Neisseria using a Ba(OH)2 indicator.

    PubMed Central

    Slifkin, M; Pouchet, G R

    1977-01-01

    The Ba(OH)2 indicator system was demonstrated to be a practical procedure in assisting clinical bacteriologists in the accurate and rapid identification of the pathogenic Neisseria from clinical specimens. This system measured the release of CO2, resulting from the metabolism of fermentable carbohydrate, as the precipitated BaCO3, by means of a spectrophotometer, The method was uncomplicated and can be performed in most clinical bacteriology laboratories. PMID:319106

  11. Severe conjunctivitis due to multidrug-resistant Neisseria gonorrhoeae and adenovirus 53 coinfection in a traveler returning from Thailand.

    PubMed

    Tappe, Dennis; Mueller, Andreas; Weißbrich, Benedikt; Schubert, Jörg; Schargus, Marc; Stich, August

    2013-01-01

    A male traveler returning from Thailand with severe bilateral conjunctivitis was tested for causative pathogens by culture and polymerase chain reaction in late 2010. The culturally grown Neisseria gonorrhoeae strain was resistant against penicillin, ciprofloxacin, and tetracycline. The patient was also found to have an eye infection with the unusual and likely recombinant adenovirus type 53. Besides multidrug-resistant gonococcal strains the unusual adenovirus strain is found circulating in Asia and both pathogens may be a risk for travelers.

  12. Trends in antimicrobial resistance in Neisseria gonorrhoeae isolated from Guangzhou, China, 2000 to 2005 and 2008 to 2013.

    PubMed

    Cao, Wen-Ling; Liang, Jing-Yao; Li, Xiao-Dong; Bi, Chao; Yang, Ri-Dong; Liang, Yan-Hua; Li, Ping; Zhong, Dao-Qing; Ye, Xing-Dong; Zhang, Xi-Bao

    2015-01-01

    A total of 1224 Neisseria gonorrhoeae isolates from Guangzhou in 2 periods (2000-2005 and 2008-2013) were subjected to antimicrobial susceptibility testing. The percentage of penicillin- and ciprofloxacin-resistant isolates increased from 71.1% (473/665) to 90.9% (508/559) and 88.9% (591/665) to 98.0% (548/559), respectively. All isolates remain susceptible to spectinomycin and ceftriaxone, with increasing minimum inhibitory concentrations.

  13. Molecular characterization of FrpB, the 70-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis.

    PubMed Central

    Pettersson, A; Maas, A; van Wassenaar, D; van der Ley, P; Tommassen, J

    1995-01-01

    The structural gene encoding the 70-kDa outer membrane protein FrpB of Neisseria meningitidis was cloned and sequenced. A mutant lacking FrpB was constructed. No difference in iron utilization between the mutant and the parental strain was observed. A minor effect of the mutation on serum resistance was observed. A topology model for FrpB in the outer membrane is proposed. PMID:7558339

  14. Genome Sequence of a Neisseria meningitidis Capsule Null Locus Strain from the Clonal Complex of Sequence Type 198

    PubMed Central

    Schork, Sabine; Schlüter, Andreas; Blom, Jochen; Schneiker-Bekel, Susanne; Pühler, Alfred; Goesmann, Alexander; Frosch, Matthias

    2012-01-01

    Neisseria meningitidis is a commensal and accidental pathogen exclusively of humans. Although the production of polysaccharide capsules is considered to be essential for meningococcal virulence, there have been reports of constitutively unencapsulated strains causing invasive meningococcal disease (IMD). Here we report the genome sequence of a capsule null locus (cnl) strain of sequence type 198 (ST-198), which is found in half of the reported cases of IMD caused by cnl meningococcal strains. PMID:22933768

  15. Neisseria meningitidis tonB, exbB, and exbD genes: Ton-dependent utilization of protein-bound iron in Neisseriae.

    PubMed Central

    Stojiljkovic, I; Srinivasan, N

    1997-01-01

    We have recently cloned and characterized the hemoglobin (Hb) receptor gene, hmbR, from Neisseria meningitidis. To identify additional proteins that are involved in Hb utilization, the N. meningitidis Hb utilization system was reconstituted in Escherichia coli. Five cosmids from N. meningitidis DNA library enabled a heme-requiring (hemA), HmbR-expressing mutant of E. coli to use Hb as both porphyrin and iron source. Nucleotide sequence analysis of DNA fragments subcloned from the Hb-complementing cosmids identified four open reading frames, three of them homologous to Pseudomonas putida, E. coli, and Haemophilus influenzae exbB, exbD, and tonB genes. The N. meningitidis TonB protein is 28.8 to 33.6% identical to other gram-negative TonB proteins, while the N. meningitidis ExbD protein shares between 23.3 and 34.3% identical amino acids with other ExbD and TolR proteins. The N. meningitidis ExbB protein was 24.7 to 36.1% homologous with other gram-negative ExbB and TolQ proteins. Complementation studies indicated that the neisserial Ton system cannot interact with the E. coli FhuA TonB-dependent outer membrane receptor. The N. meningitidis tonB mutant was unable to use Hb, Hb-haptoglobin complexes, transferrin, and lactoferrin as iron sources. Insertion of an antibiotic cassette in the 3' end of the exbD gene produced a leaky phenotype. Efficient usage of heme by N. meningitidis tonB and exbD mutants suggests the existence of a Ton-independent heme utilization mechanism. E. coli complementation studies and the analysis of N. meningitidis hmbR and hpu mutants suggested the existence of another Hb utilization mechanism in this organism. PMID:9006036

  16. Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria.

    PubMed

    Hooda, Yogesh; Lai, Christine Chieh-Lin; Judd, Andrew; Buckwalter, Carolyn M; Shin, Hyejin Esther; Gray-Owen, Scott D; Moraes, Trevor F

    2016-01-01

    Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism. PMID:27572441

  17. Fructose-1,6-bisphosphate aldolase of Neisseria meningitidis binds human plasminogen via its C-terminal lysine residue.

    PubMed

    Shams, Fariza; Oldfield, Neil J; Lai, Si Kei; Tunio, Sarfraz A; Wooldridge, Karl G; Turner, David P J

    2016-04-01

    Neisseria meningitidis is a leading cause of fatal sepsis and meningitis worldwide. As for commensal species of human neisseriae, N. meningitidis inhabits the human nasopharynx and asymptomatic colonization is ubiquitous. Only rarely does the organism invade and survive in the bloodstream leading to disease. Moonlighting proteins perform two or more autonomous, often dissimilar, functions using a single polypeptide chain. They have been increasingly reported on the surface of both prokaryotic and eukaryotic organisms and shown to interact with a variety of host ligands. In some organisms moonlighting proteins perform virulence-related functions, and they may play a role in the pathogenesis of N. meningitidis. Fructose-1,6-bisphosphate aldolase (FBA) was previously shown to be surface-exposed in meningococci and involved in adhesion to host cells. In this study, FBA was shown to be present on the surface of both pathogenic and commensal neisseriae, and surface localization and anchoring was demonstrated to be independent of aldolase activity. Importantly, meningococcal FBA was found to bind to human glu-plasminogen in a dose-dependent manner. Site-directed mutagenesis demonstrated that the C-terminal lysine residue of FBA was required for this interaction, whereas subterminal lysine residues were not involved.

  18. Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro.

    PubMed

    Gong, Zijian; Lai, Wei; Liu, Min; Hua, Zhengshuang; Sun, Yayin; Xu, Qingfang; Xia, Yue; Zhao, Yue; Xie, Xiaoyuan

    2016-04-01

    The emergence of ceftriaxone-resistantNeisseria gonorrhoeaeis currently a global public health concern. However, the mechanism of ceftriaxone resistance is not yet fully understood. To investigate the potential genes related to ceftriaxone resistance inNeisseria gonorrhoeae, we subcultured six gonococcal strains with increasing concentrations of ceftriaxone and isolated the strains that became resistant. After analyzing several frequently reported genes involved in ceftriaxone resistance, we found only a single mutation inpenA(A501V). However, differential analysis of the genomes and transcriptomes between pre- and postselection strains revealed many other mutated genes as well as up- and downregulated genes. Transformation of the mutatedpenAgene into nonresistant strains increased the MIC between 2.0- and 5.3-fold, and transformation of mutatedftsXincreased the MIC between 3.3- and 13.3-fold. Genes encoding the ABC transporters FarB, Tfq, Hfq, and ExbB were overexpressed, whilepilM,pilN, andpilQwere downregulated. Furthermore, the resistant strain developed cross-resistance to penicillin and cefuroxime, had an increased biochemical metabolic rate, and presented fitness defects such as prolonged growth time and downregulated PilMNQ. In conclusion, antimicrobial pressure could result in the emergence of ceftriaxone resistance, and the evolution of resistance ofNeisseria gonorrhoeaeto ceftriaxone is a complicated process at both the pretranscriptional and posttranscriptional levels, involving several resistance mechanisms of increased efflux and decreased entry.

  19. Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro

    PubMed Central

    Gong, Zijian; Liu, Min; Hua, Zhengshuang; Sun, Yayin; Xu, Qingfang; Xia, Yue; Zhao, Yue; Xie, Xiaoyuan

    2016-01-01

    The emergence of ceftriaxone-resistant Neisseria gonorrhoeae is currently a global public health concern. However, the mechanism of ceftriaxone resistance is not yet fully understood. To investigate the potential genes related to ceftriaxone resistance in Neisseria gonorrhoeae, we subcultured six gonococcal strains with increasing concentrations of ceftriaxone and isolated the strains that became resistant. After analyzing several frequently reported genes involved in ceftriaxone resistance, we found only a single mutation in penA (A501V). However, differential analysis of the genomes and transcriptomes between pre- and postselection strains revealed many other mutated genes as well as up- and downregulated genes. Transformation of the mutated penA gene into nonresistant strains increased the MIC between 2.0- and 5.3-fold, and transformation of mutated ftsX increased the MIC between 3.3- and 13.3-fold. Genes encoding the ABC transporters FarB, Tfq, Hfq, and ExbB were overexpressed, while pilM, pilN, and pilQ were downregulated. Furthermore, the resistant strain developed cross-resistance to penicillin and cefuroxime, had an increased biochemical metabolic rate, and presented fitness defects such as prolonged growth time and downregulated PilMNQ. In conclusion, antimicrobial pressure could result in the emergence of ceftriaxone resistance, and the evolution of resistance of Neisseria gonorrhoeae to ceftriaxone is a complicated process at both the pretranscriptional and posttranscriptional levels, involving several resistance mechanisms of increased efflux and decreased entry. PMID:26787702

  20. Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro.

    PubMed

    Gong, Zijian; Lai, Wei; Liu, Min; Hua, Zhengshuang; Sun, Yayin; Xu, Qingfang; Xia, Yue; Zhao, Yue; Xie, Xiaoyuan

    2016-04-01

    The emergence of ceftriaxone-resistantNeisseria gonorrhoeaeis currently a global public health concern. However, the mechanism of ceftriaxone resistance is not yet fully understood. To investigate the potential genes related to ceftriaxone resistance inNeisseria gonorrhoeae, we subcultured six gonococcal strains with increasing concentrations of ceftriaxone and isolated the strains that became resistant. After analyzing several frequently reported genes involved in ceftriaxone resistance, we found only a single mutation inpenA(A501V). However, differential analysis of the genomes and transcriptomes between pre- and postselection strains revealed many other mutated genes as well as up- and downregulated genes. Transformation of the mutatedpenAgene into nonresistant strains increased the MIC between 2.0- and 5.3-fold, and transformation of mutatedftsXincreased the MIC between 3.3- and 13.3-fold. Genes encoding the ABC transporters FarB, Tfq, Hfq, and ExbB were overexpressed, whilepilM,pilN, andpilQwere downregulated. Furthermore, the resistant strain developed cross-resistance to penicillin and cefuroxime, had an increased biochemical metabolic rate, and presented fitness defects such as prolonged growth time and downregulated PilMNQ. In conclusion, antimicrobial pressure could result in the emergence of ceftriaxone resistance, and the evolution of resistance ofNeisseria gonorrhoeaeto ceftriaxone is a complicated process at both the pretranscriptional and posttranscriptional levels, involving several resistance mechanisms of increased efflux and decreased entry. PMID:26787702

  1. Construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae and its expression in E. coli.

    PubMed

    Chen, Hongxiang; Tu, Yating; Lin, Nengxing; Huang, Changzheng

    2005-01-01

    In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents and study on the resistant mechanism of multiple transferable resistance (mtr) efflux system, plasmid pET-28a(+) encoding mtrC gene was constructed and the related target protein was expressed in Escherichia coli (E. coli) DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with restriction endonuclease to construct recombinant pET-mtrC which was verified by restriction endonuclease and DNA sequencing. The recombinant was transformed into E. coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuclease and DNA sequencing. Its sequence was 99.5% homologus to that published on GeneBank (U14993). A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E. coli. PMID:16463681

  2. Control of pili and sialyltransferase expression in Neisseria gonorrhoeae is mediated by the transcriptional regulator CrgA.

    PubMed

    Matthias, Kathryn A; Rest, Richard F

    2014-03-01

    Contact-regulated gene A (CrgA) is a transcriptional regulator present in the pathogenic Neisseria that functions as both an activator and a repressor of transcription following contact with host cells. While its mechanism of action has been studied extensively in Neisseria meningitidis, the specific subset of genes that CrgA targets has been debated. Although the majority of these constitute virulence genes, suggesting that CrgA is important in pathogenesis, no study to date has examined the effects of CrgA in Neisseria gonorrhoeae. In this report, we generated a knockout mutant of crgA (ΔcrgA) in the serum-sensitive gonococcal strain F62. crgA deletion resulted in a reduction in the transcript and protein levels of the primary pilin component pilE via mechanisms that were both contact-dependent and -independent. In contrast, ΔcrgA overexpressed the main determinant of serum resistance in F62, lipooligosaccharide sialyltransferase (Lst). CrgA-mediated lst repression was direct as both recombinant and native CrgA bound to the lst promoter at multiple locations in EMSA and ChIP assays respectively. The increase in Lst levels associated with crgA deletion correlated with enhanced protection against killing by normal human serum. These data suggest a role for CrgA in virulence regulation during both cell adherence and planktonic growth. PMID:24433334

  3. Sustained reduction in the carriage of Neisseria meningitidis as a result of a community meningococcal disease control programme.

    PubMed Central

    Neal, K. R.; Irwin, D. J.; Davies, S.; Kaczmarski, E. B.; Wale, M. C.

    1998-01-01

    The effect of a community intervention programme of antibiotics and meningitis vaccine on pharyngeal carriage of Neisseria meningitidis was investigated. Carriage rates were determined in pupils at both secondary schools (ages 11-18 years) included in the community intervention programme and compared with two schools outside the area matched for socio-economic status. A total of 1869 pupils were studied 6 months after the programmes, and 2457 pupils after 11 months. Six months after the programme was completed there was a 72% reduction in pharyngeal carriage of Neisseria meningitidis in pupils attending the schools in the intervention area compared with pupils in the control schools. After 11 months this difference persisted in the 11-14 age group but not in the 15-18 age group. No resistance to the antibiotics used in the programme was found. A community intervention programme of antibiotics and vaccine for the control of meningococcal disease led to a long-term reduction in Neisseria meningitidis carriage in some age groups. PMID:10030696

  4. Control of pili and sialyltransferase expression in Neisseria gonorrhoeae is mediated by the transcriptional regulator CrgA.

    PubMed

    Matthias, Kathryn A; Rest, Richard F

    2014-03-01

    Contact-regulated gene A (CrgA) is a transcriptional regulator present in the pathogenic Neisseria that functions as both an activator and a repressor of transcription following contact with host cells. While its mechanism of action has been studied extensively in Neisseria meningitidis, the specific subset of genes that CrgA targets has been debated. Although the majority of these constitute virulence genes, suggesting that CrgA is important in pathogenesis, no study to date has examined the effects of CrgA in Neisseria gonorrhoeae. In this report, we generated a knockout mutant of crgA (ΔcrgA) in the serum-sensitive gonococcal strain F62. crgA deletion resulted in a reduction in the transcript and protein levels of the primary pilin component pilE via mechanisms that were both contact-dependent and -independent. In contrast, ΔcrgA overexpressed the main determinant of serum resistance in F62, lipooligosaccharide sialyltransferase (Lst). CrgA-mediated lst repression was direct as both recombinant and native CrgA bound to the lst promoter at multiple locations in EMSA and ChIP assays respectively. The increase in Lst levels associated with crgA deletion correlated with enhanced protection against killing by normal human serum. These data suggest a role for CrgA in virulence regulation during both cell adherence and planktonic growth.

  5. A large genomic island allows Neisseria meningitidis to utilize propionic acid, with implications for colonization of the human nasopharynx.

    PubMed

    Catenazzi, Maria Chiara E; Jones, Helen; Wallace, Iain; Clifton, Jacqueline; Chong, James P J; Jackson, Matthew A; Macdonald, Sandy; Edwards, James; Moir, James W B

    2014-07-01

    Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid-generating bacteria. Data from classical microbiological and sequence-based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp-containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.

  6. A genomic approach to bacterial taxonomy: an examination and proposed reclassification of species within the genus Neisseria.

    PubMed

    Bennett, Julia S; Jolley, Keith A; Earle, Sarah G; Corton, Craig; Bentley, Stephen D; Parkhill, Julian; Maiden, Martin C J

    2012-06-01

    In common with other bacterial taxa, members of the genus Neisseria are classified using a range of phenotypic and biochemical approaches, which are not entirely satisfactory in assigning isolates to species groups. Recently, there has been increasing interest in using nucleotide sequences for bacterial typing and taxonomy, but to date, no broadly accepted alternative to conventional methods is available. Here, the taxonomic relationships of 55 representative members of the genus Neisseria have been analysed using whole-genome sequence data. As genetic material belonging to the accessory genome is widely shared among different taxa but not present in all isolates, this analysis indexed nucleotide sequence variation within sets of genes, specifically protein-coding genes that were present and directly comparable in all isolates. Variation in these genes identified seven species groups, which were robust to the choice of genes and phylogenetic clustering methods used. The groupings were largely, but not completely, congruent with current species designations, with some minor changes in nomenclature and the reassignment of a few isolates necessary. In particular, these data showed that isolates classified as Neisseria polysaccharea are polyphyletic and probably include more than one taxonomically distinct organism. The seven groups could be reliably and rapidly generated with sequence variation within the 53 ribosomal protein subunit (rps) genes, further demonstrating that ribosomal multilocus sequence typing (rMLST) is a practicable and powerful means of characterizing bacteria at all levels, from domain to strain.

  7. Targeting of Neisserial PorB to the mitochondrial outer membrane: an insight on the evolution of β-barrel protein assembly machines.

    PubMed

    Jiang, Jhih-Hang; Davies, John K; Lithgow, Trevor; Strugnell, Richard A; Gabriel, Kipros

    2011-11-01

    Mitochondria originated from Gram-negative bacteria through endosymbiosis. In modern day mitochondria, the Sorting and Assembly Machinery (SAM) is responsible for eukaryotic β-barrel protein assembly in the mitochondrial outer membrane. The SAM is the functional equivalent of the β-barrel assembly machinery found in the outer membrane of Gram-negative bacteria. In this study we examined the import pathway of a pathogenic bacterial protein, PorB, which is targeted from pathogenic Neisseria to the host mitochondria. We have developed a new method for measurement of PorB assembly into mitochondria that relies on the mobility shift exhibited by bacterial β-barrel proteins once folded and separated under semi-native electrophoretic conditions. We show that PorB is targeted to the outer mitochondrial membrane with a dependence on the intermembrane space shuttling chaperones and the core component of the SAM, Sam50, which is a functional homologue of BamA that is required for PorB assembly in bacteria. The peripheral subunits of the SAM, Sam35 and Sam37, which are essential for eukaryotic β-barrel protein assembly but do not have distinguishable functional homologues in bacteria, are not required for PorB assembly in eukaryotes. This shows that PorB uses an evolutionary conserved 'bacterial like' mechanism to infiltrate the host mitochondrial outer membrane. PMID:22032638

  8. Mosaic-like structure of penicillin-binding protein 2 Gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime.

    PubMed

    Ameyama, Satoshi; Onodera, Shoichi; Takahata, Masahiro; Minami, Shinzaburo; Maki, Nobuko; Endo, Katsuhisa; Goto, Hirokazu; Suzuki, Hiroo; Oishi, Yukihiko

    2002-12-01

    Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 micro g/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene (penA) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 micro g/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava (N. sicca), Neisseria cinerea, Neisseria flavescens, and Neisseria meningitidis. These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae.

  9. Clonal analysis of Neisseria meningitidis serogroup B strains in South Africa, 2002 to 2006: emergence of new clone ST-4240/6688.

    PubMed

    Moodley, Chivonne; du Plessis, Mignon; Ndlangisa, Kedibone; de Gouveia, Linda; Klugman, Keith P; von Gottberg, Anne

    2012-11-01

    From August 1999 through July 2002, hyperinvasive Neisseria meningitidis serogroup B (MenB) clonal complexes (CCs), namely, ST-32/ET-5 (CC32) and ST-41/44/lineage 3 (CC41/44), were predominant in the Western Cape Province of South Africa. This study analyzed MenB invasive isolates from a national laboratory-based surveillance system that were collected from January 2002 through December 2006. Isolates were characterized by pulsed-field gel electrophoresis (PFGE) (n = 302), and multilocus sequence typing (MLST) and PorA and FetA typing were performed on randomly selected isolates (34/302, 11%). In total, 2,400 cases were reported, with the highest numbers from Gauteng Province (1,307/2,400, 54%) and Western Cape Province (393/2,400, 16%); 67% (1,617/2,400) had viable isolates and 19% (307/1,617) were identified as serogroup B. MenB incidence remained stable over time (P = 0.77) (average incidence, 0.13/100,000 population [range, 0.10 to 0.16/100,000 population]). PFGE (302/307, 98%) divided isolates (206/302, 68%) into 13 clusters and 96 outliers. The largest cluster, B1, accounted for 25% of isolates (76/302) over the study period; its prevalence decreased from 43% (20/47) in 2002 to 13% (8/62) in 2006 (P < 0.001), and it was common in the Western Cape (58/76, 76%). Clusters B2 and B3 accounted for 10% (31/302) and 6% (19/302), respectively, and showed no significant change over time and were predominant in Gauteng. Randomly selected isolates from clusters B1, B2, and B3 belonged to CC32, CC41/44, and the new CC4240/6688, respectively. Overall, 15 PorA and 12 FetA types were identified. MenB isolates were mostly diverse with no single dominant clone; however, CC32 and CC41/44 accounted for 35% and the new CC4240/6688 was the third most prevalent clone. PMID:22972827

  10. Optimization of Molecular Approaches to Genogroup Neisseria meningitidis Carriage Isolates and Implications for Monitoring the Impact of New Serogroup B Vaccines.

