Intercontinental spread of Neisseria meningitidis clones of the ET-5 complex

The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 structural genes encoding enzymes in 688 isolates. Variation among strains in serogroup and serotype has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at more than half their genetic loci. Clones of one of these lineages, the ET-5 complex, have been identified as the causative agent of recent outbreaks and epidemics of meningococcal disease in Europe, South Africa, Latin America, and the United States. There is evidence that organisms of the ET-5 complex reached Florida via human immigrants from Cuba.     

Population Genetic and Evolutionary Approaches to Analysis of Neisseria meningitidis Isolates Belonging to the ET-5 Complex

Periodically, new disease-associated variants of the human pathogen Neisseria meningitidis arise. These meningococci diversify during spread, and related isolates recovered from different parts of the world have different genetic and antigenic characteristics. An example is the ET-5 complex, members of which were isolated globally from the mid-1970s onwards. Isolates from a hyperendemic outbreak of meningococcal disease in Worcester, England, during the late 1980s were characterized by multilocus sequence typing and sequence determination of antigen genes. These data established that the Worcester outbreak was caused by ET-5 complex meningococci which were not closely related to the ET-5 complex bacteria responsible for a hyperendemic outbreak in the nearby town of Stroud during the years preceding the Worcester outbreak. A comparison with other ET-5 complex meningococci established that there were at least three distinct globally distributed subpopulations within the ET-5 complex, characterized by particular housekeeping and antigen gene alleles. The Worcester isolates belonged to one of these subpopulations, the Stroud isolates belonged to another, and at least one representative of the third subpopulation identified in this work was isolated elsewhere in the United Kingdom. The sequence data demonstrated that ET-5 variants have arisen by multiple complex pathways involving the recombination of antigen and housekeeping genes and de novo mutation of antigen genes. The data further suggest that either the ET-5 complex has been in existence for many years, evolving and spreading relatively slowly until its disease-causing potential was recognized, or it has evolved and spread rapidly since its first identification in the 1970s, with each of the subpopulations attaining a distribution spanning several continents.     

Genomic Basis of a Polyagglutinating Isolate of Neisseria meningitidis

Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate ({"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.     

Preemptive Homology-Directed DNA Repair Fosters Complex Genomic Rearrangements in Hepatocellular Carcinoma

Degree of genomic instability closely correlates with poor prognosis, drug resistance as well as poor survival across human cancer of different origins. This study assessed the relationship between DNA damage response (DDR) and chromosome instability in hepatocellular carcinoma (HCC). We investigated DDR signaling in HCC cells by analyzing DNA damage-dependent redistribution of major DDR proteins to damaged chromatin using immunofluorescence microscopy and Western blotting experimentations. We also performed gene conversion and metaphase analyses to address whether dysregulated DDR may bear any biological significance during hepatocarcinogenesis. Accordingly, we found that HCC cell lines suffered from elevated spontaneous DNA double-strand breaks (DSBs). In addition, analyses of HCC metaphases revealed marked aneuploidy and frequent sister chromatid exchanges when compared to immortalized hepatocytes, the latter of which were further induced following camptothecin-induced DSBs. We propose that genomic instability in HCC may be caused by erroneous DNA repair in a desperate attempt to mend DSBs for cell survival and that such preemptive measures inadvertently foster chromosome instability and thus complex genomic rearrangements.     

Phenotypic and Genotypic Characteristics of Neisseria meningitidis Disease-Causing Strains in Argentina, 2010

Phenotypic and genotypic characterization of 133 isolates of Neisseria meningitidis obtained from meningococcal disease cases in Argentina during 2010 were performed by the National Reference Laboratory as part of a project coordinated by the PAHO within the SIREVA II network. Serogroup, serotype, serosubtype and MLST characterization were performed. Minimum Inhibitory Concentration to penicillin, ampicillin, ceftriaxone, rifampin, chloramphenicol, tetracycline and ciprofloxacin were determined and interpreted according to CLSI guidelines. Almost 49% of isolates were W135, and two serotype:serosubtype combinations, W135∶2a:P1.5,2:ST-11 and W135∶2a:P1.2:ST-11 accounted for 78% of all W135 isolates. Serogroup B accounted for 42.1% of isolates, and was both phenotypically and genotypically diverse. Serogroup C isolates represented 5.3% of the dataset, and one isolate belonging to the ST-198 complex was non-groupable. Isolates belonged mainly to the ST-11 complex (48%) and to a lesser extent to the ST-865 (18%), ST-32 (9,8%) and the ST-35 complexes (9%). Intermediate resistance to penicillin and ampicillin was detected in 35.4% and 33.1% of isolates respectively. Two W135∶2a:P1.5,2:ST-11:ST-11 isolates presented resistance to ciprofloxacin associated with a mutation in the QRDR of gyrA gene Thr91-Ile. These data show serogroup W135 was the first cause of disease in Argentina in 2010, and was strongly associated with the W135∶2a:P1.5,2:ST-11 epidemic clone. Serogroup B was the second cause of disease and isolates belonging to this serogroup were phenotypically and genotypically diverse. The presence of isolates with intermediate resistance to penicillin and the presence of fluorquinolone-resistant isolates highlight the necessity and importance of maintaining and strengthening National Surveillance Programs.     

