Ecological separation and genetic isolation of Neisseria gonorrhoeae and Neisseria meningitidis

BACKGROUND: Classifying bacteria into species is problematic. Most microbiologists consider species to be groups of isolates that share some arbitrary degree of relatedness of biochemical or molecular (such as DNA sequence) features and that, ideally, are clearly delineated from all other groups of isolates. The main problem in applying to bacteria a biological concept of species based on the ability or inability of their genes to recombine, is that recombination appears to be rare in bacteria in nature, as indicated by the strong linkage disequilibrium between alleles found in most bacterial populations. However, there are some naturally transformable bacteria in which assortative recombination appears to be so frequent that alleles are in, or close to, linkage equilibrium. For these recombining populations a biological concept of species might be applicable. RESULTS: Populations of Neisseria gonorrhoeae and Neisseria meningitidis from Spain were analysed by multilocus enzyme electrophoresis. The data indicate that assortative recombination occurs frequently within populations, but not between populations. Similarly, the sequences of two house-keeping genes show no evidence of intragenic recombination between N. gonorrhoeae and N. meningitidis. CONCLUSIONS: N. gonorrhoeae and N. meningitidis represent extremely closely related 'sexual' populations that appear to be genetically isolated in nature, and thus conform to the biological concept of species. The extreme uniformity of N. gonorrhoeae house-keeping genes suggests that this species may have arisen recently as a clone of N. meningitidis that could colonize the genital tract. Ecological isolation - of populations that can colonize the genital tract from those that can colonize the nasopharynx - may have been an important component in speciation, leading to a lower frequency of recombination between species than within species.     

Prevention of autolysis in suspensions of Neisseria gonorrhoeae by mercuric ions.

Mercuric ions were the only metal ions which prevented autolysis of Neisseria gonorrhoeae, as measured both by stabilization of optical density and by prevention of release of [3H]diaminopimelic acid.     

Peptidoglycan hydrolysis mediated by the amidase AmiC and its LytM activator NlpD is critical for cell separation and virulence in the phytopathogen Xanthomonas campestris

The essential stages of bacterial cell separation are described as the synthesis and hydrolysis of septal peptidoglycan (PG). The amidase, AmiC, which cleaves the peptide side‐chains linked to the glycan strands, contributes critically to this process and has been studied extensively in model strains of Escherichia coli. However, insights into the contribution of this protein to other processes in the bacterial cell have been limited. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogen that causes black rot disease in many economically important plants. We investigated how AmiC and LytM family regulators, NlpD and EnvC, contribute to virulence and cell separation in this organism. Biochemical analyses of purified AmiC demonstrated that it could hydrolyse PG and its activity could be potentiated by the presence of the regulator NlpD. We also established that deletion of the genes encoding amiC1 or nlpD led to a reduction in virulence as well as effects on colony‐forming units and cell morphology. Moreover, further genetic and biochemical evidence showed that AmiC1 and NlpD affect the secretion of type III effector XC3176 and hypersensitive response (HR) induction in planta. These findings indicate that, in addition to their well‐studied role(s) in cell separation, AmiC and NlpD make an important contribution to the type III secretion (T3S) and virulence regulation in this important plant pathogen.     

Cultivation of Neisseria gonorrhoeae in an L-929 cell culture

N. gonorrhoeae strain b has been found to be capable of retaining its viability in medium 199 with 10% of inactivated cattle serum added and in monolayer cell culture L-929 in the above medium. The characteristics obtained in the present investigation permit simulating the mixed association of gonococci and chlamydiae in the culture system used in this work.     

Growth pattern and cell division in Neisseria gonorrhoeae.

The gram-negative coccus Neisseria gonorrhoeae was found to grow regularly in at least two dimensions. Growth proceeded at a linear rate sequentially in each dimension. Growth in the second dimension (former width) was initiated slightly before the pole-division plane distance equalled the cell width. Penicillin treatment localized presumptive growth zones to the existing septum region. It was suggested that new growth zones were always formed perpendicular to the longitudinal axis created in the incipient daughter cells of a dividing coccus. Neither penicillin nor nalidixic acid induced filaments of N. gonorrhoeae. Such structures could nevertheless be formed in the rod-shaped species Neisseria elongata. N. gonorrhoeae divides by septation; however, complete septal structures with separated cytoplasms were rather infrequent. It is proposed that N. gonorrhoeae be regarded as a short rod which always extends parallel to the actual longitudinal axis and which never undergoes a rod-sphere-rod transition.     

Mutation of a single lytic transglycosylase causes aberrant septation and inhibits cell separation of Neisseria gonorrhoeae

The function of lytic peptidoglycan transglycosylases is poorly understood. Single lytic transglycosylase mutants of Escherichia coli have no growth phenotype. By contrast, mutation of Neisseria gonorrhoeae ltgC inhibited cell separation without affecting peptidoglycan monomer production. Thus, LtgC has a dedicated function in gonococcal cell division.     

AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system

Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.     

