The DNA-binding activity of the Neisseria gonorrhoeae LexA orthologue NG1427 is modulated by oxidation

Neisseria gonorrhoeae is a human-specific organism that is not usually exposed to UV light or chemicals but is likely to encounter reactive oxygen species during infection. Exposure of N. gonorrhoeae to sublethal hydrogen peroxide revealed that the ng1427 gene was upregulated sixfold. N. gonorrhoeae was thought to lack an SOS system, although NG1427 shows amino acid sequence similarity to the SOS response regulator LexA from Escherichia coli. Similar to LexA and other S24 peptidases, NG1427 undergoes autoproteolysis in vitro, which is facilitated by either the gonococcal or E. coli RecA proteins or high pH, and autoproteolysis requires the active and cleavage site residues conserved between LexA and NG1427. NG1427 controls a three gene regulon: itself; ng1428, a Neisseria-specific, putative integral membrane protein; and recN, a DNA repair gene known to be required for oxidative damage survival. Full NG1427 regulon de-repression requires RecA following methyl methanesulphonate or mitomycin C treatment, but is largely RecA-independent following hydrogen peroxide treatment. NG1427 binds specifically to the operator regions of the genes it controls, and DNA binding is abolished by oxidation of the single cysteine residue encoded in NG1427. We propose that NG1427 is inactivated independently of RecA by oxidation.     

Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes

Symptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40–70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase- and RecA-deficient Gc, which are more sensitive to ROS in vitro , had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.     

Properdin is critical for antibody-dependent bactericidal activity against Neisseria gonorrhoeae that recruit C4b-binding protein

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, is an important cause of morbidity worldwide. A safe and effective vaccine against gonorrhea is needed because of emerging resistance of gonococci to almost every class of antibiotic. A gonococcal lipooligosaccharide epitope defined by the mAb 2C7 is being evaluated as a candidate for development of an Ab-based vaccine. Immune Abs against N. gonorrhoeae need to overcome several subversive mechanisms whereby gonococcus evades complement, including binding to C4b-binding protein (C4BP; classical pathway inhibitor) and factor H (alternative pathway [AP] inhibitor). The role of AP recruitment and, in particular, properdin in assisting killing of gonococci by specific Abs is the subject of this study. We show that only those gonococcal strains that bind C4BP require properdin for killing by 2C7, whereas strains that do not bind C4BP are efficiently killed by 2C7 even when AP function is blocked. C3 deposition on bacteria mirrored killing. Recruitment of the AP by mAb 2C7, as measured by factor B binding, occurred in a properdin-dependent manner. These findings were confirmed using isogenic mutant strains that differed in their ability to bind to C4BP. Immune human serum that contained bactericidal Abs directed against the 2C7 lipooligosaccharide epitope as well as murine antigonococcal antiserum required functional properdin to kill C4BP-binding strains, but not C4BP-nonbinding strains. Collectively, these data point to an important role for properdin in facilitating immune Ab-mediated complement-dependent killing of gonococcal strains that inhibit the classical pathway by recruiting C4BP.     

Competition between Neisseria gonorrhoeae and Staphylococcus epidermidis during iron-limited or replete continuous culture

Neisseria gonorrhoeae strain P9-2 was grown in iron-limited or replete continuous culture at a dilution rate of 0.05 h-1, in the presence and absence of Staphylococcus epidermidis. Gonococci maintained expression of pili (P+) and the transparent colony phenotype in pure culture during transitions of iron- and cystine-limited growth. They competed well with staphylococci during iron-limited co-culture and comprised greater than 95% of the population. Transition to cystine-limited growth allowed the staphylococcus to predominate but the gonococcus did not wash out. Furthermore, the gonococcal opaque colony phenotype (O+), indicating synthesis of outer membrane proteins II, was now expressed. Restoration of iron limitation returned the co-culture to its original composition but with P+O+ gonococci dominating. These results suggest that environments might exist in Man where gonococci can compete successfully with normal indigenous bacteria during infection.     

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

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

Molecular mechanism for the antigonococcal action of lysosomal cathepsin G

Human lysosomal cathepsin G (cat G) appears to be an important mediator of non-oxidative killing of Neisseria gonorrhoeae ingested by human polymorphonuclear leucocytes (PMNLs). Nearly isogenic strains of gonococci having variations in the structure of penicillin-binding protein 2 (PBP2) also exhibit different levels of susceptibility to the lethal action of cat G in vitro. Accordingly, we examined the relationship between gonococcal susceptibility to cat G and PBP2 structure. The results of this study suggest that cat G has the capacity to interact with PBP2, as evidenced by its ability to inhibit binding of [3H]-benzylpenicillin to PBP2. We also found that changes in the amino acid sequence within the transpeptidase domain of PBP2, because of certain penA mutations, modulated such interactions. We propose that PBP2 is an intracellular target for cat G and that levels of gonococcal susceptibility to cat G may be related to PBP2 structure and/or intracellular availability.     