    PubMed

    Rojas, Eduardo; Hoyos, Johanna; Oldfield, Neil J; Lee, Philip; Flint, Mike; Jones, C Hal; Ala'Aldeen, Dlawer A A; Jansen, Kathrin U; Anderson, Annaliesa S

    2015-01-01

    The reservoir for Neisseria meningitidis (Nm) is the human oropharynx. Implementation of Nm serogroup C (NmC) glycoconjugate vaccines directly reduced NmC carriage. Prophylactic vaccines are now available to prevent disease caused by the five major Nm disease causing serogroups (ABCWY). Nm serogroup B (NmB) vaccines are composed of antigens that are conserved across Nm serogroups and therefore have the potential to impact all Nm carriage. To assess the effect of these vaccines on carriage, standardized approaches to identify and group Nm are required. Real-time PCR (rt-PCR) capsule grouping assays that were internally controlled to confirm Nm species were developed for eight serogroups associated with carriage (A, B, C, E, W, X, Y and Z). The grouping scheme was validated using diverse bacterial species associated with carriage and then used to evaluate a collection of diverse Nm carriage isolates (n=234). A scheme that also included porA and ctrA probes was able to speciate the isolates, while ctrA also provided insights on the integrity of the polysaccharide loci. Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen. The porA rt-PCR yielded positive results with all 234 of the Nm carriage isolates. Genogrouping assays classified 76.5% (179/234) of these isolates to a group, categorized 53 as nongenogroupable (NGG) and two as mixed results. Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result. Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide. These rt-PCR assays provide a comprehensive means to identify and genogroup N. meningitidis in carriage studies used to guide vaccination strategies and to assess the impact of novel fHbp containing vaccines on meningococcal carriage. PMID:26147212

  11. Optimization of Molecular Approaches to Genogroup Neisseria meningitidis Carriage Isolates and Implications for Monitoring the Impact of New Serogroup B Vaccines

    PubMed Central

    Rojas, Eduardo; Hoyos, Johanna; Oldfield, Neil J.; Lee, Philip; Flint, Mike; Jones, C. Hal; Ala’Aldeen, Dlawer A. A.; Jansen, Kathrin U.; Anderson, Annaliesa S.

    2015-01-01

    The reservoir for Neisseria meningitidis (Nm) is the human oropharynx. Implementation of Nm serogroup C (NmC) glycoconjugate vaccines directly reduced NmC carriage. Prophylactic vaccines are now available to prevent disease caused by the five major Nm disease causing serogroups (ABCWY). Nm serogroup B (NmB) vaccines are composed of antigens that are conserved across Nm serogroups and therefore have the potential to impact all Nm carriage. To assess the effect of these vaccines on carriage, standardized approaches to identify and group Nm are required. Real-time PCR (rt-PCR) capsule grouping assays that were internally controlled to confirm Nm species were developed for eight serogroups associated with carriage (A, B, C, E, W, X, Y and Z). The grouping scheme was validated using diverse bacterial species associated with carriage and then used to evaluate a collection of diverse Nm carriage isolates (n=234). A scheme that also included porA and ctrA probes was able to speciate the isolates, while ctrA also provided insights on the integrity of the polysaccharide loci. Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen. The porA rt-PCR yielded positive results with all 234 of the Nm carriage isolates. Genogrouping assays classified 76.5% (179/234) of these isolates to a group, categorized 53 as nongenogroupable (NGG) and two as mixed results. Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result. Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide. These rt-PCR assays provide a comprehensive means to identify and genogroup N. meningitidis in carriage studies used to guide vaccination strategies and to assess the impact of novel fHbp containing vaccines on meningococcal carriage. PMID:26147212

  12. Optimization of Molecular Approaches to Genogroup Neisseria meningitidis Carriage Isolates and Implications for Monitoring the Impact of New Serogroup B Vaccines.

    PubMed

    Rojas, Eduardo; Hoyos, Johanna; Oldfield, Neil J; Lee, Philip; Flint, Mike; Jones, C Hal; Ala'Aldeen, Dlawer A A; Jansen, Kathrin U; Anderson, Annaliesa S

    2015-01-01

    The reservoir for Neisseria meningitidis (Nm) is the human oropharynx. Implementation of Nm serogroup C (NmC) glycoconjugate vaccines directly reduced NmC carriage. Prophylactic vaccines are now available to prevent disease caused by the five major Nm disease causing serogroups (ABCWY). Nm serogroup B (NmB) vaccines are composed of antigens that are conserved across Nm serogroups and therefore have the potential to impact all Nm carriage. To assess the effect of these vaccines on carriage, standardized approaches to identify and group Nm are required. Real-time PCR (rt-PCR) capsule grouping assays that were internally controlled to confirm Nm species were developed for eight serogroups associated with carriage (A, B, C, E, W, X, Y and Z). The grouping scheme was validated using diverse bacterial species associated with carriage and then used to evaluate a collection of diverse Nm carriage isolates (n=234). A scheme that also included porA and ctrA probes was able to speciate the isolates, while ctrA also provided insights on the integrity of the polysaccharide loci. Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen. The porA rt-PCR yielded positive results with all 234 of the Nm carriage isolates. Genogrouping assays classified 76.5% (179/234) of these isolates to a group, categorized 53 as nongenogroupable (NGG) and two as mixed results. Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result. Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide. These rt-PCR assays provide a comprehensive means to identify and genogroup N. meningitidis in carriage studies used to guide vaccination strategies and to assess the impact of novel fHbp containing vaccines on meningococcal carriage.

  13. Recommendations for the Laboratory-Based Detection of Chlamydia trachomatis and Neisseria gonorrhoeae — 2014

    PubMed Central

    Papp, John R.; Schachter, Julius; Gaydos, Charlotte A.; Van Der Pol, Barbara

    2014-01-01

    Summary This report updates CDC's 2002 recommendations regarding screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections (CDC. Screening tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae infections—2002. MMWR 2002;51[No. RR-15]) and provides new recommendations regarding optimal specimen types, the use of tests to detect rectal and oropharyngeal C. trachomatis and N. gonorrhoeae infections, and circumstances when supplemental testing is indicated. The recommendations in this report are intended for use by clinical laboratory directors, laboratory staff, clinicians, and disease control personnel who must choose among the multiple available tests, establish standard operating procedures for collecting and processing specimens, interpret test results for laboratory reporting, and counsel and treat patients. The performance of nucleic acid amplification tests (NAATs) with respect to overall sensitivity, specificity, and ease of specimen transport is better than that of any of the other tests available for the diagnosis of chlamydial and gonococcal infections. Laboratories should use NAATs to detect chlamydia and gonorrhea except in cases of child sexual assault involving boys and rectal and oropharyngeal infections in prepubescent girls and when evaluating a potential gonorrhea treatment failure, in which case culture and susceptibility testing might be required. NAATs that have been cleared by the Food and Drug Administration (FDA) for the detection of C. trachomatis and N. gonorrhoeae infections are recommended as screening or diagnostic tests because they have been evaluated in patients with and without symptoms. Maintaining the capability to culture for both N. gonorrhoeae and C. trachomatis in laboratories throughout the country is important because data are insufficient to recommend nonculture tests in cases of sexual assault in prepubescent boys and extragenital anatomic site exposure in prepubescent girls. N

  14. Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection.

    PubMed

    Melendez, Johan H; Santaus, Tonya M; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A; Geddes, Chris D

    2016-10-01

    Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

  15. Anaerobiosis increases resistance of Neisseria gonorrhoeae to O2-independent antimicrobial proteins from human polymorphonuclear granulocytes.

    PubMed Central

    Casey, S G; Shafer, W M; Spitznagel, J K

    1985-01-01

    We investigated the in vitro resistance of Neisseria gonorrhoeae FA19 to the O2-independent antimicrobial systems of human polymorphonuclear leukocytes. Acid extracts of polymorphonuclear leukocyte granules (crude granule extracts) and a purified granule protein (57 kilodaltons) were, at low concentrations, bactericidal for gonococci under aerobic conditions that permitted growth. However, they were less effective under anaerobic conditions that imposed bacteriostasis. We found that adding sodium nitrite to reduced growth media permitted the growth of strain FA19 in an anaerobic environment. Under these conditions with nitrite, anaerobic cultures of strain FA19 were no more resistant to the crude granule extract and the 57-kilodalton protein than aerobic cultures. In contrast, Salmonella typhimurium SL-1004, a facultative anaerobe, was readily killed by both the crude granule extract and the 57-kilodalton antimicrobial protein regardless of the presence or absence of free molecular oxygen. This is the first demonstration that an isolated antimicrobial protein from polymorphonuclear leukocyte granules is active against bacteria under anaerobic conditions. Our results also indicated that the efficacy of human polymorphonuclear leukocyte O2-independent killing of N. gonorrhoeae may, in part, be inhibited by bacteriostatic conditions imposed by hypoxia. Images PMID:3917976

  16. Relative penicillin G resistance in Neisseria meningitidis and reduced affinity of penicillin-binding protein 3.

    PubMed Central

    Mendelman, P M; Campos, J; Chaffin, D O; Serfass, D A; Smith, A L; Sáez-Nieto, J A

    1988-01-01

    We examined clinical isolates of Neisseria meningitidis relatively resistant to penicillin G (mean MIC, 0.3 micrograms/ml; range, 0.1 to 0.7 micrograms/ml), which were isolated from blood and cerebrospinal fluid for resistance mechanisms, by using susceptible isolates (mean MIC, less than or equal to 0.06 micrograms/ml) for comparison. The resistant strains did not produce detectable beta-lactamase activity, otherwise modify penicillin G, or bind less total penicillin. Penicillin-binding protein (PBP) 3 of the six resistant isolates tested uniformly bound less penicillin G in comparison to the same PBP of four susceptible isolates. Reflecting the reduced binding affinity of PBP 3 of the two resistant strains tested, the amount of 3H-labeled penicillin G required for half-maximal binding was increased in comparison with that of PBP 3 of the two susceptible isolates. We conclude that the mechanism of resistance in these meningococci relatively resistant to penicillin G was decreased affinity of PBP 3. Images PMID:3134848

  17. Dynamics of the Murine Humoral Immune Response to Neisseria meningitidis Group B Capsular Polysaccharide

    PubMed Central

    Colino, Jesús; Outschoorn, Ingrid

    1998-01-01

    Immunization with Neisseria meningitidis group B capsular polysaccharide (CpsB) elicited responses in adult mice that showed the typical dynamic characteristics of the response to a thymus-independent antigen, in contrast to the thymus-dependent behavior of antibody responses to CpsC. The former had a short latent period and showed a rapid increase in serum antibodies that peaked at day 5, and immunoglobulin M (IgM) was the major isotype even though IgG (mainly IgG2a and IgG2b) was also detectable. This response was of short duration, and the specific antibodies were rapidly cleared from the circulation. The secondary responses were similar in magnitude, kinetics, IgM predominance, and IgG distribution. Nevertheless, a threefold IgG increase, a correlation between IgM and IgG levels, and dose-dependent secondary responses were observed. Hyperimmunization considerably reinforced these responses: 10-fold for IgM and 300-fold for IgG. This favored isotype switch was accompanied by a progressive change in the subclass distribution to IgG3 (62%) and IgG1 (28%), along with the possible generation of B-cell memory. The results indicate that CpsB is being strictly thymus independent and suggest that unresponsiveness to purified CpsB is due to tolerance. PMID:9453603

  18. Mannose-binding lectin enhances phagocytosis and killing of Neisseria meningitidis by human macrophages.

    PubMed

    Jack, Dominic L; Lee, Margaret E; Turner, Malcolm W; Klein, Nigel J; Read, Robert C

    2005-03-01

    Deficiency of mannose-binding lectin (MBL) is probably the most common human immunodeficiency and is associated with an increased risk of mucosally acquired infections including meningococcal disease. Tissue macrophages are an important component of mucosal defense, and so we determined the effect of MBL on uptake of meningococci by human monocyte-derived macrophages. Opsonization with MBL significantly increased the capture and doubled the amount of internalization of Neisseria meningitidis. Inhibition of f-actin polymerization indicated that MBL exerted this effect by a dose-dependent acceleration of uptake into phagosomes, which was maximal within the normal physiological concentration of MBL (1.5 microg/ml) and was independent of scavenger receptors. MBL accelerated the acquisition and subsequent loss of the early endosome marker, early endosomal antigen-1, and enhanced the acquisition of the late endosomal marker, lysosome-associated membrane protein-1. MBL reduced the survival of meningococci within macrophages by more than half, despite the increased uptake of organisms, and significantly reduced the number of viable extracellular bacteria by 80%. We conclude that MBL is a dependent opsonin able to accelerate microbial uptake and killing. These results suggest that MBL could modify disease susceptibility by modulating macrophage interactions with mucosal organisms at the site of initial acquisition.

  19. A Systematic Review of Point of Care Testing for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.

    PubMed

    Herbst de Cortina, Sasha; Bristow, Claire C; Joseph Davey, Dvora; Klausner, Jeffrey D

    2016-01-01

    Objectives. Systematic review of point of care (POC) diagnostic tests for sexually transmitted infections: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Methods. Literature search on PubMed for articles from January 2010 to August 2015, including original research in English on POC diagnostics for sexually transmitted CT, NG, and/or TV. Results. We identified 33 publications with original research on POC diagnostics for CT, NG, and/or TV. Thirteen articles evaluated test performance, yielding at least one test for each infection with sensitivity and specificity ≥90%. Each infection also had currently available tests with sensitivities <60%. Three articles analyzed cost effectiveness, and five publications discussed acceptability and feasibility. POC testing was acceptable to both providers and patients and was also demonstrated to be cost effective. Fourteen proof of concept articles introduced new tests. Conclusions. Highly sensitive and specific POC tests are available for CT, NG, and TV, but improvement is possible. Future research should focus on acceptability, feasibility, and cost of POC testing. While pregnant women specifically have not been studied, the results available in nonpregnant populations are encouraging for the ability to test and treat women in antenatal care to prevent adverse pregnancy and neonatal outcomes. PMID:27313440

  20. Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood–brain barrier

    PubMed Central

    Mairey, Emilie; Genovesio, Auguste; Donnadieu, Emmanuel; Bernard, Christine; Jaubert, Francis; Pinard, Elisabeth; Seylaz, Jacques; Olivo-Marin, Jean-Christophe; Nassif, Xavier; Duménil, Guillaume

    2006-01-01

    Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, this bacterium reaches the bloodstream and causes meningitis after crossing the blood–brain barrier by an unknown mechanism. An immunohistological study of a meningococcal sepsis case revealed that neisserial adhesion was restricted to capillaries located in low blood flow regions in the infected organs. This study led to the hypothesis that drag forces encountered by the meningococcus in the bloodstream determine its attachment site in vessels. We therefore investigated the ability of N. meningitidis to bind to endothelial cells in the presence of liquid flow mimicking the bloodstream with a laminar flow chamber. Strikingly, average blood flows reported for various organs strongly inhibited initial adhesion. As cerebral microcirculation is known to be highly heterogeneous, cerebral blood velocity was investigated at the level of individual vessels using intravital imaging of rat brain. In agreement with the histological study, shear stress levels compatible with meningococcal adhesion were only observed in capillaries, which exhibited transient reductions in flow. The flow chamber assay revealed that, after initial attachment, bacteria resisted high blood velocities and even multiplied, forming microcolonies resembling those observed in the septicemia case. These results argue that the combined mechanical properties of neisserial adhesion and blood microcirculation target meningococci to transiently underperfused cerebral capillaries and thus determine disease development. PMID:16864659

  1. Release of enzymes from human leucocytes during incubation with Neisseria gonorrhoeae

    PubMed Central

    Senff, Leah Morford; Sawyer, William D.

    1977-01-01

    The effect of Neisseria gonorrhoeae on release of enzymes from human leucocytes was determined. Supernatants from incubation mixtures containing leucocytes and gonococci were assayed for activity of the cytoplasmic enzyme, lactic acid dehydrogenase, as well as for activity of the hydrolytic enzymes, β-glucuronidase and lysozyme, which are found primarily in leucocyte granules. Thirty-minute incubation of leucocytes with pilated T1 gonococci resulted in a negligible release of lactic acid dehydrogenase and little release of β-glucuronidase even at bacteria to leucocyte ratios as high as 50 to 1. Lysozyme release, however, was significant at this ratio and at 20 to 1 but not at 5 to 1. Incubation with non-pilated T4 bacteria yielded no significant release of lactic acid dehydrogenase or β-glucuronidase, but it caused a significant release of lysozyme at bacteria to leucocyte ratios as low as 2 to 1. These results suggested that the lysozyme release might be related to the degree of phagocytic activity since, at low ratios, T4 was readily ingested but T1 was not. Consistent with this hypothesis, serum which promoted the phagocytosis of the pilated gonococci also stimulated lysozyme release at low ratios of T1 to leucocyte. Absorption of the serum with T1 abolished the opsonic effect and markedly diminished the amount of lysozyme released. PMID:414817

  2. Release of enzymes from human leucocytes during incubation with Neisseria gonorrhoeae.

    PubMed

    Senff, L M; Sawyer, W D

    1977-12-01

    The effect of Neisseria gonorrhoeae on release of enzymes from human leucocytes was determined. Supernatants from incubation mixtures containing leucocytes and gonococci were assayed for activity of the cytoplasmic enzyme, lactic acid dehydrogenase, as well as for activity of the hydrolytic enzymes, β-glucuronidase and lysozyme, which are found primarily in leucocyte granules. Thirty-minute incubation of leucocytes with pilated T1 gonococci resulted in a negligible release of lactic acid dehydrogenase and little release of β-glucuronidase even at bacteria to leucocyte ratios as high as 50 to 1. Lysozyme release, however, was significant at this ratio and at 20 to 1 but not at 5 to 1. Incubation with non-pilated T4 bacteria yielded no significant release of lactic acid dehydrogenase or β-glucuronidase, but it caused a significant release of lysozyme at bacteria to leucocyte ratios as low as 2 to 1. These results suggested that the lysozyme release might be related to the degree of phagocytic activity since, at low ratios, T4 was readily ingested but T1 was not. Consistent with this hypothesis, serum which promoted the phagocytosis of the pilated gonococci also stimulated lysozyme release at low ratios of T1 to leucocyte. Absorption of the serum with T1 abolished the opsonic effect and markedly diminished the amount of lysozyme released.

  3. Olfactory nerve--a novel invasion route of Neisseria meningitidis to reach the meninges.

    PubMed

    Sjölinder, Hong; Jonsson, Ann-Beth

    2010-11-18

    Neisseria meningitidis is a human-specific pathogen with capacity to cause septic shock and meningitis. It has been hypothesized that invasion of the central nervous system (CNS) is a complication of a bacteremic condition. In this study, we aimed to characterize the invasion route of N. meningitidis to the CNS. Using an intranasally challenged mouse disease model, we found that twenty percent of the mice developed lethal meningitis even though no bacteria could be detected in blood. Upon bacterial infection, epithelial lesions and redistribution of intracellular junction protein N-cadherin were observed at the nasal epithelial mucosa, especially at the olfactory epithelium, which is functionally and anatomically connected to the CNS. Bacteria were detected in the submucosa of the olfactory epithelium, along olfactory nerves in the cribriform plate, at the olfactory bulb and subsequently at the meninges and subarachnoid space. Furthermore, our data suggest that a threshold level of bacteremia is required for the development of meningococcal sepsis. Taken together, N. meningitidis is able to pass directly from nasopharynx to meninges through the olfactory nerve system. This study enhances our understanding how N. meningitidis invades the meninges. The nasal olfactory nerve system may be a novel target for disease prevention that can improve outcome and survival.

  4. Refolding, purification and crystallization of the FrpB outer membrane iron transporter from Neisseria meningitidis.

    PubMed

    Saleem, Muhammad; Prince, Stephen M; Patel, Hema; Chan, Hannah; Feavers, Ian M; Derrick, Jeremy P

    2012-02-01

    FrpB is an integral outer membrane protein from the human pathogen Neisseria meningitidis. It is a member of the TonB-dependent transporter family and promotes the uptake of iron across the outer membrane. There is also evidence that FrpB is an antigen and hence a potential component of a vaccine against meningococcal meningitis. FrpB incorporating a polyhistidine tag was overexpressed in Escherichia coli into inclusion bodies. The protein was then solubilized in urea, refolded and purified to homogeneity. Two separate antigenic variants of FrpB were crystallized by sitting-drop vapour diffusion. Crystals of the F5-1 variant diffracted to 2.4 Å resolution and belonged to space group C2, with unit-cell parameters a = 176.5, b = 79.4, c = 75.9 Å, β = 98.3°. Crystal-packing calculations suggested the presence of a monomer in the asymmetric unit. Crystals of the F3-3 variant also diffracted to 2.4 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 85.3, b = 104.6, c = 269.1 Å. Preliminary analysis suggested the presence of an FrpB trimer in the asymmetric unit.

  5. A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis

    PubMed Central

    Li, Yanwen; Pelicic, Vladimir; Freemont, Paul S.; Baldwin, Geoff S.; Tang, Christoph M.