A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae

We have identified a gene encoding an autolysin (atlA) from Neisseria gonorrhoeae . The deduced amino acid sequence of AtlA shows significant similarity to the peptidoglycan degrading transglycosylases (endolysins) of bacteriophages lambda and P2, suggesting that the encoded protein also functions in peptidoglycan hydrolysis. An atlA mutant was identical to the wild-type strain in exponential growth rate, but demonstrated reduced lysis and peptidoglycan turnover in the stationary phase of growth. When transferred into a buffer solution, at a pH non-permissive for other gonococcal autolysins, an autolytic activity was detectable in the wild-type strain that was not present in the mutant. The most dramatic phenotype of the mutant occurred after extended time in stationary phase. After approximately 16 h in stationary phase, both strains underwent an apparent replication event, after which the wild-type strain died rapidly whereas the atlA mutant survived considerably longer. Even after both the wild-type and mutant cells were dead, many of the mutant cells maintained intact morphology, whereas the wild-type cells were lysed. These results suggest that AtlA is a peptidoglycan transglycosylase related to bacteriophage endolysins and acts as an autolysin in the stationary phase.     

Tetrapac (tpc), a novel genotype of Neisseria gonorrhoeae affecting epithelial cell invasion, natural transformation competence and cell separation

We characterized a novel mutant phenotype (t etrap ac , tpc) of Neisseria gonorrhoeae (Ngo) associated with a distinctive rough-colony morphology and bacterial growth in clusters of four. This phenotype, suggesting a defect in cell division, was isolated from a mutant library of Ngo MS11 generated with the phoA minitransposon TnMax4. The tpc mutant shows a 30% reduction in the overall murein hydrolase activity using Escherichia coli murein as substrate. Tetrapacs can be resolved by co-cultivation with wild-type Ngo, indicating that Tpc is a diffusible protein. Interestingly, Tpc is absolutely required for the natural transformation competence of piliated Ngo. Mutants in tpc grow normally, but show a ～ 10-fold reduction in their ability to invade human epithelial cells. The tpc sequence reveals an open reading frame of ～1 kb encoding a protein (Tpc) of 37kDa. The primary gene product exhibits an N-terminal leader sequence typical of lipoproteins, but palmitoylation of Tpc could not be demonstrated. The ribosomal binding site of tpc is immediately downstream of the translational stop codon of the folC gene coding for an enzyme involved in folic acid biosynthesis and one-carbon metabolism. The tpc gene is probably co-transcribed from the folC promoter and a promoter located within the folC gene. The latter promoter sequence shares significant homology with E. coli gearbox consensus promoters. All three mutant phenotypes, i.e. the cell separation defect, the transformation deficiency and the defect in cell invasion can be restored by complementation of the mutant with an intact tpc gene. To some extent the tcp phenotype is reminiscent of iap in Listeria, lytA in Streptococcus pneumoniae and lyt in Bacillus subtilis, all of which are considered to represent murein hydrolase defects.     

Cefonicid as Therapy for Uncomplicated Gonococcal Urethritis Caused by Penicillinase-Producing Neisseria gonorrhoeae

Young men with uncomplicated gonococcal urethritis were treated with 1 gram of cefonicid given intramuscularly plus 1 gram of probenecid by mouth. Of 53 evaluable patients, 33 (62%) had penicillinase-producing Neisseria gonorrhoeae. All but one of these patients were cured. All men who had penicillin-sensitive infections were cured. Cefonicid was highly effective in the treatment of both penicillin-sensitive and penicillin-resistant N gonorrhoeae. Other than moderate pain at the site of injection, there were no adverse side effects. Cefonicid can be added to the group of newer cephalosporins that are effective in the treatment of gonococcal urethritis caused by either penicillin-sensitive or penicillin-resistant strains.     

New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis

We have created new complementation constructs for use in Neisseria gonorrhoeae and Neisseria meningitidis. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genes iga and trpB. In order to increase the available options for gene expression in Neisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-β-d-thiogalactopyranoside-inducible lac promoter, which has been widely used in Neisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcal opaB gene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter in Neisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenic Neisseria at very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy in N. gonorrhoeae as well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.     

2010年广州地区淋球菌耐药监测结果分析

目的了解广州地区淋球菌对抗生素耐药性的变化及PPNG和TRNG的流行趋势。方法用琼脂稀释法测定头孢曲松、大观霉素、环丙沙星、阿奇霉素和四环素的最低抑菌浓度（MIC）;用纸片碘量法检测β-内酰胺酶。结果83株淋球菌检出PPNG24株（28．9％）、TRNG50株（60．2％）、环丙沙星耐药率高达98．8％,高度耐药株（MIC≥16mg／L）43株（51．8％）,而76株淋球菌中阿奇霉素耐药株11株（14．5％）,均未出现对头孢曲松、大观霉素耐药的菌株,抗菌活性强。结论合理规范使用抗生素及动态监测淋球菌耐药性变迁是临床减少淋球菌耐药菌株出现的有效办法。     

Control of pilus expression in Neisseria gonorrhoeae as an original system in the family of two-component regulators