Roles of the recJ and recN Genes in Homologous Recombination and DNA Repair Pathways of Neisseria gonorrhoeae

The paradigm of homologous recombination comes from Escherichia coli, where the genes involved have been segregated into pathways. In the human pathogen Neisseria gonorrhoeae (the gonococcus), the pathways of homologous recombination are being delineated. To investigate the roles of the gonococcal recN and recJ genes in the recombination-based processes of the gonococcus, these genes were inactivated in the N. gonorrhoeae strain FA1090. We report that both recN and recJ loss-of-function mutants show decreased DNA repair ability. In addition, the recJ mutant was decreased in pilus-dependent colony morphology variation frequency but not DNA transformation efficiency, while the recN mutant was decreased in DNA transformation efficiency but not pilus-dependent variation frequency. We were able to complement all of these deficiencies by supplying an ectopic functional copy of either recJ or recN at an irrelevant locus. These results describe the role of recJ and recN in the recombination-dependent processes of the gonococcus and further define the pathways of homologous recombination in this organism.     

Detection of a gonococcal endo-beta-N-acetyl-D-glucosaminidase and its peptidoglycan cleavage site

Neisseria gonorrhoeae contains several hydrolases which may be responsible for gonococcal cell lysis. One of these enzymes, an endo-beta-N-acetyl-D-glucosaminidase, has been extracted from supernatants of sonicated gonococci and partially purified by ammonium sulfate precipitation and affinity and ion-exchange chromatography. This enzyme has a different specificity than egg white lysozyme and cleaves the beta 1 leads to 4 glycosidic linkage between N-acetylglucosamine and N-acetylmuramic acid in gonococcal peptidoglycan.     

Rapid damage to membranes of Neisseria gonorrhoeae caused by human neutrophil granule extracts

Neisseria gonorrhoeae were exposed to extracts of human neutrophil granules and effects on gonococcal growth and membranes were determined. Enumeration of gonococci by phase-contrast microscopy at 0 and 60 min revealed that they underwent very limited cell division after exposure to granule extract. At 60 min, treated gonococci tended to clump, and some lost their refractivity under phase-contrast optics, indicating membrane damage. Treated and untreated gonococci utilized oxygen at similar rates at time 0; treated gonococci utilized oxygen at a relatively constant rate for 60 min, even though colony-forming ability (i.e. viability) decreased by 90%, whereas untreated gonococci showed a steadily increasing rate of oxygen consumption over the same period, which essentially paralleled increase in colony-forming ability. Membrane ultrastructure of untreated and treated gonococci was compared in thin section by transmission electron microscopy. Extract treatment resulted in a time-related increase in disruption of the bacterial outer membrane, which became apparent almost immediately after treatment. This was accompanied by increasingly aberrant septum structure. Extract treatment also increased the resolution of peptidoglycan by electron microscopy, as early as 10 min after treatment. These data suggest that extract treatment of gonococci caused a rapid loss of the ability to form colonies on agar concomitant with alteration of gonococcal peptidoglycan and outer-membrane structure, but with little alteration of inner-membrane function.     

Immunologic classification of Neisseria gonorrhoeae with micro-immunofluorescence.

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.     

A genetic screen identifies genes and sites involved in pilin antigenic variation in Neisseria gonorrhoeae

It has previously been shown that the frequency of pilin antigenic variation in Neisseria gonorrhoeae (the gonococcus, Gc) is regulated by iron availability. To identify factors involved in pilin variation in an iron-dependent or an iron-independent manner, we conducted a genetic screen of transposon-mutated gonococci using a pilus-dependent colony morphology phenotype to detect antigenic variation deficient mutants. Forty-six total mutants representing insertions in 30 different genes were shown to have reduced colony morphology changes resulting from impaired pilin variation. Five mutants exhibited an iron-dependent decrease in pilin variation, while the remaining 41 displayed an iron-independent decrease in pilin variation. Based on the levels of antigenic variation impairment, we defined the genes as being essential for, important for, or involved in antigenic variation. DNA repair and DNA transformation frequencies of each mutant were measured to determine whether other recombination-based processes were also affected in the mutants. Each mutant was placed into one of six classes based on their pilin variation, DNA repair and DNA transformation phenotypes. Among the many genes identified, recR is shown to be an additional member of the gonococcal RecF-like recombination pathway. In addition, recG and ruvA represent the first evidence that the processing of Holliday junctions is required for pilin antigenic variation. Moreover, two independent insertions in a non-coding region upstream of the pilE gene suggest that cis-acting sequences important for pilin variation are found in that region. Finally, insertions that effect expression of the thrB and thrC genes suggest that molecules in the threonine biosynthetic pathway are important for pilin variation. Many of the other genes identified in this genetic screen do not have an obvious role in pilin variation, DNA repair, or DNA transformation.     