    2013-01-01

    Although oxidative stress is a key aspect of innate immunity, little is known about how host-restricted pathogens successfully repair DNA damage. Base excision repair (BER) is responsible for correcting nucleobases damaged by oxidative stress, and is essential for bloodstream infection caused by the human pathogen, Neisseria meningitidis. We have characterised meningococcal BER enzymes involved in the recognition and removal of damaged nucleobases, and incision of the DNA backbone. We demonstrate that the bi-functional glycosylase/lyases Nth and MutM share several overlapping activities and functional redundancy. However MutM and other members of the GO system, which deal with 8-oxoG, a common lesion of oxidative damage, are not required for survival of N. meningitidis under oxidative stress. Instead, the mismatch repair pathway provides back-up for the GO system, while the lyase activity of Nth can substitute for the meningococcal AP endonuclease, NApe. Our genetic and biochemical evidence show that DNA repair is achieved through a robust network of enzymes that provides a flexible system of DNA repair. This network is likely to reflect successful adaptation to the human nasopharynx, and might provide a paradigm for DNA repair in other prokaryotes. PMID:22296581

  6. Detection and serogroup determination of Neisseria meningitidis in CSF by polymerase chain reaction (PCR).

    PubMed

    Porritt, R J; Mercer, J L; Munro, R

    2000-02-01

    A PCR protocol for the detection and serogroup determination of Neisseria meningitidis in CSF from 85 cases of suspected meningitis was evaluated. Screening assays for both IS1106 and the ctrA gene were used to detect meningococcal DNA, and a further two assays using the siaD gene were performed to determine the serogroup. PCR results were compared with results of bacteriological culture and discrepant results resolved by analysis of clinical data and further laboratory test results. The resolved sensitivity and specificity of the PCR screening assay were 89 and 100%, and those of bacteriological culture were 37 and 100%, respectively. The siaD B/C PCR assay was able to determine a serogroup in 85% of cases positive by the PCR screening assay compared with 50% of cases where a serogroup was determined by traditional methods. PCR is a useful tool for diagnosis of meningococcal meningitis when Gram stain and culture tests are negative, a situation that may arise when antibiotic treatment has commenced prior to lumbar puncture.

  7. A vaccine carrier derived from Neisseria meningitidis with mitogenic activity for lymphocytes.

    PubMed Central

    Liu, M A; Friedman, A; Oliff, A I; Tai, J; Martinez, D; Deck, R R; Shieh, J T; Jenkins, T D; Donnelly, J J; Hawe, L A

    1992-01-01

    Protein carriers vary in their ability to increase the immunogenicity of poorly immunogenic or T-lymphocyte-independent antigens. We examined one such carrier, the outer membrane protein complex derived from Neisseria meningitidis serogroup B strain B11, in an attempt to determine why this outer membrane protein complex was more immunogenic in young infants and in relevant animal models than two other carriers used in conjugates made with Haemophilus influenzae type b polysaccharide, a T-cell-independent antigen. A single protein of the outer membrane protein complex, the class 2 porin protein, was purified and shown to function as a T-helper lymphocyte carrier protein. Unexpectedly, it was also found to have mitogenic activity for lymphocytes that was not due to lipopolysaccharide. This mitogenic activity appears to date to be unique to this carrier protein of the carrier proteins tested and may contribute to the ability of the H. influenzae type b conjugate vaccine made with the outer membrane protein complex to generate IgG anti-polysaccharide antibody responses in mice and infant monkeys and protective immune responses in infants less than 6 months of age. Images PMID:1533934

  8. Phosphoethanolamine Modification of Neisseria gonorrhoeae Lipid A Reduces Autophagy Flux in Macrophages.

    PubMed

    Zughaier, Susu M; Kandler, Justin L; Balthazar, Jacqueline T; Shafer, William M

    2015-01-01

    Autophagy, an ancient homeostasis mechanism for macromolecule degradation, performs an important role in host defense by facilitating pathogen elimination. To counteract this host defense strategy, bacterial pathogens have evolved a variety of mechanisms to avoid or otherwise dysregulate autophagy by phagocytic cells so as to enhance their survival during infection. Neisseria gonorrhoeae is a strictly human pathogen that causes the sexually transmitted infection, gonorrhea. Phosphoethanolamine (PEA) addition to the 4' position of the lipid A (PEA-lipid A) moiety of the lipooligosaccharide (LOS) produced by gonococci performs a critical role in this pathogen's ability to evade innate defenses by conferring decreased susceptibility to cationic antimicrobial (or host-defense) peptides, complement-mediated killing by human serum and intraleukocytic killing by human neutrophils compared to strains lacking this PEA decoration. Heretofore, however, it was not known if gonococci can evade autophagy and if so, whether PEA-lipid A contributes to this ability. Accordingly, by using murine macrophages and human macrophage-like phagocytic cell lines we investigated if PEA decoration of gonococcal lipid A modulates autophagy formation. We report that infection with PEA-lipid A-producing gonococci significantly reduced autophagy flux in murine and human macrophages and enhanced gonococcal survival during their association with macrophages compared to a PEA-deficient lipid A mutant. Our results provide further evidence that PEA-lipid A produced by gonococci is a critical component in the ability of this human pathogen to evade host defenses. PMID:26641098

  9. Neisseria gonorrhoeae suppresses dendritic cell-induced, antigen-dependent CD4 T cell proliferation.

    PubMed

    Zhu, Weiyan; Ventevogel, Melissa S; Knilans, Kayla J; Anderson, James E; Oldach, Laurel M; McKinnon, Karen P; Hobbs, Marcia M; Sempowski, Gregory D; Duncan, Joseph A

    2012-01-01

    Neisseria gonorrhoeae is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with N. gonorrhoeae cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to N. gonorrhoeae. N. gonorrhoeae is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. N. gonorrhoeae has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that N. gonorrhoeae also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that N. gonorrhoeae-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. N. gonorrhoeae exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that N. gonorrhoeae is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, N. gonorrhoeae also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen.

  10. Complement-enhanced immunity to infection with Neisseria gonorrhoeae in mice.

    PubMed

    Arko, R J; Wong, K H; Steurer, F J; Schalla, W O

    1979-05-01

    Subcutaneous chambers were implanted in mice, injected with Neisseria gonorrhoeae, and supplemented with complement as a model for studying the immunogenicity and strain diversity of N. gonorrhoeae. Immunotypic resistance to N. gonorrhoeae in immunized mice was significantly (P less than 0.01) increased by injection of exogenous guinea pig complement into the host before challenge with gonococci. By using this model to test gonococcal isolates from various geographical areas, two highly immunogenic but immunotypically different gonococcal strains were identified. The piliated cells of these strains induced both complement-enhanced immunity and a degree of exogenous complement-independent immunity. The immunity in mice not treated with complement developed more slowly, was less effective, and waned earlier than that which was complement-dependent. Pretreatment with complement, although highly effective in preventing infection in immunized mice, was much less beneficial in terminating already established infections, even though bactericidal antibodies were present at the time of complement treatment. The mouse chamber model in which both complement-mediated and complement-independent mechanisms of protection can be evaluated may provide an additional tool for elucidating the immunology of gonococcal or other microbial infections.

  11. Estradiol-Treated Female Mice as Surrogate Hosts for Neisseria gonorrhoeae Genital Tract Infections.

    PubMed

    Jerse, Ann E; Wu, Hong; Packiam, Mathanraj; Vonck, Rachel A; Begum, Afrin A; Garvin, Lotisha E

    2011-01-01

    Historically, animal modeling of gonorrhea has been hampered by the exclusive adaptation of Neisseria gonorrhoeae to humans. Genital tract infection can be established in female mice that are treated with 17β-estradiol, however, and many features of experimental murine infection mimic human infection. Here we review the colonization kinetics and host response to experimental murine gonococcal infection, including mouse strain differences and evidence that IL-17 responses, toll-like receptor 4, and T regulatory cells play a role in infection. We also discuss the strengths and limitations of the mouse system and the potential of transgenic mice to circumvent host restrictions. Additionally, we review studies with genetically defined mutants that demonstrated a role for sialyltransferase and the MtrC-MtrD-MtrE active efflux pump in evading innate defenses in vivo, but not for factors hypothesized to protect against the phagocytic respiratory burst and H(2)O(2)-producing lactobacilli. Studies using estradiol-treated mice have also revealed the existence of non-host-restricted iron sources in the female genital tract and the influence of hormonal factors on colonization kinetics and selection for opacity (Opa) protein expression. Recent work by others with estradiol-treated mice that are transgenic for human carcinoembryonic adhesion molecules (CEACAMs) supports a role for Opa proteins in enhancing cellular attachment and thus reduced shedding of N. gonorrhoeae. Finally we discuss the use of the mouse model in product testing and a recently developed gonorrhea chlamydia coinfection model.

  12. Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells.

    PubMed

    Nassif, X; Lowy, J; Stenberg, P; O'Gaora, P; Ganji, A; So, M

    1993-05-01

    Pili have been shown to play an essential role in the adhesion of Neisseria meningitidis to epithelial cells. However, among piliated strains, both inter- and intrastrain variability exist with respect to their degree of adhesion to epithelial cells in vitro (Virji et al., 1992). This suggests that factors other than the presence of pili per se are involved in this process. The N. meningitidis pilin subunit undergoes extensive antigenic variation. Piliated low- and high-adhesive derivatives of the same N. meningitidis strain were selected and the nucleotide sequence of the pilin gene expressed in each was determined. The highly adhesive derivatives had the same pilin sequence. The alleles encoding the pilin subunit of the low-adhesive derivatives were completely different from the one found in the high-adhesive isolates. Using polyclonal antibodies raised against one hyperadhesive variant, it was confirmed that the low-adhesive piliated derivatives expressed pilin variants antigenically different from the highly adhesive strains. The role of antigenic variation in the adhesive process of N. meningitidis was confirmed by performing allelic exchanges of the pilE locus between low- and high-adhesive isolates. Antigenic variation has been considered a means by which virulent bacteria evade the host immune system. This work provides genetic proof that a bacterial pathogen, N. meningitidis, can use antigenic variation to modulate their degree of virulence.

  13. Neisseria gonorrhoeae induces a tolerogenic phenotype in macrophages to modulate host immunity.

    PubMed

    Escobar, Alejandro; Candia, Enzo; Reyes-Cerpa, Sebastian; Villegas-Valdes, Bélgica; Neira, Tanya; Lopez, Mercedes; Maisey, Kevin; Tempio, Fabián; Ríos, Miguel; Acuña-Castillo, Claudio; Imarai, Mónica

    2013-01-01

    Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world. N. gonorrhoeae does not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. N. gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whether N. gonorrhoeae directly conditions the phenotype of RAW 264.7 murine macrophage cell line and its response. We established that gonococcus was effectively phagocytosed by the RAW 264.7 cells and upregulates production of immunoregulatory cytokines (IL-10 and TGF- β 1) but not the production of proinflammatory cytokine TNF- α , indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover, N. gonorrhoeae did not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed that N. gonorrhoeae infected macrophage cell line fails to elicit proliferative CD4+ response. This implies that macrophage that can phagocytose gonococcus do not display proper antigen-presenting functions. These results indicate that N. gonorrhoeae induces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response.

  14. Antibiotic resistance in Neisseria gonorrhoeae: origin, evolution, and lessons learned for the future.

    PubMed

    Unemo, Magnus; Shafer, William M

    2011-08-01

    The strict human pathogen Neisseria gonorrhoeae has caused gonorrhea for thousands of years, and currently gonorrhea is the second most prevalent bacterial sexually transmitted infection worldwide. Given the ancient nature of N. gonorrhoeae and its unique obligate relationship with humankind over the millennia, its remarkable ability to adapt to the host immune system and cause repeated infections, and its propensity to develop resistance to all clinically useful antibiotics, the gonococcus is an ideal pathogen on which to study the evolution of bacterial pathogenesis, including antimicrobial resistance, over the long term and within the host during infection. Recently, the first gonococcus displaying high-level resistance to ceftriaxone, identified in Japan, was characterized in detail. Ceftriaxone is the last remaining option for empirical first-line treatment, and N. gonorrhoeae now seems to be evolving into a true "superbug." In the near future, gonorrhea may become untreatable in certain circumstances. Herein, the history of antibiotics used for treatment of gonorrhea, the evolution of resistance emergence in N. gonorrhoeae, the linkage between resistance and biological fitness of N. gonorrhoeae, lessons learned, and future perspectives are reviewed and discussed.

  15. Spread of a chromosomal cefixime-resistant penA gene among different Neisseria gonorrhoeae lineages.

    PubMed

    Ohnishi, Makoto; Watanabe, Yuko; Ono, Emi; Takahashi, Chieko; Oya, Hitomi; Kuroki, Toshiro; Shimuta, Ken; Okazaki, Norio; Nakayama, Shu-ichi; Watanabe, Haruo

    2010-03-01

    In Neisseria gonorrhoeae, the mosaic type of penA, which encodes penicillin-binding protein 2 (PBP 2), is associated with reduced susceptibility to oral cephalosporins. To investigate the relatedness of N. gonorrhoeae clinical isolates with reduced susceptibility, we sequenced the penA genes of 32 isolates. Five different amino acid sequence types of PBP 2 were identified, but all seemed to be derivatives of pattern X of PBP 2 (PBP 2-X). However, multilocus sequence typing of the isolates showed that the isolates belonged to six different sequence types. As PBP 2-X was identified in three different sequence types, horizontal transfer of the penA allele encoding PBP2-X was suggested. We demonstrated that the penA gene could be transferred from an isolate with reduced susceptibility to a sensitive isolate by natural transformation. Comparison of the sequence of the penA-flanking regions of 12 transformants with those of the donor and the recipient suggested that at least a 4-kb DNA segment, including the penA gene, was transferred. During horizontal transfer, some of the penA alleles also acquired variations due to point mutations and genetic exchange within the allele. Our results provide evidence that the capacity for natural transformation in N. gonorrhoeae plays a role in the spread of chromosomal antibiotic resistance genes and the generation of diversity in such genes.

  16. Neisserial Heparin Binding Antigen (NHBA) Contributes to the Adhesion of Neisseria meningitidis to Human Epithelial Cells

    PubMed Central

    Vacca, Irene; Del Tordello, Elena; Gasperini, Gianmarco; Pezzicoli, Alfredo; Di Fede, Martina; Rossi Paccani, Silvia; Marchi, Sara; Mubaiwa, Tsisti D.; Hartley-Tassell, Lauren E.; Jennings, Michael P.; Seib, Kate L.; Masignani, Vega; Pizza, Mariagrazia; Serruto, Davide; Aricò, Beatrice; Delany, Isabel

    2016-01-01

    Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by Neisseria meningitidis strains and an antigen of the Bexsero® vaccine. NHBA binds heparin through a conserved Arg-rich region that is the target of two proteases, the meningococcal NalP and human lactoferrin (hLf). In this work, in vitro studies showed that recombinant NHBA protein was able to bind epithelial cells and mutations of the Arg-rich tract abrogated this binding. All N-terminal and C-terminal fragments generated by NalP or hLf cleavage, regardless of the presence or absence of the Arg-rich region, did not bind to cells, indicating that a correct positioning of the Arg-rich region within the full length protein is crucial. Moreover, binding was abolished when cells were treated with heparinase III, suggesting that this interaction is mediated by heparan sulfate proteoglycans (HSPGs). N. meningitidis nhba knockout strains showed a significant reduction in adhesion to epithelial cells with respect to isogenic wild-type strains and adhesion of the wild-type strain was inhibited by anti-NHBA antibodies in a dose-dependent manner. Overall, the results demonstrate that NHBA contributes to meningococcal adhesion to epithelial cells through binding to HSPGs and suggest a possible role of anti-Bexsero® antibodies in the prevention of colonization. PMID:27780200

  17. Structural characterization of the lipid A component of pathogenic Neisseria meningitidis.

    PubMed Central

    Kulshin, V A; Zähringer, U; Lindner, B; Frasch, C E; Tsai, C M; Dmitriev, B A; Rietschel, E T

    1992-01-01

    The lipid A component of meningococcal lipopolysaccharide was structurally characterized by using chemical modification methods, methylation analysis, 31P nuclear magnetic resonance, and laser desorption mass spectroscopy. It was shown that Neisseria meningitidis lipid A consists of a 1,4'-bisphosphorylated beta(1'----6)-linked D-glucosamine disaccharide (lipid A backbone), both phosphate groups being largely replaced by O-phosphorylethanolamine. This disaccharide harbors two nonsubstituted hydroxyl groups at positions 4 and 6', the latter representing the attachment site of the oligosaccharide portion in lipopolysaccharide. In addition, it is substituted by up to six fatty acid residues. In the major lipid A component, representing a hexaacyl species, the hydroxyl groups at positions 3 and 3' carry (R)-3-hydroxydodecanoic acid [12:0(3-OH)], whereas the amino groups at positions 2 and 2' are substituted by (R)-3-(dodecanoyloxy)tetradecanoic acid [3-O(12:0)-14:0]. A minor portion was present as a tetraacyl lipid A component lacking either dodecanoic acid (12:0) or 12:0 and 12:0(3-OH). N. meningitidis lipid A, therefore, significantly differs from Escherichia coli lipid A by the nature and locations of fatty acids and the substitution of O-phosphorylethanolamine for the nonglycosyl (4'-P) and glycosyl phosphate. PMID:1548229

  18. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae

    PubMed Central

    Gong, Zheng; Tang, M. Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A.; Fan, Huizhou

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis. PMID:26808268

  19. Polyamines Can Increase Resistance of Neisseria gonorrhoeae to Mediators of the Innate Human Host Defense ▿

    PubMed Central

    Goytia, Maira; Shafer, William M.

    2010-01-01

    Polyamines are biogenic polycationic molecules involved in key cellular functions. Extracellular polyamines found in bodily fluids or laboratory media can be imported by bacteria or bind to negatively charged bacterial surface structures, where they can impair binding of antimicrobials. We hypothesized that the presence of polyamines in fluids that bathe urogenital mucosal surfaces could alter the susceptibility of the sexually transmitted strict human pathogen Neisseria gonorrhoeae to mediators of the innate host defense. Herein we report that polyamines can significantly increase gonococcal resistance to two structurally diverse cationic antimicrobial peptides (polymyxin B and LL-37) but not to antibiotics that exert activity in the cytosol or periplasm (e.g., ciprofloxacin, spectinomycin, or penicillin). The capacity of polyamines to increase gonococcal resistance to cationic antimicrobial peptides was dose dependent, correlated with the degree of cationicity, independent of a polyamine transport system involving the polyamine permeases PotH and PotI, and was reversible. In addition, we found that polyamines increase gonococcal resistance to complement-mediated killing by normal human serum. We propose that polyamines in genital mucosal fluids may enhance gonococcal survival during infection by reducing bacterial susceptibility to host-derived antimicrobials that function in innate host defense. PMID:20439477

  20. A Systematic Review of Point of Care Testing for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis

    PubMed Central

    Herbst de Cortina, Sasha; Bristow, Claire C.; Joseph Davey, Dvora; Klausner, Jeffrey D.

    2016-01-01

    Objectives. Systematic review of point of care (POC) diagnostic tests for sexually transmitted infections: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Methods. Literature search on PubMed for articles from January 2010 to August 2015, including original research in English on POC diagnostics for sexually transmitted CT, NG, and/or TV. Results. We identified 33 publications with original research on POC diagnostics for CT, NG, and/or TV. Thirteen articles evaluated test performance, yielding at least one test for each infection with sensitivity and specificity ≥90%. Each infection also had currently available tests with sensitivities <60%. Three articles analyzed cost effectiveness, and five publications discussed acceptability and feasibility. POC testing was acceptable to both providers and patients and was also demonstrated to be cost effective. Fourteen proof of concept articles introduced new tests. Conclusions. Highly sensitive and specific POC tests are available for CT, NG, and TV, but improvement is possible. Future research should focus on acceptability, feasibility, and cost of POC testing. While pregnant women specifically have not been studied, the results available in nonpregnant populations are encouraging for the ability to test and treat women in antenatal care to prevent adverse pregnancy and neonatal outcomes. PMID:27313440

  1. A Systematic Review of Point of Care Testing for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.

    PubMed

    Herbst de Cortina, Sasha; Bristow, Claire C; Joseph Davey, Dvora; Klausner, Jeffrey D

    2016-01-01

    Objectives. Systematic review of point of care (POC) diagnostic tests for sexually transmitted infections: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Methods. Literature search on PubMed for articles from January 2010 to August 2015, including original research in English on POC diagnostics for sexually transmitted CT, NG, and/or TV. Results. We identified 33 publications with original research on POC diagnostics for CT, NG, and/or TV. Thirteen articles evaluated test performance, yielding at least one test for each infection with sensitivity and specificity ≥90%. Each infection also had currently available tests with sensitivities <60%. Three articles analyzed cost effectiveness, and five publications discussed acceptability and feasibility. POC testing was acceptable to both providers and patients and was also demonstrated to be cost effective. Fourteen proof of concept articles introduced new tests. Conclusions. Highly sensitive and specific POC tests are available for CT, NG, and TV, but improvement is possible. Future research should focus on acceptability, feasibility, and cost of POC testing. While pregnant women specifically have not been studied, the results available in nonpregnant populations are encouraging for the ability to test and treat women in antenatal care to prevent adverse pregnancy and neonatal outcomes.

  2. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-01-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)—CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications. PMID:26782639

  3. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells.

    PubMed

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-03-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications. PMID:26782639

  4. Neisseria gonorrhoeae Induces a Tolerogenic Phenotype in Macrophages to Modulate Host Immunity

    PubMed Central

    Candia, Enzo; Reyes-Cerpa, Sebastian; Villegas-Valdes, Bélgica; Neira, Tanya; Lopez, Mercedes; Maisey, Kevin; Tempio, Fabián; Ríos, Miguel; Acuña-Castillo, Claudio; Imarai, Mónica

    2013-01-01

    Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world. N. gonorrhoeae does not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. N. gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whether N. gonorrhoeae directly conditions the phenotype of RAW 264.7 murine macrophage cell line and its response. We established that gonococcus was effectively phagocytosed by the RAW 264.7 cells and upregulates production of immunoregulatory cytokines (IL-10 and TGF-β1) but not the production of proinflammatory cytokine TNF-α, indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover, N. gonorrhoeae did not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed that N. gonorrhoeae infected macrophage cell line fails to elicit proliferative CD4+ response. This implies that macrophage that can phagocytose gonococcus do not display proper antigen-presenting functions. These results indicate that N. gonorrhoeae induces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response. PMID:24204097

  5. Neisseria gonorrhoeae pilin glycan contributes to CR3 activation during challenge of primary cervical epithelial cells

    PubMed Central

    Jennings, Michael P.; Jen, Freda E.-C.; Roddam, Louise F.; Apicella, Michael A.; Edwards, Jennifer L.