We have previously reported the identification of two genes, pilA and pilB, which act in trans to regulate pilus expression in Neisseria gonorrhoeae. Here we show that PilA and PilB have amino acid sequence similarities with members of the two component 'sensor-regulator' family of proteins. PilB has homology with histidine kinase sensors. Alkaline phosphatase fusions to the predicted sensor and transmitter domains are described. Their PhoA activity and cellular location suggest that PilB is inserted in the cytoplasmic membrane and predict periplasmic and cytoplasmic locations for the sensor and the transmitter domains, respectively. PilA has homology with response regulators in its N-terminal part, and with components of the eukaryotic protein secretory apparatus (SRP 54 and SRP receptor) as well as two Escherichia coli gene products in its C-terminal part. In particular, it contains a putative GTP-binding site. Mini-transposon insertions into different regions of pilA were obtained. The phenotypes and genotypes of these mutants and preliminary biochemical studies of the gene products of two of these mutants lend further support to the hypothesis that PilA is a DNA-binding response regulator and confirm that it participates in an essential function in the bacterium.     

DNA methylation in Neisseria gonorrhoeae and other Neisseriae

It has been reported in the literature that Neisseria gonorrhoeae DNA is modified by the methyltransferases (MTases) M.NgoI, M.NgoII, and M.NgoIII, as well as three other cytosine MTases and one adenine MTase, even if the corresponding restriction endonucleases are not present. We envisioned the possibility of cloning one of the N. gonorrhoeae MTase-encoding genes for use as a species-specific DNA probe. We therefore undertook a survey of methylation patterns of several clinical isolates of N. gonorrhoeae and N. meningitidis as well as ATCC strains of other Neisseriae. We found, from digestion patterns with isoschizomers, one N. gonorrhoeae strain that lacked M.NgoII and two that lacked M.NgoIII. All N. meningitidis strains (save one) were resistant to digestion with NlaIV thus possessing an MTase like NgoV, and one was resistant to SstII, thus having an NgoIII-like MTase. None were resistant to isoschizomers of NgoI, NgoIII and NgoIV. Some other Neisseriae had an MTase with NlaIV (NgoV) specificity, but none had NgoI, II, III or IV specificity, except for the Branhamella-like N. caviae-ovis group and N. lactamica where these specificities were present in at least one strain of this group. Therefore, among the Neisseriae other than N. caviae only M.NgoI is N. gonorrhoeae-specific.     

Changes in host cell membrane activities in response to adhesion of Neisseria gonorrhoeae

Physiological changes in host cell model membranes (intact human erythrocytes and ghosts) as a consequence of bacterial adhesion were studied with special reference to Neisseria gonorrhoeae. Membrane activities examined were transport of K+, Cl- ions, pyruvate kinase, Na-K-dependent ATPase, and cAMP. We found that K+ and Cl- transport were affected, more so in membranes with attached pilated (P+) organisms than in those with apilated (P-) isogenic strains. In N. gonorrhoeae and in several other species of gram-negative bacteria studied, hemagglutination titres were directly correlated with effects on anion transport, suggesting that perturbations in anion transport are an immediate result of adhesion. Of three P+ gonococcus strains tested, two depressed Na-K-ATPase activity in the membrane, indicating a possible effect on the Na-K pump. Pyruvate kinase activity associated with the membrane appeared to be stimulated by attached gonococci, again by P+ strains to higher levels than P- organisms. Clearly, some enzyme properties of host membranes are intrinsically affected by bacterial adhesion. Human polymorphonuclear neutrophils were also investigated, and with some exceptions, changes observed in leukocyte enzyme activities tended to parallel those in erythrocytes. Since hypochlorous acid production is considered to be an important microbicidal mechanism in neutrophils, interference with Cl- transport could jeopardize their role in host defense.     

Syndecan-1 and syndecan-4 can mediate the invasion of OpaHSPG-expressing Neisseria gonorrhoeae into epithelial cells

Neisseria gonorrhoeae ( Ngo ) expressing the outer membrane protein Opa_HSPG can adhere to and invade epithelial cells via binding to heparan sulphate proteoglycan (HSPG) receptors. In this study, we have investigated the role of syndecan-1 and syndecan-4, two members of the HSPG family, in the uptake of Ngo by epithelial cells. When overexpressed in HeLa cells, both syndecans co-localize with adherent Ngo on the host cell surface. This overexpression of syndecan-1 and syndecan-4 leads to a three- and sevenfold increase in Ngo invasion respectively. In contrast, transfection with the syndecan-1 and syndecan-4 mutant constructs lacking the intracellular domain results in an abrogation of the invasion process, characteristic of a dominant-negative mode of action. A concomitant loss of the capacity to mediate Ngo uptake was also observed with syndecan-4 mutant constructs carrying lesions in the dimerization motif necessary for the binding of protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PIP₂), and mutants that are deficient in a C-terminal EFYA amino acid motif responsible for binding to syntenin or CASK. We conclude that syndecan-1 and syndecan-4 can both mediate Ngo uptake into epithelial cells, and that their intracellular domains play a crucial role in this process, perhaps by mediating signal transduction or anchorage to the cytoskeleton.