AmiC functions as an N-acetylmuramyl-l-alanine amidase necessary for cell separation and can promote autolysis in Neisseria gonorrhoeae

Neisseria gonorrhoeae is prone to undergo autolysis under many conditions not conducive to growth. The role of autolysis during gonococcal infection is not known, but possible advantages for the bacterial population include provision of nutrients to a starving population, modulation of the host immune response by released cell components, and donation of DNA for natural transformation. Biochemical studies indicated that an N-acetylmuramyl-l-alanine amidase is responsible for cell wall breakdown during autolysis. In order to better understand autolysis and in hopes of creating a nonautolytic mutant, we mutated amiC, the gene for a putative peptidoglycan-degrading amidase in N. gonorrhoeae. Characterization of peptidoglycan fragments released during growth showed that an amiC mutant did not produce free disaccharide, consistent with a role for AmiC as an N-acetylmuramyl-l-alanine amidase. Compared to the wild-type parent, the mutant exhibited altered growth characteristics, including slowed exponential-phase growth, increased turbidity in stationary phase, and increased colony opacity. Thin-section electron micrographs showed that mutant cells did not fully separate but grew as clumps. Complementation of the amiC deletion mutant with wild-type amiC restored wild-type growth characteristics and transparent colony morphology. Overexpression of amiC resulted in increased cell lysis, supporting AmiC's purported function as a gonococcal autolysin. However, amiC mutants still underwent autolysis in stationary phase, indicating that other gonococcal enzymes are also involved in this process.     

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.     

A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhoea. Most gonococcal infections remain localized to the genital tract but, in a small proportion of untreated cases, the bacterium becomes systemic to produce the serious complication of disseminated gonococcal infection (DGI). We have identified a large region of chromosomal DNA in N. gonorrhoeae that is not found in a subset of gonococcal isolates (a genetic island), in the closely related pathogen, Neisseria meningitidis or in commensal Neisseria that do not usually cause disease. Certain versions of the island carry a serum resistance locus and a gene for the production of a cytotoxin; these versions of the island are found preferentially in DGI isolates. All versions of the genetic island encode homologues of F factor conjugation proteins, suggesting that, like some other pathogenicity islands, this region encodes a conjugation-like secretion system. Consistent with this hypothesis, a wild-type strain released large amounts of DNA into the medium during exponential growth without cell lysis, whereas an isogenic strain mutated in a peptidoglycan hydrolase gene (atlA) was drastically reduced in its ability to donate DNA for transformation during growth. This genetic island constitutes the first major discriminating factor between the gonococcus and the other Neisseria and carries genes for providing DNA for genetic transformation.     

Induction of reaginic (IgE) gonococcal antibodies in the rat by a common antigen of Neisseria gonorrhoeae.

An antigen (ZAB) common to Neisseria gonorrhoeae was prepared by stepwise elution of a crude gonococcal antigen (ZA) from columns of diethylaminoethyl cellulose employing 0.02 M phosphate buffers, pH 7.6, containing increasing concentrations of sodium chloride. Rats immunized with ZAB produced reaginic (IgE) antibody which cross-reacted with ZA prepared from eight gonococcal strains by the passive cutaneous anaphylaxis (PCA) test. Heating of the sera at 56 degrees C for 4 h destroyed the PCA activity. The PCA activity of the anti-ZAB rat serum was removed after absorption with ZAB antigen or with rabbit anti-rat IgE but not after absorption with gonococcal lipopolysaccharide or with heat-killed or formalinized gonococci. Treatment of ZAB with trypsin or heating at 100 degrees C for 30 min destroyed or reduced the antigenic activity respectively. Further purification of ZAB by filtration through Sephadex G-100 gave a preparation (ZAB2) which contained the common antigen as shown by the cross-reactivity of anti-ZAB2 rat serum with seven stains of N. gonorrhoeae. Fraction ZAB2 contained material which had a molecular weight less than 13,700 and was associated with the presence of material absorbing at 260 nm. The results of this study indicate that a low molecular weight antigen, which appears to be protein in nature and associated with nuclei acid, is common to the gonococcus and is the main antigenic component inducing reaginic (IgE) antibody in the rat.