    2013-01-01

    Summary Expression of type IV pili by Neisseria gonorrhoeae plays a critical role in mediating adherence to human epithelial cells. Gonococcal pilin is modified with an O-linked glycan, which may be present as a di- or monosaccharide because of phase variation of select pilin glycosylation genes. It is accepted that bacterial proteins may be glycosylated; less clear is how the protein glycan may mediate virulence. Using primary, human, cervical epithelial (i.e. pex) cells, we now provide evidence to indicate that the pilin glycan mediates productive cervical infection. In this regard, pilin glycan-deficient mutant gonococci exhibited an early hyper-adhesive phenotype but were attenuated in their ability to invade pex cells. Our data further indicate that the pilin glycan was required for gonococci to bind to the I-domain region of complement receptor 3, which is naturally expressed by pex cells. Comparative, quantitative, infection assays revealed that mutant gonococci lacking the pilin glycan did not bind to the I-domain when it is in a closed, low-affinity conformation and cannot induce an active conformation to complement receptor 3 during pex cell challenge. To our knowledge, these are the first data to directly demonstrate how a protein-associated bacterial glycan may contribute to pathogenesis. PMID:21371235

  6. Pilin regulation in the pilT mutant of Neisseria gonorrhoeae strain MS11

    PubMed Central

    Dietrich, Manuela; Mollenkopf, Hans; So, Magdalene; Friedrich, Alexandra

    2009-01-01

    The ATPase protein PilT mediates retraction of type IV pili (Tfp). Tfp retraction of Neisseria gonorrhoeae causes many signal transduction events and changes in gene expression in infected epithelial cells. To find out whether a pilT mutation and lack of Tfp retraction, respectively, lead also to gene regulation in bacteria we performed microarrays comparing the transcriptional profiles of the N. gonorrhoeae parent strain MS11 and its isogenic pilT mutant during growth in vitro. A loss-of-function-mutation in pilT led to altered transcript levels of 63 open reading frames. Levels of pilE transcripts and its deduced protein the major Tfp subunit pilin, were increased most markedly by a mutation in pilT. Further studies revealed that pilE expression was also controlled by two other genes encoding Tfp biogenesis proteins, pilD and pilF. Our studies strongly suggest that pilE expression is a finely-tuned process. PMID:19486161

  7. Induction and repression of outer membrane proteins by anaerobic growth of Neisseria gonorrhoeae.

    PubMed Central

    Clark, V L; Campbell, L A; Palermo, D A; Evans, T M; Klimpel, K W

    1987-01-01

    Neisseria gonorrhoeae is generally considered to be an obligate aerobe; it can, however, grow in the absence of oxygen by anaerobic respiration by using nitrite as a terminal electron acceptor. The outer membrane protein compositions of aerobically and anaerobically grown N. gonorrhoeae strains were compared by one- and two-dimensional polyacrylamide gel electrophoresis. Anaerobically grown strains expressed at least three proteins (Pan 1 to Pan 3) at much higher levels than did aerobically grown cells. Conversely, at least five other proteins (Pox 1 to Pox 5) were found to be expressed at significantly higher levels in aerobically grown cells. None of the Pan or Pox proteins were heat modifiable, and none of the heat-modifiable protein IIs or other major outer membrane proteins (protein I, protein III, pilin, or H-8 protein) were significantly altered in expression by anaerobic growth. There were also no apparent differences in lipopolysaccharide composition in aerobically and anaerobically grown gonococci. The regulation of protein expression by oxygen availability suggests that anaerobic growth is a physiologically significant state for this organism. Images PMID:3106220

  8. Multiple protein differences exist between Neisseria gonorrhoeae type 1 and type 4.

    PubMed Central

    Klimpel, K W; Clark, V L

    1988-01-01

    Neisseria gonorrhoeae undergoes a spontaneous conversion from a form which is virulent, competent for DNA-mediated transformation, and piliated (type 1) to a form which is avirulent and neither piliated nor competent (type 4). This phase variation has become thought of as simply a conversion from piliated to nonpiliated. Using the techniques of cell fractionation, two-dimensional electrophoresis, and nonequilibrium pH gradient gel electrophoresis, we identified differences in the expression levels of multiple proteins between type 1 and type 4 cells. A total of 26 type 1-specific (T1S) and 23 type 4-specific (T4S) cytoplasmic or cytoplasmic membrane proteins were identified in O'Farrell two-dimensional gels. Using nonequilibrium pH gradient gel electrophoresis, we detected a minimum of eight T1S outer membrane proteins and four T4S outer membrane proteins which were not detected in the O'Farrell gels. Thus, the conversion from type 1 to type 4 is a complex event involving many different proteins of all cellular locations. Images PMID:3126144

  9. Unusual genetic organization of a functional type I protein secretion system in Neisseria meningitidis.

    PubMed

    Wooldridge, Karl G; Kizil, Murat; Wells, Damien B; Ala'aldeen, Dlawer A A

    2005-09-01

    Proteins secreted by Neisseria meningitidis are thought to play important roles in the pathogenesis of meningococcal disease. These proteins include the iron-repressible repeat-in-toxin (RTX) exoprotein FrpC. Related proteins in other pathogens are secreted via a type I secretion system (TOSS), but such a system has not been demonstrated in N. meningitidis. An in silico search of the group B meningococcal genome suggested the presence of a uniquely organized TOSS. Genes encoding homologs of the Escherichia coli HlyB (ATP-binding), HlyD (membrane fusion), and TolC (outer membrane channel) proteins were identified. In contrast to the cistronic organization of the secretion genes in most other rtx operons, the hlyD and tolC genes were adjacent but unlinked to hlyB; neither locus was part of an operon containing genes encoding putative TOSS substrates. Both loci were flanked by genes normally associated with mobile genetic elements. The three genes were shown to be expressed independently. Mutation at either locus resulted in an inability to secrete FrpC and a related protein, here called FrpC2. Successful complementation of these mutations at an ectopic site confirmed the observed phenotypes were caused by loss of function of the putative TOSS genes. We show that genes scattered in the meningococcal genome encode a functional TOSS required for secretion of the meningococcal RTX proteins.

  10. Antigen-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein.

    PubMed Central

    Buchanan, T M; Hildebrandt, J F

    1981-01-01

    Principal outer membrane protein (protein I) of Neisseria gonorrhoeae was prepared nearly free of lipopolysaccharide (LPS) and substantially purified from other membrane proteins by chromatography of partially purified gonococcal outer membranes over Sepharose 6B in the presence of deoxycholate at pH 9.0. This protein I of nine separate antigenic types was coated to polystyrene tubes and used in the enzyme-linked immunosorbent assay (ELISA) to measure antibody to protein I or in inhibition tests to quantitate protein I antigen. No significant inhibition of the ELISA test was produced by purified LPS from the strain used to prepare each of the protein I types or by whole gonococci bearing the same LPS but different protein I antigens as the strain used to produce a given protein I antigen. Of 125 strains of gonococci used as whole organisms to inhibit the protein I ELISA, 124 (99%) typed with one or more of the nine protein I types, and 35% of these typed with a single protein I serotype. Sixty-one of 65 (94%) strains from Seattle and Atlanta patients with disseminated gonococcal infection contained protein I serotype 1, and 16 of 24 (64%) strains from Seattle patients with salpingitis bore one or both of protein I serotypes 1 and 2. Images PMID:6166568

  11. Methods for Identifying Neisseria meningitidis Carriers: A Multi-Center Study in the African Meningitis Belt

    PubMed Central

    Basta, Nicole E.; Stuart, James M.; Nascimento, Maria C.; Manigart, Olivier; Trotter, Caroline; Hassan-King, Musa; Chandramohan, Daniel; Sow, Samba O.; Berthe, Abdoulaye; Bedru, Ahmed; Tekletsion, Yenenesh K.; Collard, Jean-Marc; Jusot, Jean-François; Diallo, Aldiouma; Basséne, Hubert; Daugla, Doumagoum M.; Gamougam, Khadidja; Hodgson, Abraham; Forgor, Abudulai A.; Omotara, Babatunji A.; Gadzama, Galadima B.; Watkins, Eleanor R.; Rebbetts, Lisa S.; Diallo, Kanny; Weiss, Noel S.; Halloran, M. Elizabeth; Maiden, Martin C. J.; Greenwood, Brian

    2013-01-01

    Objective Detection of meningococcal carriers is key to understanding the epidemiology of Neisseria meningitidis, yet no gold standard has been established. Here, we directly compare two methods for collecting pharyngeal swabs to identify meningococcal carriers. Methods We conducted cross-sectional surveys of schoolchildren at multiple sites in Africa to compare swabbing the posterior pharynx behind the uvula (U) to swabbing the posterior pharynx behind the uvula plus one tonsil (T). Swabs were cultured immediately and analyzed using molecular methods. Results One thousand and six paired swab samples collected from schoolchildren in four countries were analyzed. Prevalence of meningococcal carriage was 6.9% (95% CI: 5.4-8.6%) based on the results from both swabs, but the observed prevalence was lower based on one swab type alone. Prevalence based on the T swab or the U swab alone was similar (5.2% (95% CI: 3.8-6.7%) versus 4.9% (95% CI: 3.6-6.4%) respectively (p=0.6)). The concordance between the two methods was 96.3% and the kappa was 0.61 (95% CI: 0.50-0.73), indicating good agreement. Conclusions These two commonly used methods for collecting pharyngeal swabs provide consistent estimates of the prevalence of carriage, but both methods misclassified carriers to some degree, leading to underestimates of the prevalence. PMID:24194921

  12. Polysaccharide Production in Pilot Scale Bioreactor Cultivations of Neisseria meningitidis Serogroup C

    PubMed Central

    Baruque-Ramos, Julia; Juncioni de Arauz, Luciana; Fossa da Paz, Marcelo; Vicentin, Marcio Alberto; Hiss, Haroldo

    2016-01-01

    Serogroup C polysaccharide from Neisseria meningitidis (PS) constitutes the antigen for the respective vaccine production. In order to investigate the enhancement of the final PS concentration (Pf), as well as the overall yield factor (PS/biomass) (YP/X), 13 total cultivations distributed in 6 series (from A to F) were carried out in Frantz medium (40 L plus inoculum) in a 80L bioreactor at 35oC, 0.4 atm, 120 rpm, airflow rate of 5 L/min and KLa = 4.2 h-1. The series (A-F) correspond to different experimental conditions as follows: A) without pH and dissolved O2 controls; B) pH control at 6.5; C) pH control at 6.5 and glucose pulse at the 10th hour; D) dissolved O2 control at 10% saturation value; E) pH control at 7.4; F) dissolved O2 limitation (set rotation at 55 rpm). Concentrations of dry biomass, PS, cellular nitrogen, residual glucose, organic and inorganic nitrogen in the medium were measured. The best results were represented by series A (averages of Pf = 0.15 g/L and YP/X = 107 mg/g). The presented findings could be useful for a proper Frantz medium reformulation in order to obtain a greater amount of PS and improve the vaccine development in industrial scale-up production.

  13. In vitro potency and combination testing of antimicrobial agents against Neisseria gonorrhoeae.

    PubMed

    Bharat, Amrita; Martin, Irene; Zhanel, George G; Mulvey, Michael R

    2016-03-01

    Antimicrobial resistant Neisseria gonorrhoeae is a major concern to public health due to decreased susceptibility to frontline antimicrobials. To find agents that are active against N. gonorrhoeae, we tested antimicrobials alone or in combination by Etest gradient strips. The potencies (as assessed by minimum inhibitory concentrations) of twenty-five antimicrobials were evaluated against nine reference strains of N. gonorrhoeae (WHO F, G, K, L, M, N, O, P and ATCC 49226). Potency was greatest for netilmicin, quinupristin-dalfopristin, ceftriaxone, ertapenem and piperacillin-tazobactam. Combinations of azithromycin, moxifloxacin, or gentamicin with ceftriaxone, doripenem, or aztreonam were tested against reference isolates and the fractional inhibitory concentration index (FICI) was calculated. All nine combinations resulted in indifference (>0.5 FICI ≤ 4). Combinations with FICI < 1 were further evaluated in nine clinical isolates which supported the finding of indifference. No antagonism was observed in any of the combinations tested. This is the first report in which the six combinations of azithromycin, moxifloxacin or gentamcin in combination with doripenem or aztreonam were tested in N. gonorrhoeae. These data on antimicrobials with higher potency and combinations that did not show antagonism can help to guide larger scale susceptibility studies for antimicrobial resistant N. gonorrhoeae.

  14. Polymerase chain reaction-based screening for the ceftriaxone-resistant Neisseria gonorrhoeae F89 strain.

    PubMed

    Goire, N; Lahra, M M; Ohnishi, M; Hogan, T; Liminios, A E; Nissen, M D; Sloots, T P; Whiley, D M

    2013-04-04

    Emergence and spread of Neisseria gonorrhoeae resistant to extended spectrum cephalosporins is a major problem threatening treatment of gonorrhoea and is further highlighted by the recent report of a second ceftriaxone-resistant N. gonorrhoeae strain (F89) in Europe, initially observed in France and subsequently identified in Spain. N. gonorrhoeae antimicrobial resistance (AMR) surveillance has acquired new importance and molecular tools have the potential to enhance bacterial culture-based methods. In this study, we established a polymerase chain reaction (PCR) protocol for direct detection of the F89 strain. A key component of this screening protocol was the development of a hybridisation probe-based melting curve analysis assay (mosaic501-hybPCR) to detect the presence of an A501P substitution on the N. gonorrhoeae mosaic penicillin binding protein 2 (PBP2) sequence, an important characteristic of the F89 strain. The mosaic501-hybPCR was evaluated using plasmid-derived positive controls (n=3) and characterised gonococcal (n=33) and non-gonococcal (n=58) isolates. The protocol was then applied to 159 clinical specimens from Sydney, Australia, collected during the first half of the year 2012 that were N. gonorrhoeae PCR-positive. Overall, the results indicate that the PCR-based protocol is suitable for direct detection of the N. gonorrhoeae F89 strain in non-cultured clinical samples. It therefore provides an additional tool to aid investigations into the potential spread of F89 strain throughout Europe and elsewhere.

  15. Uncovering the Mechanism of Trapping and Cell Orientation during Neisseria gonorrhoeae Twitching Motility

    PubMed Central

    Zaburdaev, Vasily; Biais, Nicolas; Schmiedeberg, Michael; Eriksson, Jens; Jonsson, Ann-Beth; Sheetz, Michael P.; Weitz, David A.

    2014-01-01

    Neisseria gonorrheae bacteria are the causative agent of the second most common sexually transmitted infection in the world. The bacteria move on a surface by means of twitching motility. Their movement is mediated by multiple long and flexible filaments, called type IV pili, that extend from the cell body, attach to the surface, and retract, thus generating a pulling force. Moving cells also use pili to aggregate and form microcolonies. However, the mechanism by which the pili surrounding the cell body work together to propel bacteria remains unclear. Understanding this process will help describe the motility of N. gonorrheae bacteria, and thus the dissemination of the disease which they cause. In this article we track individual twitching cells and observe that their trajectories consist of alternating moving and pausing intervals, while the cell body is preferably oriented with its wide side toward the direction of motion. Based on these data, we propose a model for the collective pili operation of N. gonorrheae bacteria that explains the experimentally observed behavior. Individual pili function independently but can lead to coordinated motion or pausing via the force balance. The geometry of the cell defines its orientation during motion. We show that by changing pili substrate interactions, the motility pattern can be altered in a predictable way. Although the model proposed is tangibly simple, it still has sufficient robustness to incorporate further advanced pili features and various cell geometries to describe other bacteria that employ pili to move on surfaces. PMID:25296304

  16. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future.

    PubMed

    Unemo, Magnus; Shafer, William M

    2014-07-01

    Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhea, which is a major public health concern globally. Given the global nature of gonorrhea, the high rate of usage of antimicrobials, suboptimal control and monitoring of antimicrobial resistance (AMR) and treatment failures, slow update of treatment guidelines in most geographical settings, and the extraordinary capacity of the gonococci to develop and retain AMR, it is likely that the global problem of gonococcal AMR will worsen in the foreseeable future and that the severe complications of gonorrhea will emerge as a silent epidemic. By understanding the evolution, emergence, and spread of AMR in N. gonorrhoeae, including its molecular and phenotypic mechanisms, resistance to antimicrobials used clinically can be anticipated, future methods for genetic testing for AMR might permit region-specific and tailor-made antimicrobial therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. This review focuses on the history and evolution of gonorrhea treatment regimens and emerging resistance to them, on genetic and phenotypic determinants of gonococcal resistance to previously and currently recommended antimicrobials, including biological costs or benefits; and on crucial actions and future advances necessary to detect and treat resistant gonococcal strains and, ultimately, retain gonorrhea as a treatable infection.

  17. Structure and function of Neisseria gonorrhoeae MtrF illuminates a class of antimetabolite efflux pumps.

    PubMed

    Su, Chih-Chia; Bolla, Jani Reddy; Kumar, Nitin; Radhakrishnan, Abhijith; Long, Feng; Delmar, Jared A; Chou, Tsung-Han; Rajashankar, Kanagalaghatta R; Shafer, William M; Yu, Edward W

    2015-04-01

    Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs.

  18. Antimicrobial susceptibility and genotyping analysis of Hungarian Neisseria gonorrhoeae strains in 2013.

    PubMed

    Nemes-Nikodém, Éva; Brunner, Alexandra; Pintér, Dóra; Mihalik, Noémi; Lengyel, György; Marschalkó, Márta; Kárpáti, Sarolta; Szabó, Dóra; Ostorházi, Eszter

    2014-12-01

    Emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern worldwide. The current study aims to determine the antimicrobial resistance in N. gonorrhoeae and associated molecular typing to enhance gonococcal antimicrobial surveillance in Hungary. In the National N. gonorrhoeae Reference Laboratory of Hungary 187 N. gonorrhoeae infections were detected in 2013, antibiograms were determined for all the isolated strains, and 52 (one index strain from every sexually contact related group) of them were also analysed by the N. gonorrhoeae multi-antigen sequence typing (NG-MAST) method. Twenty-two different NG-MAST sequence types (STs) were identified, of which 8 STs had not been previously described. In Hungary, the highly diversified gonococcal population displayed high resistance to penicillin, ciprofloxacin and tetracycline (the antimicrobials previously recommended for gonorrhoea treatment). Resistance to the currently recommended extended spectrum cephalosporines were rare: only two of the expected strains, an ST 1407 and an ST 210, had cefixime MIC above the resistance breakpoint. By the revision of our National Treatment Guideline, it must be considered, that the azithromycin resistance is about 60% among the four most frequently isolated STs in Hungary.

  19. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

    PubMed

    Gong, Zheng; Tang, M Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A; Fan, Huizhou

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.

  20. Antibacterial activity of resazurin-based compounds against Neisseria gonorrhoeae in vitro and in vivo.

    PubMed

    Schmitt, Deanna M; Connolly, Kristie L; Jerse, Ann E; Detrick, Melinda S; Horzempa, Joseph

    2016-10-01

    Neisseria gonorrhoeae is the cause of the second most common sexually transmitted bacterial infection, with ca. 80 million new cases of gonorrhoea reported annually. The recent emergence of clinical isolates resistant to the last monotherapy against this bacterium, the cephalosporins, illustrates the need for new antigonococcal agents. Here we have characterised a new group of antimicrobials based on the compound resazurin that exhibits robust activity against N. gonorrhoeae in vitro. Resazurin inhibits the growth of a broad range of N. gonorrhoeae isolates, including those resistant to multiple antibiotics. Furthermore, treatment of human endometrial cells infected with N. gonorrhoeae with resazurin significantly reduces the number of intracellular bacteria. Whilst resazurin exhibited potent in vitro antimicrobial activity, in vivo resazurin did not limit the colonisation of mice with N. gonorrhoeae following vaginal infection. The ineffectiveness of resazurin in vivo is likely due to its interaction with serum albumin, which completely diminishes its antimicrobial activity. However, treatment of mice with a resazurin analogue (resorufin pentyl ether) that maintains its antimicrobial activity in the presence of serum albumin approached a significant decrease in the percentage of mice vaginally colonised. This treatment also decreased vaginal colonisation by N. gonorrhoeae over time. Together, these data suggest that resazurin derivatives have potential for the treatment of gonorrhoea.

  1. The Development of an Experimental Multiple Serogroups Vaccine for Neisseria meningitidis

    PubMed Central

    Pinto, Valerian B.; Burden, Robert; Wagner, Allyn; Moran, Elizabeth E.; Lee, Che-Hung

    2013-01-01

    A native outer membrane vesicles (NOMV) vaccine was developed from three antigenically diverse strains of Neisseria meningitidis that express the L1,8, L2, and L3,7 lipooligosaccharide (LOS) immunotypes, and whose synX, and lpxL1 genes were deleted.. Immunogenicity studies in mice showed that the vaccine induced bactericidal antibody against serogroups B, C, W, Y and X N. meningitidis strains. However, this experimental NOMV vaccine was not effective against serogroup A N. meningitidis strains. N. meningitidis capsular polysaccharide (PS) from serogroups A, C, W and Y were effective at inducing bactericidal antibody when conjugated to either tetanus toxoid or the fHbp1-fHbp2 fusion protein fHbp(1+2). The combination of the NOMV vaccine and the N. meningitidis serogroup A capsular polysaccharide (MAPS) protein conjugate was capable of inducing bactericidal antibodies against a limited number of N. meningitidis strains from serogroups A, B, C, W, Y and X tested in this study. PMID:24244473

  2. Lipoprotein NMB0928 from Neisseria meningitidis serogroup B as a novel vaccine candidate.

    PubMed

    Delgado, Maité; Yero, Daniel; Niebla, Olivia; González, Sonia; Climent, Yanet; Pérez, Yusleydis; Cobas, Karem; Caballero, Evelín; García, Darien; Pajón, Rolando

    2007-12-01

    Polysaccharide-based vaccines for serogroup B Neisseria meningitidis have failed to induce protective immunity. As a result, efforts to develop vaccines for serogroup B meningococcal disease have mostly focused on outer membrane proteins (OMP). Vaccine candidates based on meningococcal OMP have emerged in the form of outer membrane vesicles (OMVs) or, more recently, purified recombinant proteins, as alternative strategies for serogroup B vaccine development. In our group, the protein composition of the Cuban OMVs-based vaccine VA-MENGOC-BC was elucidated using two-dimensional gel electrophoresis and mass spectrometry. The proteomic map of this product allowed the identification of new putative protective proteins not previously reported as components of an antimeningococcal vaccine. In the present study, we have determined the immunogenicity and protective capacity of NMB0928, one of those proteins present in the OMVs. The antigen was obtained as a recombinant protein in Escherichia coli, purified and used to immunize mice. The antiserum produced against the protein was capable to recognize the natural protein in different meningococcal strains by whole-cell ELISA and Western blotting. After immunization, recombinant NMB0928 induced bactericidal antibodies, and when the protein was administered inserted into liposomes, the elicited antibodies were protective in the infant rat model. These results suggest that NMB0928 is a novel antigen worth to be included in a broadly protective meningococcal vaccine.

  3. Neisseria meningitidis antigen NMB0088: sequence variability, protein topology and vaccine potential.

    PubMed

    Sardiñas, Gretel; Yero, Daniel; Climent, Yanet; Caballero, Evelin; Cobas, Karem; Niebla, Olivia

    2009-02-01

    The significance of Neisseria meningitidis serogroup B membrane proteins as vaccine candidates is continually growing. Here, we studied different aspects of antigen NMB0088, a protein that is abundant in outer-membrane vesicle preparations and is thought to be a surface protein. The gene encoding protein NMB0088 was sequenced in a panel of 34 different meningococcal strains with clinical and epidemiological relevance. After this analysis, four variants of NMB0088 were identified; the variability was confined to three specific segments, designated VR1, VR2 and VR3. Secondary structure predictions, refined with alignment analysis and homology modelling using FadL of Escherichia coli, revealed that almost all the variable regions were located in extracellular loop domains. In addition, the NMB0088 antigen was expressed in E. coli and a procedure for obtaining purified recombinant NMB0088 is described. The humoral immune response elicited in BALB/c mice was measured by ELISA and Western blotting, while the functional activity of these antibodies was determined in a serum bactericidal assay and an animal protection model. After immunization in mice, the recombinant protein was capable of inducing a protective response when it was administered inserted into liposomes. According to our results, the recombinant NMB0088 protein may represent a novel antigen for a vaccine against meningococcal disease. However, results from the variability study should be considered for designing a cross-protective formulation in future studies.

  4. Lectin characterization of gonococci from an outbreak caused by penicillin-resistant Neisseria gonorrhoeae.

    PubMed Central

    Schalla, W O; Rice, R J; Biddle, J W; Jeanlouis, Y; Larsen, S A; Whittington, W L

    1985-01-01

    A total of 40 Neisseria gonorrhoeae isolates, representing 19 penicillin-resistant isolates (from 8 heterosexual patients and 11 homosexual patients) and 21 penicillin-susceptible isolates (from 15 heterosexual patients and 6 homosexual patients) and obtained from the same geographic area, were examined. Lectin agglutination patterns were based on the reactivity of the isolates with the following 14 lectins: concanavalin A, Lens culinaris, Trichosanthes kinlowii, Griffonia simplicifolia I, Arachis hypogeae (peanut agglutinin), Glycine max (soybean agglutinin), Dolichos bifloris, Griffonia simplicifolia II, Solanum tuberosum (potato starch agglutinin), Triticum vulgaris (wheat germ agglutinin), Limax flavus, Phaseolus vulgaris, Ulex europaeus I, and Lotus tetragonolobus. All isolates were serotyped with monoclonal antibodies specific for gonococcal outer membrane protein I and auxotyped, and the plasmid content was determined. Resistant patient isolates were selected for their decreased penicillin susceptibility, and control isolates were selected for their penicillin susceptibility. Even though the patient isolates demonstrated resistance to penicillin, no phenotypic differences in lectin-grouping patterns were demonstrated between the two study groups; i.e., two predominant lectin groups were observed. No resistance-associated plasmids were detected. All patient isolates were serogroup IB (serovars IB-1, IB-2, and IB-4), whereas 12 of 21 control isolates were serogroup IA (P less than 0.05). Isolates obtained from different anatomical sites in the same patient (cervical and rectal) agreed with regard to lectin patterns and serovars but not auxotypes. PMID:3935658

  5. Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

    PubMed

    Gong, Zheng; Tang, M Matt; Wu, Xueliang; Phillips, Nancy; Galkowski, Dariusz; Jarvis, Gary A; Fan, Huizhou

    2016-01-01

    Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis. PMID:26808268

  6. Mutation in ribosomal protein S5 leads to spectinomycin resistance in Neisseria gonorrhoeae.

    PubMed

    Ilina, Elena N; Malakhova, Maya V; Bodoev, Ivan N; Oparina, Nina Y; Filimonova, Alla V; Govorun, Vadim M

    2013-01-01

    Spectinomycin remains a useful reserve option for therapy of gonorrhea. The emergence of multidrug-resistant Neisseria gonorrhoeae strains with decreased susceptibility to cefixime and to ceftriaxone makes it the only medicine still effective for treatment of gonorrhea infection in analogous cases. However, adoption of spectinomycin as a routinely used drug of choice was soon followed by reports of spectinomycin resistance. The main molecular mechanism of spectinomycin resistance in N. gonorrhoeae was C1192T substitution in 16S rRNA genes. Here we reported a Thr-24→Pro mutation in ribosomal protein S5 (RPS5) found in spectinomycin resistant clinical N. gonorrhoeae strain, which carried no changes in 16S rRNA. In a series of experiments, the transfer of rpsE gene allele encoding the mutant RPS5 to the recipient N. gonorrhoeae strains was analyzed. The relatively high rate of transformation [ca. 10(-5) colony-forming units (CFUs)] indicates the possibility of spread of spectinonycin resistance within gonococcal population due to the horizontal gene transfer (HGT). PMID:23847609

  7. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    PubMed

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  8. Serotype and serovar distribution of Neisseria gonorrhoeae isolated from high-risk populations in Bangladesh.

    PubMed

    Alam, M A; Chowdhury, M Z; Ahmed, F; Alam, A; Hossain, M A

    2012-12-01

    Neisseria gonorrhoeae, the causative agent of gonococcal infection, is known to frequently change their characteristics to evade host immune mechanism. Characterization of the clinical isolates of the organism can lead to identification of the circulating strains and often a sexual network in a community to help in designing the control strategy. Keeping in mind the above consideration, a total of 239 N. gonorrhoeae, isolated from high-risk populations, were characterized for serotypes and serovars by monoclonal antibodies against protein 1 of the organism. Majority of the serotypes were serotype B (142, 59.4%). Majority of the isolates showing resistance to at least one of the antibiotics tested were also serotype B (139, 59.2%), whereas, majority of the isolates showing resistance to any three of the antibiotics (multidrug resistant, MDR) (63%) was serotype A. A total of 41 different serovars were also identified and five of which (Arst, Bropt, Bopt, Arost, and Brop) included the highest percent (49.3%) of the isolates. Many serovars (23/41, 56.1%) were new emergent and included 58 (24.3%) of the isolates investigated. All of the new serovars were resistant to at least one of the antibiotics tested and the highest rate (40/102, 39.2%) was MDR. Serotyping and serovar determination was found contributory to understand the microepidemics of the N. gonorrhoeae isolates. Further studies including antibiogram and contact tracing can efficiently help in control of the disease. PMID:23540188

  9. Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils.

    PubMed

    Johnson, M Brittany; Criss, Alison K

    2013-08-01

    Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome-primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome-granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule-negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule-negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome-granule fusion. Ectopically increasing primary granule-phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule-phagosome fusion, thus preventing formation of a degradative phagolysosome. PMID:23374609

  10. Uncovering the mechanism of trapping and cell orientation during Neisseria gonorrhoeae twitching motility.

    PubMed

    Zaburdaev, Vasily; Biais, Nicolas; Schmiedeberg, Michael; Eriksson, Jens; Jonsson, Ann-Beth; Sheetz, Michael P; Weitz, David A

    2014-10-01

    Neisseria gonorrheae bacteria are the causative agent of the second most common sexually transmitted infection in the world. The bacteria move on a surface by means of twitching motility. Their movement is mediated by multiple long and flexible filaments, called type IV pili, that extend from the cell body, attach to the surface, and retract, thus generating a pulling force. Moving cells also use pili to aggregate and form microcolonies. However, the mechanism by which the pili surrounding the cell body work together to propel bacteria remains unclear. Understanding this process will help describe the motility of N. gonorrheae bacteria, and thus the dissemination of the disease which they cause. In this article we track individual twitching cells and observe that their trajectories consist of alternating moving and pausing intervals, while the cell body is preferably oriented with its wide side toward the direction of motion. Based on these data, we propose a model for the collective pili operation of N. gonorrheae bacteria that explains the experimentally observed behavior. Individual pili function independently but can lead to coordinated motion or pausing via the force balance. The geometry of the cell defines its orientation during motion. We show that by changing pili substrate interactions, the motility pattern can be altered in a predictable way. Although the model proposed is tangibly simple, it still has sufficient robustness to incorporate further advanced pili features and various cell geometries to describe other bacteria that employ pili to move on surfaces. PMID:25296304

  11. DC-SIGN (CD209) recognition of Neisseria gonorrhoeae is circumvented by lipooligosaccharide variation.

    PubMed

    Zhang, Pei; Schwartz, Olivier; Pantelic, Milica; Li, Geling; Knazze, Quita; Nobile, Cinzia; Radovich, Milan; He, Johnny; Hong, Soon-Cheol; Klena, John; Chen, Tie

    2006-04-01

    Neisseria gonorrhoeae (GC) or Escherichia coli HB101 (hereafter referred to as E. coli) expressing opacity (Opa) proteins adhere to human host cells and stimulate phagocytosis as a result of the interaction of certain Opa proteins to carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1; CD66a) receptors. Our experiments show that the Opa-CEACAM1 interaction does not play a significant role in adherence between these bacteria and dendritic cells (DCs). Instead, phagocytosis of GC and E. coli by DCs is mediated by the DC-specific intercellular adhesion molecule-grabbing nonintegrin, (SIGN; CD209) receptor. DC-SIGN recognition and subsequent phagocytosis of GC are limited, however, to a lipooligosaccharide (LOS) mutant (lgtB) of GC. This conclusion is supported by experiments demonstrating that HeLa cells expressing human DC-SIGN (HeLa-DC-SIGN) bind exclusively to and engulf an lgtB mutant of GC, and this interaction is blocked specifically by an anti-DC-SIGN antibody. The experiments suggest that LOS variation may have evolved as a mechanism for GC to avoid phagocytosis by DCs. PMID:16461738

  12. Enhancement of adaptive immunity to Neisseria gonorrhoeae by local intravaginal administration of microencapsulated interleukin 12.

    PubMed

    Liu, Yingru; Egilmez, Nejat K; Russell, Michael W

    2013-12-01

    Gonorrhea remains one of the most frequent infectious diseases, and Neisseria gonorrhoeae is emerging as resistant to most available antibiotics, yet it does not induce a state of specific protective immunity against reinfection. Our recent studies have demonstrated that N. gonorrhoeae proactively suppresses host T-helper (Th) 1/Th2-mediated adaptive immune responses, which can be manipulated to generate protective immunity. Here we show that intravaginally administered interleukin 12 (IL-12) encapsulated in sustained-release polymer microspheres significantly enhanced both Th1 and humoral immune responses in a mouse model of genital gonococcal infection. Treatment of mice with IL-12 microspheres during gonococcal challenge led to faster clearance of infection and induced resistance to reinfection, with the generation of gonococcus-specific circulating immunoglobulin G and vaginal immunoglobulin A and G antibodies. These results suggest that local administration of microencapsulated IL-12 can serve as a novel therapeutic and prophylactic strategy against gonorrhea, with implications for the development of an effective vaccine. PMID:24048962

  13. Non-cytotoxic nanomaterials enhance antimicrobial activities of cefmetazole against multidrug-resistant Neisseria gonorrhoeae.

    PubMed

    Li, Lan-Hui; Yen, Muh-Yong; Ho, Chao-Chi; Wu, Ping; Wang, Chien-Chun; Maurya, Pawan Kumar; Chen, Pai-Shan; Chen, Wei; Hsieh, Wan-Yu; Chen, Huei-Wen

    2013-01-01

    The emergence and spread of antibiotic-resistant Neisseria gonorrhoeae has led to difficulties in treating patients, and novel strategies to prevent and treat this infection are urgently needed. Here, we examined 21 different nanomaterials for their potential activity against N. gonorrhoeae (ATCC 49226). Silver nanoparticles (Ag NPs, 120 nm) showed the greatest potency for reducing N. gonorrhoeae colony formation (MIC: 12.5 µg/ml) and possessed the dominant influence on the antibacterial activity with their properties of the nanoparticles within a concentration range that did not induce cytotoxicity in human fibroblasts or epithelial cells. Electron microscopy revealed that the Ag NPs significantly reduced bacterial cell membrane integrity. Furthermore, the use of clinical isolates of multidrug-resistant N. gonorrhoeae showed that combined treatment with 120 nm Ag NPs and cefmetazole produced additive effects. This is the first report to screen the effectiveness of nanomaterials against N. gonorrhoeae, and our results indicate that 120 nm Ag NPs deliver low levels of toxicity to human epithelial cells and could be used as an adjuvant with antibiotic therapy, either for topical use or as a coating for biomaterials, to prevent or treat multidrug-resistant N. gonorrhoeae. PMID:23705013

  14. In silico identification of candidate drug and vaccine targets from various pathways in Neisseria gonorrhoeae.

    PubMed

    Barh, Debmalya; Kumar, Anil

    2009-01-01

    Neisseria gonorrhoeae is responsible for causing gonorrhea, one of the most common sexually transmitted diseases prevailing globally. Although extensive researches are in progress in order to control the transmission of the disease and to develop drug(s) against the pathogen, till date no effective vaccine or specific drug could be developed and only antibiotic treatment is in use. Perhaps, due to excess use of antibiotics, several resistant strains have been found. In the present study, metabolic pathways-related candidate drug and vaccine targets have been identified in N. gonorrhoeae virulent strain FA 1090 using an in silico subtractive genomics approach. 106 putative drug targets out of 537 essential genes have been predicted. 67 cytoplasmic and 9 membrane enzymes, along with 10 membrane transporters are found to be the potential drug targets from the host-pathogen common metabolic pathways. Among these targets, competence lipoproteins (NGO0277) and cysW have been identified as candidate vaccine targets. 20 drug targets have been identified from pathogen specific unique metabolic pathways. Out of these, 6 enzymes are involved in dual metabolic pathways and 2 are expressed in cell wall and fimbrium. These gonococci-specific proteins are expected to be better possible drug targets. Screening of the functional inhibitors against these novel targets may result in discovery of novel therapeutic compounds that can be effective against antibiotic resistant strains. PMID:20109152

  15. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future.

    PubMed

    Unemo, Magnus; Shafer, William M

    2014-07-01

    Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhea, which is a major public health concern globally. Given the global nature of gonorrhea, the high rate of usage of antimicrobials, suboptimal control and monitoring of antimicrobial resistance (AMR) and treatment failures, slow update of treatment guidelines in most geographical settings, and the extraordinary capacity of the gonococci to develop and retain AMR, it is likely that the global problem of gonococcal AMR will worsen in the foreseeable future and that the severe complications of gonorrhea will emerge as a silent epidemic. By understanding the evolution, emergence, and spread of AMR in N. gonorrhoeae, including its molecular and phenotypic mechanisms, resistance to antimicrobials used clinically can be anticipated, future methods for genetic testing for AMR might permit region-specific and tailor-made antimicrobial therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. This review focuses on the history and evolution of gonorrhea treatment regimens and emerging resistance to them, on genetic and phenotypic determinants of gonococcal resistance to previously and currently recommended antimicrobials, including biological costs or benefits; and on crucial actions and future advances necessary to detect and treat resistant gonococcal strains and, ultimately, retain gonorrhea as a treatable infection. PMID:24982323

  16. Carriage of Neisseria meningitidis in the Hajj and Umrah mass gatherings.

    PubMed

    Yezli, Saber; Wilder-Smith, Annelies; Bin Saeed, Abdulaziz A

    2016-06-01

    Meningococcal disease is a serious public health threat, especially during mass gatherings such as Hajj and Umrah which provide optimal conditions for disease transmission. The disease is caused by Neisseria meningitidis and transmitted mainly via asymptomatic carriers. A review of the literature on asymptomatic N. meningitidis carriage among Hajj and Umrah pilgrims and their household contacts was performed. Carriage studies reported carriage rates to be higher in Hajj pilgrims compared to Umrah pilgrims and that these events promote acquisition of carriage among pilgrims. With some outliers, most studies found carriage rates among pilgrims to be comparable to those in populations under non-epidemic settings. However, these results should be interpreted with caution, taking into account the limitations within the studies identified. A wide variety of N. meningitidis serogroups appear to be circulating among Hajj and Umrah pilgrims, with serogroups W135 and B being most prominent. Current Hajj and Umrah meningococcal disease preventative measures do not necessarily prevent carriage and transmission, which may result in local and international outbreaks among susceptible populations. Monitoring carriage states of visitors and local inhabitants in the Kingdom of Saudi Arabia, as well as the implementation of preventive measures that impact carriage, are warranted to reduce the risk of Hajj and Umrah-related meningococcal disease outbreaks. PMID:26612675

  17. Effectiveness of immunoprophylaxis in suppressing carriage of Neisseria meningitidis in the military environment.

    PubMed

    Korzeniewski, K; Skoczyńska, A; Guzek, A; Konior, M; Chciałowski, A; Waśko, I; Markowska, M; Zwolińska, E

    2015-01-01

    Neisseria meningitidis, etiological factor of invasive meningococcal disease, is a human commensal that colonizes the nasopharynx. Colonization is usually asymptomatic, but it is a prerequisite for disease. Asymptomatic carriers are the major source of infection. In the present study, a survey of N. meningitidis carriage was conducted between January and March 2013 in a military unit in Poland. Single-time throat culture samples were collected from professional 559 soldiers (302 unvaccinated vs. 257 vaccinated individuals with the quadrivalent conjugate vaccine ACYW-135). Bacterial identification was performed with classic microbiological methods (culture, incubation, identification). Non-culture method (PCR) was used for confirmation of detected strains of N. meningitidis and determination of serogroups. We found 29 carriers in the group of unvaccinated soldiers (9.6 % of examined individuals) whereas among vaccinated soldiers only 3 persons were carriers of N. meningitidis (1.2 %). The most frequently identified serogroups among the carriers serving in the same military facility were serogroup B (28 %), followed by Y (25 %), and C (22 %). In conclusion, the initiation of mass vaccination with the quadrivalent conjugate vaccine ACYW-135 in the military environment seems an effective method of suppressing N. meningitidis carriage. PMID:25310944

  18. Human monocytes/macrophages are a target of Neisseria meningitidis Adhesin A (NadA).

    PubMed

    Franzoso, Susanna; Mazzon, Cristina; Sztukowska, Maryta; Cecchini, Paola; Kasic, Tihana; Capecchi, Barbara; Tavano, Regina; Papini, Emanuele

    2008-05-01

    Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA(Delta351-405), a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.

  19. Penicillin resistance compromises Nod1-dependent proinflammatory activity and virulence fitness of neisseria meningitidis.

    PubMed

    Zarantonelli, Maria Leticia; Skoczynska, Anna; Antignac, Aude; El Ghachi, Meriem; Deghmane, Ala-Eddine; Szatanik, Marek; Mulet, Céline; Werts, Catherine; Peduto, Lucie; d'Andon, Martine Fanton; Thouron, Françoise; Nato, Faridabano; Lebourhis, Lionel; Philpott, Dana J; Girardin, Stephen E; Vives, Francina Langa; Sansonetti, Philippe; Eberl, Gérard; Pedron, Thierry; Taha, Muhamed-Kheir; Boneca, Ivo G

    2013-06-12

    Neisseria meningitidis is a life-threatening human bacterial pathogen responsible for pneumonia, sepsis, and meningitis. Meningococcal strains with reduced susceptibility to penicillin G (Pen(I)) carry a mutated penicillin-binding protein (PBP2) resulting in a modified peptidoglycan structure. Despite their antibiotic resistance, Pen(I) strains have failed to expand clonally. We analyzed the biological consequences of PBP2 alteration among clinical meningococcal strains and found that peptidoglycan modifications of the Pen(I) strain resulted in diminished in vitro Nod1-dependent proinflammatory activity. In an influenza virus-meningococcal sequential mouse model mimicking human disease, wild-type meningococci induced a Nod1-dependent inflammatory response, colonizing the lungs and surviving in the blood. In contrast, isogenic Pen(I) strains were attenuated for such response and were out-competed by meningococci sensitive to penicillin G. Our results suggest that antibiotic resistance imposes a cost to the success of the pathogen and may potentially explain the lack of clonal expansion of Pen(I) strains. PMID:23768497

  20. Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide.

    PubMed

    Stohl, Elizabeth A; Seifert, H Steven

    2006-11-01

    The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H(2)O(2)) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H(2)O(2) damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2). PMID:16936020

  1. The number of Neisseria meningitidis type IV pili determines host cell interaction

    PubMed Central

    Imhaus, Anne-Flore; Duménil, Guillaume

    2014-01-01

    As mediators of adhesion, autoaggregation and bacteria-induced plasma membrane reorganization, type IV pili are at the heart of Neisseria meningitidis infection. Previous studies have proposed that two minor pilins, PilV and PilX, are displayed along the pilus structure and play a direct role in mediating these effects. In contrast with this hypothesis, combining imaging and biochemical approaches we found that PilV and PilX are located in the bacterial periplasm rather than along pilus fibers. Furthermore, preventing exit of these proteins from the periplasm by fusing them to the mCherry protein did not alter their function. Deletion of the pilV and pilX genes led to a decrease in the number, but not length, of pili displayed on the bacterial surface indicating a role in the initiation of pilus biogenesis. By finely regulating the expression of a central component of the piliation machinery, we show that the modest reductions in the number of pili are sufficient to recapitulate the phenotypes of the pilV and pilX mutants. We further show that specific type IV pili-dependent functions require different ranges of pili numbers. PMID:24864127

  2. Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae.

    PubMed Central

    Goodman, S D; Scocca, J J

    1988-01-01

    DNA segments from Neisseria gonorrhoeae, cloned and propagated in Escherichia coli, were tested for the ability to competitively inhibit gonococcal transformation. The nucleotide sequences of active segments were determined and compared; these sequences contained the sequence 5' GCCGTCTGAA 3' in common. Subcloning studies confirmed the identity of this sequence as the gonococcal DNA recognition site. The three instances of the recognition sequence isolated from N. gonorrhoeae chromosomal DNA contain the sequence in the immediate neighborhood of its inverted repeat. Because a single copy of the sequence functions as a recognition site, the inverted duplication is not required for specific binding. The dyad symmetric arrangements of the chromosomal recognition sequences may form stable stem-loop structures that can function as terminators or attenuators of transcription. These inverted repeats are located at the boundaries of long open reading frames. The recognition sequence also constitutes part of two other probable terminators of gonococcal genes. We conclude that the signal for recognition of transforming DNA by gonococci is a frequent component of transcriptional terminator sequences. This regulatory function might account for the origin and maintenance of recognition sequences in the chromosomes of Gram-negative transformable bacteria. PMID:3137581

  3. Unusual Genetic Organization of a Functional Type I Protein Secretion System in Neisseria meningitidis

    PubMed Central

    Wooldridge, Karl G.; Kizil, Murat; Wells, Damien B.; Ala'Aldeen, Dlawer A. A.

    2005-01-01

    Proteins secreted by Neisseria meningitidis are thought to play important roles in the pathogenesis of meningococcal disease. These proteins include the iron-repressible repeat-in-toxin (RTX) exoprotein FrpC. Related proteins in other pathogens are secreted via a type I secretion system (TOSS), but such a system has not been demonstrated in N. meningitidis. An in silico search of the group B meningococcal genome suggested the presence of a uniquely organized TOSS. Genes encoding homologs of the Escherichia coli HlyB (ATP-binding), HlyD (membrane fusion), and TolC (outer membrane channel) proteins were identified. In contrast to the cistronic organization of the secretion genes in most other rtx operons, the hlyD and tolC genes were adjacent but unlinked to hlyB; neither locus was part of an operon containing genes encoding putative TOSS substrates. Both loci were flanked by genes normally associated with mobile genetic elements. The three genes were shown to be expressed independently. Mutation at either locus resulted in an inability to secrete FrpC and a related protein, here called FrpC2. Successful complementation of these mutations at an ectopic site confirmed the observed phenotypes were caused by loss of function of the putative TOSS genes. We show that genes scattered in the meningococcal genome encode a functional TOSS required for secretion of the meningococcal RTX proteins. PMID:16113272

  4. Patterns of meningococcal infection in Sudan with emergence of Neisseria meningitidis serogroup W135.

    PubMed

    Karsany, M S; Elshayeb, A A; Saeed, E S; Elaagib, R; Ibrahim, S A; Elsamani, E; Hussien, K; Salih, K M

    2013-10-01

    This study was conducted during the 2004-2006 epidemic of meningitis in Sudan to follow-up the frequency of disease outbreak or endemic waves and to evaluate the new quadrivalent vaccine for actual application. Samples were collected from Darfur, El Gedaref, Kassala and Khartoum States and transported to the National Health Central Laboratory in Khartoum. Of 196 patients with clinical symptoms and signs of meningitis, conventional culture identified Neisseria meningitidis in 37 (189%), confirmed by polymerase chain reaction. N. meningitidis type A was identified in 29 (78.4%) patients, type C in 3 (8.1%) and N. meningitidis W135 in 5 (15.5%). The serotyping and molecular diagnosis patterns of N. meningitidis showed the emergence of the new strain, W135, in 5 (15.5%). The patients from the borders of Sudan, 3 from the West Darfur, and 1 each from El Gedaref and Kassala. These could be related to the movement of pilgrims through the borders to Saudi Arabia in the Hajj season. Serious consideration may be needed for quadrivalent vaccination to prevent seasonal and Hajj season outbreaks. PMID:24313147

  5. Antimicrobial Resistance in Neisseria gonorrhoeae in the 21st Century: Past, Evolution, and Future

    PubMed Central

    Unemo, Magnus

    2014-01-01

    SUMMARY Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhea, which is a major public health concern globally. Given the global nature of gonorrhea, the high rate of usage of antimicrobials, suboptimal control and monitoring of antimicrobial resistance (AMR) and treatment failures, slow update of treatment guidelines in most geographical settings, and the extraordinary capacity of the gonococci to develop and retain AMR, it is likely that the global problem of gonococcal AMR will worsen in the foreseeable future and that the severe complications of gonorrhea will emerge as a silent epidemic. By understanding the evolution, emergence, and spread of AMR in N. gonorrhoeae, including its molecular and phenotypic mechanisms, resistance to antimicrobials used clinically can be anticipated, future methods for genetic testing for AMR might permit region-specific and tailor-made antimicrobial therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. This review focuses on the history and evolution of gonorrhea treatment regimens and emerging resistance to them, on genetic and phenotypic determinants of gonococcal resistance to previously and currently recommended antimicrobials, including biological costs or benefits; and on crucial actions and future advances necessary to detect and treat resistant gonococcal strains and, ultimately, retain gonorrhea as a treatable infection. PMID:24982323

  6. Adherence of clinically isolated lactobacilli to human cervical cells in competition with Neisseria gonorrhoeae.

    PubMed

    Vielfort, Katarina; Sjölinder, Hong; Roos, Stefan; Jonsson, Hans; Aro, Helena

    2008-10-01

    Lactobacilli are normal inhabitants of our microbiota and are known to protect against pathogens. Neisseria gonorrhoeae is a human specific pathogenic bacterium that colonises the urogenital tract where it causes gonorrhoea. In this study we analysed early interactions between lactobacilli and gonococci and investigated how they compete for adherence to human epithelial cervical cells. We show that lactobacilli adhere at various levels and that the number of adherent bacteria does not correlate to the level of protection against gonococcal infection. Protection against gonococcal adhesion varied between Lactobacillus species. Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus reuteri were capable of reducing gonococcal adherence while Lactobacillus rhamnosus was not. Lactobacillus strains of vaginal origin had the best capacity to remain attached to the host cell during gonococcal adherence. Further, we show that gonococci and lactobacilli interact with each other with resultant lactobacilli incorporation into the gonococcal microcolony. Hence, gonococci bind to colonised lactobacilli and this complex frequently detaches from the epithelial cell surface, resulting in reduced bacterial colonisation. Also, purified gonococcal pili are capable of removing adherent lactobacilli from the cell surface. Taken together, we reveal novel data regarding gonococcal and lactobacilli competition for adherence that will benefit future gonococcal prevention and treatments.

  7. Bacteriocins and other bioactive substances of probiotic lactobacilli as biological weapons against Neisseria gonorrhoeae.

    PubMed

    Ruíz, Francisco O; Pascual, Liliana; Giordano, Walter; Barberis, Lucila

    2015-04-01

    In the search of new antimicrobial agents against Neisseria gonorrhoeae, the bacteriocins-producing probiotic lactobacilli deserve special attention. The inhibitory effects of biosubstances such as organic acids, hydrogen peroxide and each bacteriocin-like inhibitory substance (BLIS) L23 and L60 on the growth of different gonococcal strains were investigated. Different non-treated and treated cell-free supernatants of two probiotic lactobacilli containing these metabolites were used. The aims of this work were (i) to evaluate the antimicrobial activity of the biosubstances produced by two probiotic lactobacilli, estimating the proportion in which each of them is responsible for the inhibitory effect, (ii) to define their minimum inhibitory concentrations (MICs) and, (iii) to determine the potential interactions between these biosubstances against N. gonorrhoeae. The main antimicrobial metabolites were the BLIS-es L23 and L60 in comparison with other biosubstances. Proportionally, their contributions to the inhibition on the gonococcal growth were 87.28% and 80.66%, respectively. The MIC values of bacteriocins were promising since these substances, when diluted, showed considerable inhibitory activity for all gonococci. In the interaction between bacteriocins, 100% of synergism was found on the gonococcal growth. In summary, this study indicates that both L23 and L60 could potentially serve to design new bioproducts against N. gonorrhoeae.

  8. Fur-Mediated Activation of Gene Transcription in the Human Pathogen Neisseria gonorrhoeae

    PubMed Central

    Yu, Chunxiao

    2012-01-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  9. Molecular epidemiology of Neisseria gonorrhoeae in Amsterdam, The Netherlands, shows distinct heterosexual and homosexual networks.

    PubMed

    Kolader, Marion-Eliëtte; Dukers, Nicole H T M; van der Bij, Akke K; Dierdorp, Mirjam; Fennema, Johan S A; Coutinho, Roel A; Bruisten, Sylvia M

    2006-08-01

    Molecular typing, added to epidemiological data, can better identify transmission patterns of gonorrhea in Western countries, where the incidence has recently been rising. From September 2002 to September 2003, patients with a laboratory-confirmed diagnosis of gonorrhea at the Clinic for Sexually Transmitted Infections in Amsterdam, The Netherlands, were subjected to a questionnaire pertaining to sexual risk behavior and sexual partners in the 6 months prior to the diagnosis. The Neisseria gonorrhoeae isolates were all genotyped using PCR-restriction fragment length polymorphism of the porin and opacity genes. All patients with a completed questionnaire and genotyped isolates were included in the study. We obtained 885 N. gonorrhoeae isolates from 696 patients that revealed 88 clusters and 46 unique genotypes. Patients infected at multiple anatomical sites with one or more strains and patients infected several times during the study period were shown to pursue high-risk sexual behavior and were considered core groups. There were 11 clusters of > or =20 patients; in seven clusters, 81% to 100% of patients were men who have sex with men (MSM), three clusters contained 87 to 100% heterosexual men and women, and one cluster was formed by equal proportions of MSM and heterosexual male and female patients. However, the various clusters differed in characteristics such as types of coinfections, numbers of sexual partners, Internet use to seek sexual partners, and locations of sexual encounters. Molecular epidemiology of gonococcal isolates in Amsterdam revealed core groups and clusters of MSM and heterosexual patients that probably indicate distinct transmission networks.

  10. Purification and characterization of an esterase involved in cellulose acetate degradation by Neisseria sicca SB.

    PubMed

    Moriyoshi, K; Ohmoto, T; Ohe, T; Sakai, K

    1999-10-01

    An esterase catalyzing the hydrolysis of acetyl ester moieties in cellulose acetate was purified 1,110-fold to electrophoretic homogeneity from the culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The purified enzyme was a monomeric protein with a molecular mass of 40 kDa and the isoelectric point was 5.3. The pH and temperature optima of the enzyme were 8.0-8.5 and 45 degrees C. The enzyme catalyzed the hydrolysis of acetyl saccharides, p-nitrophenyl esters of short-chain fatty acids, and was slightly active toward aliphatic and aromatic esters. The K(m) and Vmax for cellulose acetate (degree of substitution, 0.88) and p-nitrophenyl acetate were 0.0162% (716 microM as acetyl content in the polymer) and 36.0 microM, and 66.8 and 39.1 mumol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, which indicated that the enzyme was a serine esterase.

  11. Characterization of invasive Neisseria meningitidis strains isolated at the Children's Hospital of Tunis, Tunisia.

    PubMed

    Saguer, A; Smaoui, H; Taha, M-K; Kechrid, A

    2016-05-01

    Neisseria meningitidis, a leading cause of bacterial meningitis and other serious infections, is responsible for approximately one-third of cases of bacterial meningitis in the Children's Hospital of Tunis. The serogroup distribution, antibiotic susceptibility and antigenic and molecular characteristics of N. meningitidis isolates were determined in patients aged 3 days-13 years between February 1998 and June 2013. In all 107 invasive strains of N. meningitidis were isolated. Reduced susceptibility to penicillin G was seen in 55.7% of isolates, with a low level of resistance in all cases; 28.4% showed a low level of resistance to amoxicillin. Serogroup B isolates were the most frequent (80.4%), followed by serogroups C (12.2%) and A (5.6%). Isolates of serogroup A had the same antigenic formula (A:4:P1.9), the same variable regions VR1, VR2 and VR3, and belonged to the same clonal complex (CC5). Isolates of serogroups B and C were more heterogeneous with several antigenic formulae. The most frequent clonal complex in these isolates was CC35. Serogroup B accounted for a large percentage of our isolates with marked diversity. PMID:27553401

  12. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells.

    PubMed

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-03-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications.

  13. Antibacterial activity of resazurin-based compounds against Neisseria gonorrhoeae in vitro and in vivo.

    PubMed

    Schmitt, Deanna M; Connolly, Kristie L; Jerse, Ann E; Detrick, Melinda S; Horzempa, Joseph

    2016-10-01

    Neisseria gonorrhoeae is the cause of the second most common sexually transmitted bacterial infection, with ca. 80 million new cases of gonorrhoea reported annually. The recent emergence of clinical isolates resistant to the last monotherapy against this bacterium, the cephalosporins, illustrates the need for new antigonococcal agents. Here we have characterised a new group of antimicrobials based on the compound resazurin that exhibits robust activity against N. gonorrhoeae in vitro. Resazurin inhibits the growth of a broad range of N. gonorrhoeae isolates, including those resistant to multiple antibiotics. Furthermore, treatment of human endometrial cells infected with N. gonorrhoeae with resazurin significantly reduces the number of intracellular bacteria. Whilst resazurin exhibited potent in vitro antimicrobial activity, in vivo resazurin did not limit the colonisation of mice with N. gonorrhoeae following vaginal infection. The ineffectiveness of resazurin in vivo is likely due to its interaction with serum albumin, which completely diminishes its antimicrobial activity. However, treatment of mice with a resazurin analogue (resorufin pentyl ether) that maintains its antimicrobial activity in the presence of serum albumin approached a significant decrease in the percentage of mice vaginally colonised. This treatment also decreased vaginal colonisation by N. gonorrhoeae over time. Together, these data suggest that resazurin derivatives have potential for the treatment of gonorrhoea. PMID:27451856

  14. Regulation of catalase in Neisseria gonorrhoeae. Effects of oxidant stress and exposure to human neutrophils.

    PubMed

    Zheng, H Y; Hassett, D J; Bean, K; Cohen, M S

    1992-09-01

    We studied the effects of oxidant stress on the catalase activity and hydrogen peroxide sensitivity of Neisseria gonorrhoeae. N. gonorrhoeae is an obligate pathogen of man that evokes a remarkable but ineffective neutrophil response. Gonococci make no superoxide dismutase but express high catalase activity. Gonococcal catalase activity increased threefold when organisms were subjected to 1.0 mM hydrogen peroxide. This increase in catalase activity was marked by a parallel increase in protein concentration recognized by a rabbit polyclonal antibody raised against the purified gonococcal enzyme. Catalase was primarily localized to the gonococcal cytoplasm in the presence or absence of stress; only a single isoenzyme of catalase could be identified. Exposure of gonococci to neutrophil-derived oxidants was accomplished by stimulating neutrophils with phorbol myristate acetate or by using gonococcal Opa variants that interacted with neutrophils with different degrees of efficiency. Gonococci exposed to neutrophils demonstrated a twofold increase in catalase activity in spite of some reduction in viability. Exposure of gonococci to 1.0 mM hydrogen peroxide made the organisms significantly more resistant to higher concentrations of hydrogen peroxide and to neutrophils than control organisms. These results suggest that catalase is an important defense for N. gonorrhoeae during attack by human neutrophils. The rapid response of this enzyme to hydrogen peroxide should be taken into consideration in studies designed to evaluate the interaction between neutrophils and gonococci. PMID:1522209

  15. Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase.

    PubMed

    Daudé, David; Topham, Christopher M; Remaud-Siméon, Magali; André, Isabelle

    2013-12-01

    The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases that naturally catalyzes the synthesis of α-glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite -1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure-based approach was followed to reshape the subsite -1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site-directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8 °C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite -1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes.

  16. Essential role of amino acid position 226 in oligosaccharide elongation by amylosucrase from Neisseria polysaccharea.

    PubMed

    Cambon, Emmanuelle; Barbe, Sophie; Pizzut-Serin, Sandra; Remaud-Simeon, Magali; André, Isabelle

    2014-09-01

    Amylosucrase from Neisseria polysaccharea is a remarkable transglucosylase that synthesizes an insoluble amylose-like polymer from sole substrate sucrose. One particular amino acid, Arg226, was proposed from molecular modeling studies to play an important role in the formation of the active site topology and in the accessibility of ligands to the catalytic site. The systematic mutation of this Arg residue by all 19 other possible amino acids revealed that all single-mutants had a higher activity on sucrose compared to the wild-type enzyme. An extensive kinetic investigation showed that catalytic efficiencies are greatly impacted by the presence of natural acceptors in the reaction media, their chain length and the nature of the amino acid at position 226. Compared to the wild-type enzyme, the R226N mutant showed a 10-fold enhancement in the catalytic efficiency and a nearly twofold higher production of an insoluble amylose-like polymer that can be of interest for biotechnological applications.

  17. Molecular Engineering of Ghfp, the Gonococcal Orthologue of Neisseria meningitidis Factor H Binding Protein.

    PubMed

    Rippa, Valentina; Santini, Laura; Lo Surdo, Paola; Cantini, Francesca; Veggi, Daniele; Gentile, Maria Antonietta; Grassi, Eva; Iannello, Giulia; Brunelli, Brunella; Ferlicca, Francesca; Palmieri, Emiliano; Pallaoro, Michele; Aricò, Beatrice; Banci, Lucia; Pizza, Mariagrazia; Scarselli, Maria

    2015-07-01

    Knowledge of the sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility of unraveling the mechanisms of pathogenesis at the molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability, and variability. In the present study, we used epitope mapping information on the well-characterized antigen of Neisseria meningitidis factor H binding protein (fHbp) to engineer its gonococcal homologue, Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy for modulating immune responses, and it is a powerful tool for investigating the extension and localization of immunodominant epitopes.

  18. Notes from the Field: Increase in Neisseria meningitidis-Associated Urethritis Among Men at Two Sentinel Clinics - Columbus, Ohio, and Oakland County, Michigan, 2015.

    PubMed

    Bazan, Jose A; Peterson, Amy S; Kirkcaldy, Robert D; Briere, Elizabeth C; Maierhofer, Courtney; Turner, Abigail Norris; Licon, Denisse B; Parker, Nicole; Dennison, Amanda; Ervin, Melissa; Johnson, Laura; Weberman, Barbara; Hackert, Pamela; Wang, Xin; Kretz, Cecilia B; Abrams, A Jeanine; Trees, David L; Del Rio, Carlos; Stephens, David S; Tzeng, Yih-Ling; DiOrio, Mary; Roberts, Mysheika Williams

    2016-01-01

    Neisseria meningitidis (Nm) urogenital infections, although less common than infections caused by Neisseria gonorrhoeae (Ng), have been associated with urethritis, cervicitis, proctitis, and pelvic inflammatory disease. Nm can appear similar to Ng on Gram stain analysis (gram-negative intracellular diplococci) (1-5). Because Nm colonizes the nasopharynx, men who receive oral sex (fellatio) can acquire urethral Nm infections (1,3,5). This report describes an increase in Nm-associated urethritis in men attending sexual health clinics in Columbus, Ohio, and Oakland County, Michigan.

  19. Notes from the Field: Increase in Neisseria meningitidis-Associated Urethritis Among Men at Two Sentinel Clinics - Columbus, Ohio, and Oakland County, Michigan, 2015.

    PubMed

    Bazan, Jose A; Peterson, Amy S; Kirkcaldy, Robert D; Briere, Elizabeth C; Maierhofer, Courtney; Turner, Abigail Norris; Licon, Denisse B; Parker, Nicole; Dennison, Amanda; Ervin, Melissa; Johnson, Laura; Weberman, Barbara; Hackert, Pamela; Wang, Xin; Kretz, Cecilia B; Abrams, A Jeanine; Trees, David L; Del Rio, Carlos; Stephens, David S; Tzeng, Yih-Ling; DiOrio, Mary; Roberts, Mysheika Williams

    2016-01-01

    Neisseria meningitidis (Nm) urogenital infections, although less common than infections caused by Neisseria gonorrhoeae (Ng), have been associated with urethritis, cervicitis, proctitis, and pelvic inflammatory disease. Nm can appear similar to Ng on Gram stain analysis (gram-negative intracellular diplococci) (1-5). Because Nm colonizes the nasopharynx, men who receive oral sex (fellatio) can acquire urethral Nm infections (1,3,5). This report describes an increase in Nm-associated urethritis in men attending sexual health clinics in Columbus, Ohio, and Oakland County, Michigan. PMID:27254649

  20. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing. PMID:27225407

  1. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

  2. Effect of Variants of Penicillin-Binding Protein 2 on Cephalosporin and Carbapenem Susceptibilities in Neisseria gonorrhoeae

    PubMed Central

    Bharat, Amrita; Demczuk, Walter; Martin, Irene

    2015-01-01

    To characterize the relationship between penicillin-binding protein 2 (PBP2/penA) and susceptibility to extended-spectrum cephalosporins (ESCs) and carbapenem antibiotics, we compared 17 PBP2 variants in Neisseria gonorrhoeae. Nonmosaic and mosaic variants of PBP2 caused decreased susceptibility to ESCs and, to a lesser extent, to carbapenems. An A501P substitution in mosaic XXXIV_A501P conferred decreased susceptibility to ESCs but restored carbapenem susceptibility to wild-type levels. These results could aid the molecular surveillance of antimicrobial resistance to these agents. PMID:25987627

  3. Effect of anti-pilus antisera on virulence of variants of Neisseria gonorrhoeae for cultured epithelial cells.

    PubMed

    Virji, M; Everson, J S; Lambden, P R

    1982-05-01

    Variants of Neisseria gonorrhoeae P9 possessing a, gamma or delta pili were shown to vary in their toxicity and virulence for human epithelial cells. Studies with antisera raised against purified pili showed that attachment and virulence were reduced to a significant degree in the presence of antisera to homologous pili. Heterologous antisera, while capable of agglutinating whole organisms, were largely ineffective in reducing attachment and cytotoxicity. Using an enzyme-linked immunosorbent assay (ELISA) system, the cross-reactivity between pili and heterologous antisera was estimated to be no more than 10-20%.

  4. New Ceftriaxone- and Multidrug-Resistant Neisseria gonorrhoeae Strain with a Novel Mosaic penA Gene Isolated in Japan.

    PubMed

    Nakayama, Shu-Ichi; Shimuta, Ken; Furubayashi, Kei-Ichi; Kawahata, Takuya; Unemo, Magnus; Ohnishi, Makoto

    2016-07-01

    We have characterized in detail a new ceftriaxone- and multidrug-resistant Neisseria gonorrhoeae strain (FC428) isolated in Japan in 2015. FC428 differed from previous ceftriaxone-resistant strains and contained a novel mosaic penA allele encoding a new mosaic penicillin-binding protein 2 (PBP 2). However, the resistance-determining 3'-terminal region of penA was almost identical to the regions of two previously reported ceftriaxone-resistant strains from Australia and Japan, indicating that both ceftriaxone-resistant strains and conserved ceftriaxone resistance-determining PBP 2 regions might spread.

  5. Effect of Variants of Penicillin-Binding Protein 2 on Cephalosporin and Carbapenem Susceptibilities in Neisseria gonorrhoeae.

    PubMed

    Bharat, Amrita; Demczuk, Walter; Martin, Irene; Mulvey, Michael R

    2015-08-01

    To characterize the relationship between penicillin-binding protein 2 (PBP2/penA) and susceptibility to extended-spectrum cephalosporins (ESCs) and carbapenem antibiotics, we compared 17 PBP2 variants in Neisseria gonorrhoeae. Nonmosaic and mosaic variants of PBP2 caused decreased susceptibility to ESCs and, to a lesser extent, to carbapenems. An A501P substitution in mosaic XXXIV_A501P conferred decreased susceptibility to ESCs but restored carbapenem susceptibility to wild-type levels. These results could aid the molecular surveillance of antimicrobial resistance to these agents.

  6. New Ceftriaxone- and Multidrug-Resistant Neisseria gonorrhoeae Strain with a Novel Mosaic penA Gene Isolated in Japan.

    PubMed

    Nakayama, Shu-Ichi; Shimuta, Ken; Furubayashi, Kei-Ichi; Kawahata, Takuya; Unemo, Magnus; Ohnishi, Makoto

    2016-07-01

    We have characterized in detail a new ceftriaxone- and multidrug-resistant Neisseria gonorrhoeae strain (FC428) isolated in Japan in 2015. FC428 differed from previous ceftriaxone-resistant strains and contained a novel mosaic penA allele encoding a new mosaic penicillin-binding protein 2 (PBP 2). However, the resistance-determining 3'-terminal region of penA was almost identical to the regions of two previously reported ceftriaxone-resistant strains from Australia and Japan, indicating that both ceftriaxone-resistant strains and conserved ceftriaxone resistance-determining PBP 2 regions might spread. PMID:27067334

  7. Importance of multidrug efflux pumps in the antimicrobial resistance property of clinical multidrug-resistant isolates of Neisseria gonorrhoeae.

    PubMed

    Golparian, Daniel; Shafer, William M; Ohnishi, Makoto; Unemo, Magnus

    2014-06-01

    The contribution of drug efflux pumps in clinical isolates of Neisseria gonorrhoeae that express extensively drug-resistant or multidrug-resistant phenotypes has heretofore not been examined. Accordingly, we assessed the effect on antimicrobial resistance of loss of the three gonococcal efflux pumps associated with a known capacity to export antimicrobials (MtrC-MtrD-MtrE, MacA-MacB, and NorM) in such clinical isolates. We report that the MIC of several antimicrobials, including seven previously and currently recommended for treatment was significantly impacted. PMID:24733458

  8. Identification of TEM-135 β-lactamase in Neisseria gonorrhoeae strains carrying African and Toronto plasmids in Argentina.

    PubMed

    Gianecini, R; Oviedo, C; Littvik, A; Mendez, E; Piccoli, L; Montibello, S; Galarza, P

    2015-01-01

    One hundred forty-three penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates obtained in Argentina from 2008 and 2012 were examined to detect blaTEM-135 genes and to investigate plasmid profiles and multiantigen sequence types. Forty-two PPNG isolates were found to carry TEM-135, and two contained a new TEM derivative characterized as TEM-220. The blaTEM-135 allele was carried by the Toronto/Rio and African plasmids. Molecular epidemiology revealed that two blaTEM-135 isolates were related to previously described isolates from Thailand and China, indicating a common evolutionary origin.

  9. Identification of TEM-135 β-Lactamase in Neisseria gonorrhoeae Strains Carrying African and Toronto Plasmids in Argentina

    PubMed Central

    Gianecini, R.; Oviedo, C.; Littvik, A.; Mendez, E.; Piccoli, L.; Montibello, S.

    2014-01-01

    One hundred forty-three penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates obtained in Argentina from 2008 and 2012 were examined to detect blaTEM-135 genes and to investigate plasmid profiles and multiantigen sequence types. Forty-two PPNG isolates were found to carry TEM-135, and two contained a new TEM derivative characterized as TEM-220. The blaTEM-135 allele was carried by the Toronto/Rio and African plasmids. Molecular epidemiology revealed that two blaTEM-135 isolates were related to previously described isolates from Thailand and China, indicating a common evolutionary origin. PMID:25367903

  10. [Detection of Neisseria gonorrhoeae(NG), Chlamydia trachomatis(CT) and Ureaplasma urealyticum(UU) by multiple primer PCR].

    PubMed

    Lu, Y; Li, J; Chen, Y

    2000-04-01

    For the detection of Neisseria gonorrhoeae(NG), Chlamydia trachomat is(CT) and Ureaplasma urealyticum(UU) in urogenital infection in one specimen, multiple primer PCR was used in 156 sex transmitted disease patients, it was shown that in 156 specimens there were 40 NG positive and 31 CT positive and 27 UU positive, 10 NG + CT positive, 4 NG + UU positive, 3 CT + UU positive and 2 NG + CT + UU positive respectively. The results were in correspondence with each other. It is suggested that multiple primer PCR is a quick, sensitive, simple and specific technique which can be applied in clinical test.

  11. Chemical characterization of binding properties of opacity-associated protein II from Neisseria gonorrhoeae.

    PubMed Central

    Bessen, D; Gotschlich, E C

    1987-01-01

    Binding of an opacity-associated protein II (PIIop) from Neisseria gonorrhoeae to eucaryotic macromolecules was studied. HeLa cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose, and purified PIIop bound to approximately 50 distinct molecular species. The binding of PIIop to HeLa cell components was stable in high salt and nonionic detergent and was not inhibited by a variety of monosaccharides and polyionic substances. PIIop binding behavior was compared with that of two model carbohydrate-binding proteins, wheat germ agglutinin (WGA) and concanavalin A (ConA). Model glycoproteins (ovomucoid, fetuin, mucin, ovalbumin) inhibited binding by PIIop, WGA, and ConA to various degrees. HeLa cell glycopeptides, generated by pronase digestion of chloroform-methanol-extracted cells, were tested for their ability to inhibit binding by PIIop to Western blots of HeLa cell macromolecules. HeLa cell extracts inhibited PIIop binding before pronase treatment, but inhibitory activity was lost as a result of pronase digestion. Direct binding to defined glycosylated and nonglycosylated proteins revealed that ConA and WGA bound only glycoproteins, whereas PIIop bound to proteins lacking carbohydrate as well. PIIop binding to human and bovine serum albumins was of high affinity and required partial unfolding of albumin; native albumin was not bound by PIIop; however, both the denatured, reduced form of albumin and the compact, nonreduced form of carboxymethylated albumin were bound strongly by PIIop. Albumin-PIIop interaction did not involve covalent bond formation through sulfhydryl groups. The predominant binding interactions of PIIop found in this study were with protein rather than carbohydrate, and the chemical nature of the interactions is more complex than involvement of purely ionic or hydrophobic forces. Images PMID:3098683

  12. Proteomics of Neisseria gonorrhoeae: the treasure hunt for countermeasures against an old disease.

    PubMed

    Baarda, Benjamin I; Sikora, Aleksandra E

    2015-01-01

    Neisseria gonorrhoeae is an exquisitely adapted, strictly human pathogen and the causative agent of the sexually transmitted infection gonorrhea. This ancient human disease remains a serious problem, occurring at high incidence globally and having a major impact on reproductive and neonatal health. N. gonorrhoeae is rapidly evolving into a superbug and no effective vaccine exists to prevent gonococcal infections. Untreated or inadequately treated gonorrhea can lead to severe sequelae, including pelvic inflammatory disease and infertility in women, epididymitis in men, and sight-threatening conjunctivitis in infants born to infected mothers. Therefore, there is an immediate need for accelerated research toward the identification of molecular targets for development of drugs with new mechanisms of action and preventive vaccine(s). Global proteomic approaches are ideally suited to guide these studies. Recent quantitative proteomics (SILAC, iTRAQ, and ICAT) have illuminated the pathways utilized by N. gonorrhoeae to adapt to different lifestyles and micro-ecological niches within the host, while comparative 2D SDS-PAGE analysis has been used to elucidate spectinomycin resistance mechanisms. Further, high-throughput examinations of cell envelopes and naturally released membrane vesicles have unveiled the ubiquitous and differentially expressed proteins between temporally and geographically diverse N. gonorrhoeae isolates. This review will focus on these different approaches, emphasizing the role of proteomics in the search for vaccine candidates. Although our knowledge of N. gonorrhoeae has been expanded, still far less is known about this bacterium than the closely related N. meningitidis, where genomics- and proteomics-driven studies have led to the successful development of vaccines. PMID:26579097

  13. Specificity of antibodies against Neisseria gonorrhoeae that stimulate neutrophil chemotaxis. Role of antibodies directed against lipooligosaccharides.

    PubMed Central

    Densen, P; Gulati, S; Rice, P A

    1987-01-01

    Five strains each of Neisseria gonorrhoeae sensitive or resistant to complement (C) dependent killing by normal human serum (NHS) were examined for their ability to stimulate chemotaxis of polymorphonuclear leukocytes (PMNs) after preincubation with NHS; or IgM or IgG derived from NHS. Serum-sensitive N. gonorrhoeae stimulated C-dependent chemotaxis when opsonized with IgM, but not IgG, however, serum-resistant strains, taken as a whole, failed to promote chemotaxis when opsonized with either isotype. IgM titers in NHS against lipooligosaccharide (LOS) antigens from individual serum-sensitive, but not serum-resistant strains, correlated with the magnitude of chemotaxis generated by the corresponding opsonized strains (r = 0.99). Western blots demonstrated that IgM and IgG from NHS recognized different antigenic determinants on LOS from serum-sensitive gonococci. IgM from NHS immunopurified against serum-sensitive LOS accounted for two-thirds of the chemotaxis promoting activity present in whole serum. IgG titers in NHS against LOS antigens from individual serum-resistant strains also correlated with magnitude of chemotaxis generated by the corresponding opsonized strains (r = 0.87), although most opsonized serum-resistant strains did not generate significantly higher magnitudes of chemotaxis than controls. In contrast, a serum-resistant isolate from a patient with disseminated gonococcal infection (DGI) stimulated chemotaxis when opsonized with IgG obtained from the patient's convalescent serum. By Western blot, convalescent IgG antibody recognized an additional determinant on serum-resistant LOS not seen by normal IgG. Images PMID:2439546

  14. Characterization of epidemic and nonepidemic Neisseria meningitidis serogroup A strains from Sudan and Sweden.

    PubMed Central

    Salih, M A; Danielsson, D; Bäckman, A; Caugant, D A; Achtman, M; Olcén, P

    1990-01-01

    A random selection of 25 strains isolated during an epidemic caused by serogroup A Neisseria meningitidis in Sudan (1988), 3 preepidemic meningococcal strains (1985), and 26 serogroup A strains isolated from sporadic cases of meningitis in Sweden (1973 to 1987) were assessed for multilocus enzyme genotypes (ETs), DNA restriction enzyme patterns, outer membrane proteins, lipopolysaccharides, pilus formation, and antibiograms. All of the 25 Sudanese epidemic isolates and 22 of the Swedish strains were of the same or closely related ETs (ETs 3, 4, and 5), corresponding to clone III-1, which has been responsible for two pandemic waves in the last three decades. The earlier pandemic involved Scandinavia, and the last one caused an outbreak during the pilgrimage to Mecca, Saudi Arabia (August 1987), spreading to Sudan, Chad, and Ethiopia. The three Sudanese preepidemic isolates (1985) were clone IV-1 (sulfonamide susceptible), which has been resident in the African meningitis belt for the last 25 years. The uniformity of clone III-1 strains (all sulfonamide resistant) from Sudan and Sweden was confirmed by DNA restriction enzyme patterns. ETs 3, 4, and 5 from Sudan and Sweden had 86 to 100% similarity to a Swedish clone III-1 reference strain, whereas ETs 1, 2, 6, and 7 showed 50 to 80% similarity. Class 1 protein for clone III-1 showed serosubtype antigens P1.9 and P1.x, whereas ET6 strains (clone IV-1) had serosubtype P1.7. Lipopolysaccharides were variable in the Sudanese and Swedish strains. Pili were expressed in all clone III-1 isolates from Sudan and Sweden but in none of the clone IV-1 isolates (Sudan, 1985). Images PMID:1975593

  15. A Novel Factor H-Fc Chimeric Immunotherapeutic Molecule against Neisseria gonorrhoeae.

    PubMed

    Shaughnessy, Jutamas; Gulati, Sunita; Agarwal, Sarika; Unemo, Magnus; Ohnishi, Makoto; Su, Xia-Hong; Monks, Brian G; Visintin, Alberto; Madico, Guillermo; Lewis, Lisa A; Golenbock, Douglas T; Reed, George W; Rice, Peter A; Ram, Sanjay

    2016-02-15

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including N. gonorrhoeae, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18-20 (FH18-20). We explored the use of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic. FH18-20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18-20 that eliminated complement activation on host cells, yet maintained binding to N. gonorrhoeae, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18-20/Fc but, unlike FH18-20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical N. gonorrhoeae isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10 of 15 (67%) strains, and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant N. gonorrhoeae. PMID:26773149

  16. Molecular dynamics simulations of a bacterial autotransporter: NalP from Neisseria meningitidis.

    PubMed

    Khalid, Syma; Sansom, Mark S P

    2006-01-01

    NalP is an autotransporter secretory protein found in the outer membrane of Neisseria meningitidis. The crystal structure of the NalP translocator domain revealed a transmembrane beta-barrel containing a central alpha-helix. The role of this alpha-helix, and of the conformational dynamics of the beta-barrel pore have been studied via atomistic molecular dynamics simulations. Three simulations, each of 10 ns duration, of NalP embedded within a solvated DMPC bilayer were performed. The helix was removed from the barrel interior in one simulation. The conformational stability of the protein is similar to that of other outer membrane proteins, e.g., OmpA, in comparable simulations. The transmembrane beta-barrel is stable even in the absence of the alpha-helix. Removal of the helix results in an influx of water into the pore region, suggesting the helix acts as a 'plug'. Water molecules entering the resultant pore form hydrogen bonds with the barrel lining that compensate for the loss of helix-barrel hydrogen bonds. The dimensions of the pore fluctuate over the course of the simulation revealing it to be flexible, but only wide enough to allow transport of the passenger domain in an unfolded or extended conformation. The simulations help us to understand the role of the central helix in plugging the pore and in maintaining the width of the barrel, and show that the NalP monomer is sufficient for the transport of the passenger domain in an unfolded or extended conformation. PMID:17127622

  17. Extragenital Infections Caused by Chlamydia trachomatis and Neisseria gonorrhoeae: A Review of the Literature.

    PubMed

    Chan, Philip A; Robinette, Ashley; Montgomery, Madeline; Almonte, Alexi; Cu-Uvin, Susan; Lonks, John R; Chapin, Kimberle C; Kojic, Erna M; Hardy, Erica J

    2016-01-01

    In the United States, sexually transmitted diseases due to Chlamydia trachomatis and Neisseria gonorrhoeae continue to be a major public health burden. Screening of extragenital sites including the oropharynx and rectum is an emerging practice based on recent studies highlighting the prevalence of infection at these sites. We reviewed studies reporting the prevalence of extragenital infections in women, men who have sex with men (MSM), and men who have sex only with women (MSW), including distribution by anatomical site. Among women, prevalence was found to be 0.6-35.8% for rectal gonorrhea (median reported prevalence 1.9%), 0-29.6% for pharyngeal gonorrhea (median 2.1%), 2.0-77.3% for rectal chlamydia (median 8.7%), and 0.2-3.2% for pharyngeal chlamydia (median 1.7%). Among MSM, prevalence was found to be 0.2-24.0% for rectal gonorrhea (median 5.9%), 0.5-16.5% for pharyngeal gonorrhea (median 4.6%), 2.1-23.0% for rectal chlamydia (median 8.9%), and 0-3.6% for pharyngeal chlamydia (median 1.7%). Among MSW, the prevalence was found to be 0-5.7% for rectal gonorrhea (median 3.4%), 0.4-15.5% for pharyngeal gonorrhea (median 2.2%), 0-11.8% for rectal chlamydia (median 7.7%), and 0-22.0% for pharyngeal chlamydia (median 1.6%). Extragenital infections are often asymptomatic and found in the absence of reported risk behaviors, such as receptive anal and oral intercourse. We discuss current clinical recommendations and future directions for research. PMID:27366021

  18. Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae and repeat infection among pregnant urban adolescents

    PubMed Central

    Berggren, Erica K.; Patchen, Loral

    2013-01-01

    BACKGROUND Negative maternal and fetal consequences associated with Chlamydia trachomatis and Neisseria gonorrhoeae during pregnancy make diagnosis essential. The CDC recommends routine screening for sexually transmitted infections at the first prenatal visit, and third trimester repeat screening, specifically for C. trachomatis, is recommended for women under age 25 or at increased infection risk. The impact of repeat screening on diagnosis during pregnancy is not well documented among adolescents. METHODS A prospective cohort of 125 pregnant adolescents with at least one prenatal screen for C. trachomatis and N. gonorrhoeae was analyzed. All participants received prenatal care and delivered at one urban teaching hospital in Washington, DC. Screening results were documented for both sexually transmitted infections. Descriptive and univariate analyses were performed to describe disease prevalence. RESULTS Thirty-one percent of pregnant adolescents were diagnosed with either C. trachomatis or N. gonorrhoeae infection during pregnancy. Of the 75% (95/125) of patients who had more than one screening test, 11% (10/95) had a re-infection, and 7% (7/95) had a new infection on repeat testing. Nine percent (9/95) had recurrent C. trachomatis, while 4% (4/95) had a new diagnosis. Three percent (3/95) had recurrent N. gonorrhoeae, while 4% (4/95) had a new diagnosis. Some experienced co-infection at either initial or repeat testing. CONCLUSIONS Screening for C. trachomatis and N. gonorrhoeae is recommended during pregnancy. In this sample of pregnant adolescents, the overall high incidence and recurrence of C. trachomatis and N. gonorrhoeae support CDC screening and re-screening recommendations, regardless of initial test results. PMID:20938375

  19. Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system.

    PubMed

    Hamilton, Holly L; Domínguez, Nadia M; Schwartz, Kevin J; Hackett, Kathleen T; Dillard, Joseph P

    2005-03-01

    The process of DNA donation for natural transformation of bacteria is poorly understood and has been assumed to involve bacterial cell death. Recently in Neisseria gonorrhoeae we found that mutations in three genes in the gonococcal genetic island (GGI) reduced the ability of a strain to act as a donor in transformation and to release DNA into the culture. To better characterize the GGI and the process of DNA donation, the 57 kb genetic island was cloned, sequenced and subjected to insertional mutagenesis. DNA sequencing revealed that the GGI has characteristics of a horizontally acquired genomic island and encodes homologues of type IV secretion system proteins. The GGI was found to be incorporated near the chromosomal replication terminus at the dif site, a sequence targeted by the site-specific recombinase XerCD. Using a plasmid carrying a small region of the GGI and the associated dif site, we demonstrated that this model island could be integrated at the dif site in strains not carrying the GGI and was spontaneously excised from that site. Also, we were able to delete the entire 57 kb region by transformation with DNA from a strain lacking the GGI. Thus the GGI was likely acquired and integrated into the gonococcal chromosome by site-specific recombination and may be lost by site-specific recombination or natural transformation. We made mutations in six putative type IV secretion system genes and assayed these strains for the ability to secrete DNA. Five of the mutations greatly reduced or completely eliminated DNA secretion. Our data indicate that N. gonorrhoeae secretes DNA via a specific process. Donated DNA may be used in natural transformation, contributing to antigenic variation and the spread of antibiotic resistance, and it may modulate the host immune response.

  20. Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies.

    PubMed

    Forest, K T; Bernstein, S L; Getzoff, E D; So, M; Tribbick, G; Geysen, H M; Deal, C D; Tainer, J A

    1996-02-01

    The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber.

  1. Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission.

    PubMed

    Klotman, Mary E; Rapista, Aprille; Teleshova, Natalia; Micsenyi, Amanda; Jarvis, Gary A; Lu, Wuyuan; Porter, Edith; Chang, Theresa L

    2008-05-01

    Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.

  2. Azithromycin Resistance and Its Mechanism in Neisseria gonorrhoeae Strains in Hyogo, Japan

    PubMed Central

    Osawa, Kayo; Miura, Makiko; Tanaka, Kazushi; Arakawa, Soichi; Shirakawa, Toshiro; Fujisawa, Masato

    2015-01-01

    Therapeutic options are limited for Neisseria gonorrhoeae infection, especially for oral drugs. The purpose of this study was to investigate the susceptibility of N. gonorrhoeae to oral azithromycin (AZM) and the correlation between AZM resistance-related gene mutations and MIC. We examined the AZM MICs of clinical strains of N. gonorrhoeae, sequenced the peptidyltransferase loop in domain V of 23S rRNA, and investigated the statistical correlation between AZM MIC and the presence and number of the mutations. Among 59 N. gonorrhoeae strains, our statistical data showed that a deletion mutation was seen significantly more often in the higher-MIC group (0.5 μg/ml or higher) (35/37; 94.6%) than in the lower-MIC group (0.25 μg/ml or less) (4/22; 18.2%) (P < 0.0001). However, a mutation of codon 40 (Ala→Asp) in the mtrR gene (helix-turn-helix) was seen significantly more often in the lower-MIC group (12/22; 54.5%) (P < 0.0001). In N. gonorrhoeae multiantigen sequence typing (NG-MAST) analyses, ST4777 was representative of the lower-MIC group and ST1407, ST6798, and ST6800 were representative of the higher-MIC group. NG-MAST type 1407 was detected as the most prevalent type in AZM-resistant or -intermediate strains, as previously described. In conclusion, a deletion mutation in the mtrR promoter region may be a significant indicator for higher MIC (0.5 μg/ml or higher). ST4777 was often seen in the lower-MIC group, and ST1407, ST6798, and ST6800 were characteristic of the higher-MIC group. Further research with a greater number of strains would help elucidate the mechanism of AZM resistance in N. gonorrhoeae infection. PMID:25712352

  3. Cephalosporin susceptibility among Neisseria gonorrhoeae isolates--United States, 2000-2010.

    PubMed

    2011-07-01

    Neisseria gonorrhoeae is a major cause of pelvic inflammatory disease, ectopic pregnancy, and infertility, and it can facilitate human immunodeficiency virus (HIV) transmission. Emergence of gonococcal resistance to penicillin and tetracycline occurred during the 1970s and became widespread during the early 1980s. More recently, resistance to fluoroquinolones developed. Resistance was documented first in Asia, then emerged in the United States in Hawaii followed by other western states. It then became prevalent in all other regions of the United States. In Hawaii, fluoroquinolone resistance was first noted among heterosexuals; however, resistance in the United States initially became prevalent among men who have sex with men (MSM) before generalizing to heterosexuals. This emergence of resistance led CDC, in 2007, to discontinue recommending any fluoroquinolone regimens for the treatment of gonorrhea. CDC now recommends dual therapy for gonorrhea with a cephalosporin (ceftriaxone 250 mg) plus either azithromycin or doxycycline. This report summarizes trends in cephalosporin susceptibility among N. gonorrhoeae isolates in the United States during 2000-2010 using data from the Gonococcal Isolate Surveillance Project (GISP). During that period, the percentage of isolates with elevated minimum inhibitory concentrations (MICs) to cephalosporins (≥0.25 µg/mL for cefixime and ≥0.125 µg/mL for ceftriaxone) increased from 0.2% in 2000 to 1.4% in 2010 for cefixime and from 0.1% in 2000 to 0.3% in 2010 for ceftriaxone. Although cephalosporins remain an effective treatment for gonococcal infections, health-care providers should be vigilant for treatment failure and are requested to report its occurrence to state and local health departments. State and local public health departments should promote maintenance of laboratory capability to culture N. gonorrhoeae to allow testing of isolates for cephalosporin resistance. They also should develop enhanced surveillance and

  4. Emergence of decreased susceptibility and resistance to extended-spectrum cephalosporins in Neisseria gonorrhoeae in Korea

    PubMed Central

    Lee, Hyukmin; Unemo, Magnus; Kim, Hyo Jin; Seo, Younghee; Lee, Kyungwon; Chong, Yunsop

    2015-01-01

    Objectives Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern globally; however, no comprehensive AMR data for gonococcal isolates cultured after 2006 in Korea have been published internationally. We determined the susceptibility of N. gonorrhoeae isolates cultured in 2011–13, the mechanism of extended-spectrum cephalosporin (ESC) resistance and the molecular epidemiology of gonococcal strains in Korea. Methods In 2011–13, 210 gonococcal isolates were collected in Korea and their AMR profiles were examined by the agar dilution method. The penA, mtrR, penB, ponA and pilQ genes were sequenced in 25 isolates that were resistant to ESCs and 70 randomly selected isolates stratified by year. For molecular epidemiology, N. gonorrhoeae multiantigen sequence typing and MLST were performed. Results None of the N. gonorrhoeae isolates was susceptible to penicillin G and most were resistant to tetracycline (50%) and ciprofloxacin (97%). The rates of resistance to ceftriaxone, azithromycin, cefpodoxime and cefixime were 3%, 5%, 8% and 9%, respectively. However, all isolates were susceptible to spectinomycin. Twenty-one (84%) of the 25 ESC-resistant isolates contained the non-mosaic PBP2 XIII allele; however, the remaining 4 (16%) possessed the mosaic PBP2 X allele, which has been previously associated with ESC resistance including treatment failures. Conclusions In Korea, susceptibility to spectinomycin remains high. However, the recent emergence of ESC-resistant N. gonorrhoeae strains, including strains possessing the PBP2 mosaic X and non-mosaic XIII alleles, is a major concern and enhanced AMR surveillance is necessary to prevent transmission of these strains. PMID:26084303

  5. DNA Methylation Assessed by SMRT Sequencing Is Linked to Mutations in Neisseria meningitidis Isolates

    PubMed Central

    Sater, Mohamad R. Abdul; Lamelas, Araceli; Wang, Guilin; Clark, Tyson A.; Röltgen, Katharina; Mane, Shrikant; Korlach, Jonas; Pluschke, Gerd; Schmid, Christoph D.

    2015-01-01

    The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of

  6. Intracellular Survival of Neisseria gonorrhoeae in Male Urethral Epithelial Cells: Importance of a Hexaacyl Lipid A

    PubMed Central

    Post, Deborah M. B.; Phillips, Nancy J.; Shao, Jian Q.; Entz, David D.; Gibson, Bradford W.; Apicella, Michael A.

    2002-01-01

    Neisseria gonorrhoeae is a strict human pathogen that invades and colonizes the urogenital tracts of males and females. Lipooligosaccharide (LOS) has been shown to play a role in gonococcal pathogenesis. The acyl transferase MsbB is involved in the biosynthesis of the lipid A portion of the LOS. In order to determine the role of an intact lipid A structure on the pathogenesis of N. gonorrhoeae, the msbB gene was cloned and sequenced, a deletion and insertion mutation was introduced into N. gonorrhoeae, and the mutant strain was designated 1291A11K3. Mass spectrometric analyses of 1291A11K3 LOS determined that this mutation resulted in a pentaacyl rather than a hexaacyl lipid A structure. These analyses also demonstrated an increase in the phosphorylation of lipid A and an increase in length of the oligosaccharide of a minor species of the msbB LOS. The interactions of this mutant with male urethral epithelial cells (uec) were examined. Transmission and scanning electron microscopy studies indicated that the msbB mutants formed close associations with and were internalized by the uec at levels similar to those of the parent strain. Gentamicin survival assays performed with 1291A11K3 and 1291 bacteria demonstrated that there was no difference in the abilities of the two strains to adhere to uec; however, significantly fewer 1291A11K3 bacteria than parent strain bacteria were recovered from gentamicin-treated uec. These studies suggest that the lipid A modification in the N. gonorrhoeae msbB mutant may render it more susceptible to innate intracellular killing mechanisms when internalized by uec. PMID:11796626

  7. Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation

    PubMed Central

    Gault, Joseph; Ferber, Mathias; Machata, Silke; Imhaus, Anne-Flore; Malosse, Christian; Charles-Orszag, Arthur; Millien, Corinne; Bouvier, Guillaume; Bardiaux, Benjamin; Péhau-Arnaudet, Gérard; Klinge, Kelly; Podglajen, Isabelle; Ploy, Marie Cécile; Seifert, H. Steven; Nilges, Michael; Chamot-Rooke, Julia; Duménil, Guillaume

    2015-01-01

    The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences. PMID:26367394

  8. Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation*S⃞

    PubMed Central

    Faridmoayer, Amirreza; Fentabil, Messele A.; Haurat, M. Florencia; Yi, Wen; Woodward, Robert; Wang, Peng George; Feldman, Mario F.

    2008-01-01

    Neisseria meningitidis PglL belongs to a novel family of bacterial oligosaccharyltransferases (OTases) responsible for O-glycosylation of type IV pilins. Although members of this family are widespread among pathogenic bacteria, there is little known about their mechanism. Understanding the O-glycosylation process may uncover potential targets for therapeutic intervention, and can open new avenues for the exploitation of these pathways for biotechnological purposes. In this work, we demonstrate that PglL is able to transfer virtually any glycan from the undecaprenyl pyrophosphate (UndPP) carrier to pilin in engineered Escherichia coli and Salmonella cells. Surprisingly, PglL was also able to interfere with the peptidoglycan biosynthetic machinery and transfer peptidoglycan subunits to pilin. This represents a previously unknown post-translational modification in bacteria. Given the wide range of glycans transferred by PglL, we reasoned that substrate specificity of PglL lies in the lipid carrier. To test this hypothesis we developed an in vitro glycosylation system that employed purified PglL, pilin, and the lipid farnesyl pyrophosphate (FarPP) carrying a pentasaccharide that had been synthesized by successive chemical and enzymatic steps. Although FarPP has different stereochemistry and a significantly shorter aliphatic chain than the natural lipid substrate, the pentasaccharide was still transferred to pilin in our system. We propose that the primary roles of the lipid carrier during O-glycosylation are the translocation of the glycan into the periplasm, and the positioning of the pyrophosphate linker and glycan adjacent to PglL. The unique characteristics of PglL make this enzyme a promising tool for glycoengineering novel glycan-based vaccines and therapeutics. PMID:18930921

  9. H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae.

    PubMed

    Masters, Thao L; Wachter, Shaun; Wachter, Jenny; Hill, Stuart A

    2016-01-01

    Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single -35/ -10 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pil-specific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative reverse transcriptase PCR (qRT-PCR) analysis, we have identified three additional non-canonical promoter elements within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE ORF. Using strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. By their nature, promoter sequences tend to be AT-rich. In Escherichia coli, the small DNA-binding protein H-NS binds to AT-rich sequences and inhibits intragenic transcription. In N. gonorrhoeae hns mutants, pilE antisense transcription was increased twofold, with a concomitant decrease in sense transcript levels. However, most noticeably in these mutants, the absence of H-NS protein caused pilE/pilS recombination to increase dramatically when compared with WT values. Consequently, H-NS protein suppresses pilE intragenic transcription as well as antigenic variation through the pilE/pilS recombination system.

  10. The β-Barrel Outer Membrane Protein Assembly Complex of Neisseria meningitidis▿

    PubMed Central

    Volokhina, Elena B.; Beckers, Frank; Tommassen, Jan; Bos, Martine P.

    2009-01-01

    The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the β-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly. PMID:19767435

  11. Extragenital Infections Caused by Chlamydia trachomatis and Neisseria gonorrhoeae: A Review of the Literature

    PubMed Central

    Chan, Philip A.; Montgomery, Madeline; Almonte, Alexi; Lonks, John R.; Chapin, Kimberle C.; Kojic, Erna M.; Hardy, Erica J.

    2016-01-01

    In the United States, sexually transmitted diseases due to Chlamydia trachomatis and Neisseria gonorrhoeae continue to be a major public health burden. Screening of extragenital sites including the oropharynx and rectum is an emerging practice based on recent studies highlighting the prevalence of infection at these sites. We reviewed studies reporting the prevalence of extragenital infections in women, men who have sex with men (MSM), and men who have sex only with women (MSW), including distribution by anatomical site. Among women, prevalence was found to be 0.6–35.8% for rectal gonorrhea (median reported prevalence 1.9%), 0–29.6% for pharyngeal gonorrhea (median 2.1%), 2.0–77.3% for rectal chlamydia (median 8.7%), and 0.2–3.2% for pharyngeal chlamydia (median 1.7%). Among MSM, prevalence was found to be 0.2–24.0% for rectal gonorrhea (median 5.9%), 0.5–16.5% for pharyngeal gonorrhea (median 4.6%), 2.1–23.0% for rectal chlamydia (median 8.9%), and 0–3.6% for pharyngeal chlamydia (median 1.7%). Among MSW, the prevalence was found to be 0–5.7% for rectal gonorrhea (median 3.4%), 0.4–15.5% for pharyngeal gonorrhea (median 2.2%), 0–11.8% for rectal chlamydia (median 7.7%), and 0–22.0% for pharyngeal chlamydia (median 1.6%). Extragenital infections are often asymptomatic and found in the absence of reported risk behaviors, such as receptive anal and oral intercourse. We discuss current clinical recommendations and future directions for research. PMID:27366021

  12. Assessment of Etest as an alternative to agar dilution for antimicrobial susceptibility testing of Neisseria gonorrhoeae.

    PubMed

    Liu, Hsi; Taylor, Thomas H; Pettus, Kevin; Trees, David

    2014-05-01

    We studied whether the Etest can be used as an alternative to agar dilution to determine antimicrobial susceptibilities of ceftriaxone, cefixime, and cefpodoxime in Neisseria gonorrhoeae surveillance. One hundred fifteen clinical and laboratory isolates of N. gonorrhoeae were tested following the Clinical Laboratory Improvement Amendments (CLIA)-approved CLSI standard agar dilution method and, separately, by the Etest according to the manufacturer's recommendations. The MICs were determined and compared. Ten laboratory-generated mutants were used to simulate substantially nonsusceptible specimens. The Etest and agar dilution methods were well correlated. Statistical tests produced regression R2 values of 88%, 82%, and 85% and Pearson correlation coefficients of 92%, 91%, and 92% for ceftriaxone, cefixime, and cefpodoxime, respectively. When paired comparisons were made, the two tests were 88.7%, 80%, and 87% within 1 log2 dilution from each other for ceftriaxone, cefixime, and cefpodoxime, respectively. The within-2-log2 agreements were 99.1%, 98.3%, and 94.8% for ceftriaxone, cefixime, and cefpodoxime, respectively. Notwithstanding the good correlations and the within-2-log2 general agreement, the Etest results produced slightly lower MICs than the agar dilution results. In conclusion, we found that the Etest can be effectively used as an alternative to agar dilution testing to determine the susceptibility of N. gonorrhoeae to ceftriaxone, cefixime, and cefpodoxime, although we recommend further research into extremely resistant isolates. For isolates within the typical range of clinical MICs, reexamination of the Etest interpretation of susceptible and nonsusceptible categories would likely allow for successful transition from agar dilution to the Etest.

  13. A Novel Factor H-Fc Chimeric Immunotherapeutic Molecule against Neisseria gonorrhoeae.

    PubMed

    Shaughnessy, Jutamas; Gulati, Sunita; Agarwal, Sarika; Unemo, Magnus; Ohnishi, Makoto; Su, Xia-Hong; Monks, Brian G; Visintin, Alberto; Madico, Guillermo; Lewis, Lisa A; Golenbock, Douglas T; Reed, George W; Rice, Peter A; Ram, Sanjay

    2016-02-15

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including N. gonorrhoeae, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18-20 (FH18-20). We explored the use of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic. FH18-20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18-20 that eliminated complement activation on host cells, yet maintained binding to N. gonorrhoeae, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18-20/Fc but, unlike FH18-20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical N. gonorrhoeae isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10 of 15 (67%) strains, and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant N. gonorrhoeae.

  14. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners

    PubMed Central

    Bonhoeffer, Sebastian; Low, Nicola; Althaus, Christian L.

    2016-01-01

    The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y−1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y−1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y−1 in HMW and 3.12 y−1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population’s treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread. PMID:27196299

  15. Inhibition of Neisseria gonorrhoeae Type II Topoisomerases by the Novel Spiropyrimidinetrione AZD0914*

    PubMed Central

    Kern, Gunther; Palmer, Tiffany; Ehmann, David E.; Shapiro, Adam B.; Andrews, Beth; Basarab, Gregory S.; Doig, Peter; Fan, Jun; Gao, Ning; Mills, Scott D.; Mueller, John; Sriram, Shubha; Thresher, Jason; Walkup, Grant K.

    2015-01-01

    We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467–474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and β. PMID:26149691

  16. Inhibition of Neisseria gonorrhoeae Type II Topoisomerases by the Novel Spiropyrimidinetrione AZD0914.

    PubMed

    Kern, Gunther; Palmer, Tiffany; Ehmann, David E; Shapiro, Adam B; Andrews, Beth; Basarab, Gregory S; Doig, Peter; Fan, Jun; Gao, Ning; Mills, Scott D; Mueller, John; Sriram, Shubha; Thresher, Jason; Walkup, Grant K

    2015-08-21

    We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and β.

  17. Antibiotic-Resistant Neisseria gonorrhoeae Spread Faster with More Treatment, Not More Sexual Partners.

    PubMed

    Fingerhuth, Stephanie M; Bonhoeffer, Sebastian; Low, Nicola; Althaus, Christian L

    2016-05-01

    The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y-1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y-1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y-1 in HMW and 3.12 y-1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population's treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread.

  18. Azithromycin resistance and its mechanism in Neisseria gonorrhoeae strains in Hyogo, Japan.

    PubMed

    Shigemura, Katsumi; Osawa, Kayo; Miura, Makiko; Tanaka, Kazushi; Arakawa, Soichi; Shirakawa, Toshiro; Fujisawa, Masato

    2015-05-01

    Therapeutic options are limited for Neisseria gonorrhoeae infection, especially for oral drugs. The purpose of this study was to investigate the susceptibility of N. gonorrhoeae to oral azithromycin (AZM) and the correlation between AZM resistance-related gene mutations and MIC. We examined the AZM MICs of clinical strains of N. gonorrhoeae, sequenced the peptidyltransferase loop in domain V of 23S rRNA, and investigated the statistical correlation between AZM MIC and the presence and number of the mutations. Among 59 N. gonorrhoeae strains, our statistical data showed that a deletion mutation was seen significantly more often in the higher-MIC group (0.5 μg/ml or higher) (35/37; 94.6%) than in the lower-MIC group (0.25 μg/ml or less) (4/22; 18.2%) (P < 0.0001). However, a mutation of codon 40 (Ala → Asp) in the mtrR gene (helix-turn-helix) was seen significantly more often in the lower-MIC group (12/22; 54.5%) (P < 0.0001). In N. gonorrhoeae multiantigen sequence typing (NG-MAST) analyses, ST4777 was representative of the lower-MIC group and ST1407, ST6798, and ST6800 were representative of the higher-MIC group. NG-MAST type 1407 was detected as the most prevalent type in AZM-resistant or -intermediate strains, as previously described. In conclusion, a deletion mutation in the mtrR promoter region may be a significant indicator for higher MIC (0.5 μg/ml or higher). ST4777 was often seen in the lower-MIC group, and ST1407, ST6798, and ST6800 were characteristic of the higher-MIC group. Further research with a greater number of strains would help elucidate the mechanism of AZM resistance in N. gonorrhoeae infection.

  19. In Vivo Adaptation and Persistence of Neisseria meningitidis within the Nasopharyngeal Mucosa

    PubMed Central

    Johswich, Kay O.; McCaw, Shannon E.; Islam, Epshita; Sintsova, Anna; Gu, Angel; Shively, John E.; Gray-Owen, Scott D.

    2013-01-01

    Neisseria meningitidis (Nme) asymptomatically colonizes the human nasopharynx, yet can initiate rapidly-progressing sepsis and meningitis in rare instances. Understanding the meningococcal lifestyle within the nasopharyngeal mucosa, a phase of infection that is prerequisite for disease, has been hampered by the lack of animal models. Herein, we compare mice expressing the four different human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) that can bind the neisserial Opa protein adhesins, and find that expression of human CEACAM1 is necessary and sufficient to establish intranasal colonization. During infection, in vivo selection for phase variants expressing CEACAM1-specific Opa proteins occurs, allowing mucosal attachment and entry into the subepithelial space. Consistent with an essential role for Opa proteins in this process, Opa-deficient meningococci were unable to colonize the CEACAM1-humanized mice. While simple Opa-mediated attachment triggered an innate response regardless of meningococcal viability within the inoculum, persistence of viable Opa-expressing bacteria within the CEACAM1-humanized mice was required for a protective memory response to be achieved. Parenteral immunization with a capsule-based conjugate vaccine led to the accumulation of protective levels of Nme-specific IgG within the nasal mucus, yet the sterilizing immunity afforded by natural colonization was instead conferred by Nme-specific IgA without detectable IgG. Considered together, this study establishes that the availability of CEACAM1 helps define the exquisite host specificity of this human-restricted pathogen, displays a striking example of in vivo selection for the expression of desirable Opa variants, and provides a novel model in which to consider meningococcal infection and immunity within the nasopharyngeal mucosa. PMID:23935487

  20. Cross-reactive polyclonal antibodies to the inner core of lipopolysaccharide from Neisseria meningitidis.

    PubMed

    Andersen, Svein Rune; Guthrie, Terry; Guile, Geoffrey R; Kolberg, Jan; Hou, Sam; Hyland, Lisa; Wong, Simon Y C

    2002-03-01

    Sera from mice immunized with native or detergent-extracted outer membrane vesicles derived from lipopolysaccharide (LPS) mutant 44/76(Mu-4) of Neisseria meningitidis were analyzed for antibodies to LPS. The carbohydrate portion of 44/76(Mu-4) LPS consists of the complete inner core, Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->5KDO[4-->2 alpha KDO]. Immunoblot analysis revealed that some sera contained antibodies to wild-type LPS which has a fully extended carbohydrate chain of immunotype L3,7, as well as to the homologous LPS. Sera reacted only weakly to LPS from 44/76(Mu-3), which lacks the terminal glucose of the inner core. No binding to more truncated LPS was observed. Consequently, the cross-reactive epitopes are expressed mainly by the complete inner core. Dephosphorylation of wild-type LPS abolished antibody binding to LPS in all but one serum. Thus, at least two specificities of cross-reactive antibodies exist: one is dependent on phosphoethanolamine groups in LPS, and one is not. Detection of these cross-reactive antibodies strongly supports the notion that epitopes expressed by meningococcal LPS inner core are also accessible to antibodies when the carbohydrate chain is fully extended. Also, these inner core epitopes are sufficiently immunogenic to induce antibody levels detectable in polyclonal antibody responses. Meningococci can escape being killed by antibodies to LPS that bind only to a specific LPS variant, by altering the carbohydrate chain length. Cross-reactive antibodies may prevent such escape. Therefore, inner core LPS structures may be important antigens in future vaccines against meningococcal disease. PMID:11854213