Complex Genomic Rearrangements at the PLP1 Locus Include Triplication and Quadruplication

Inverted repeats (IRs) can facilitate structural variation as crucibles of genomic rearrangement. Complex duplication—inverted triplication—duplication (DUP-TRP/INV-DUP) rearrangements that contain breakpoint junctions within IRs have been recently associated with both MECP2 duplication syndrome (MIM#300260) and Pelizaeus-Merzbacher disease (PMD, MIM#312080). We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals—16/17 were found to have a DUP-TRP/INV-DUP rearrangement product. An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals. We show that a homology—or homeology—driven replicative mechanism of DNA repair can apparently mediate template switches within stretches of microhomology. Moreover, we provide evidence that quadruplication and potentially higher order amplification of a genomic interval can occur in a manner consistent with rolling circle amplification as predicted by the microhomology-mediated break induced replication (MMBIR) model.     

Structure of the L5 lipopolysaccharide core oligosaccharides of Neisseria meningitidis

Three different oligosaccharides were isolated by mild acid hydrolysis of the lipopolysaccharides, obtained from Neisseria meningitidis serotype 5, and their structures were elucidated by combined chemical and physical techniques. The use of 500-MHz 1H NMR in both one-dimensional and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the purified oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The largest of the three original oligosaccharides is a triantennary partially O-acetylated decasaccharide in which the largest antenna terminates in a lacto-N-neotetraose unit. The smaller oligosaccharides (heptasaccharide and octasaccharide) except for terminal glycose deletions from the longest antenna are structural replicas of the larger.     

Cloning and expression of the Neisseria meningitidis 5C outer membrane protein

The 5C outer membrane protein, one of the N. meningitidis class 5 proteins, was preferably expressed in bacteria isolated from the nasopharynx and its role in adhering to the mucosal cells and invading them as well as the development of anti-5C antibodies in healthy carriers was demonstrated. Anti-5C monoclonal antibodies are bactericidal in the presence of the human complement. The immunodominant region of the 5C protein is highly conserved among the different strains of N. meningitidis, and the opc gene, which encodes the protein, does not seem to show antigenic variations. Here the isolation of the opc gene from the Cuban strain B:4:P1.15 by PCR (Polymerase Chain Reaction) is presented. Under the regulation of the tryptophan promoter, the gene was cloned and sequenced in E. coli with a high level of expression and fused to the amino-terminal end of the interleukin-2 gene. In the dot-blot experiments, the presence of the gene in those strains which did not express the protein in the whole cell ELISA was also detectable.     

Genetic and Structural Characterization of L11 Lipooligosaccharide from Neisseria meningitidis Serogroup A Strains

The lipooligosaccharide (LOS) of immunotype L11 is unique within serogroup A meningococci. In order to resolve its molecular structure, we conducted LOS genotyping by PCR analysis of genes responsible for α-chain sugar addition (lgtA, -B, -C, -E, -H, and -F) and inner core substituents (lgtG, lpt-3, and lpt-6). For this study, we selected seven strains belonging to subgroup III, a major clonal complex responsible for meningococcal meningitis epidemics in Africa. In addition, we sequenced the homopolymeric tract regions of three phase-variable genes (lgtA, lgtG, and lot-3) to predict gene functionality. The fine structure of the L11 LOS of each strain was determined using composition and glycosyl linkage analyses, NMR, and mass spectrometry. The masses of the dephosphorylated oligosaccharides were consistent with an oligosaccharide composed of two hexoses, one N-acetyl-hexosamine, two heptoses, and one KDO, as proposed previously. The molar composition of LOS showed two glucose residues to be present, in agreement with lgtH sequence prediction. Despite phosphoethanolaminetransferase genes lpt-3 and lpt-6 being present in all seven Neisseria meningitidis strains, phosphoethanolamine (PEtn) was found at both O-3 and O-6 of HepII among the three ST-5 strains, whereas among the four ST-7 strains, only one PEtn was found and located at O-3 of the HepII. The L11 LOS was found to be O-acetylated, as was indicated by the presence of the lot-3 gene being in-frame in all of the seven N. meningitidis strains. To our knowledge, these studies represent the first full genetic and structural characterization of the L11 LOS of N. meningitidis. These investigations also suggest the presence of further regulatory mechanisms affecting LOS structure microheterogeneity in N. meningitidis related to PEtn decoration of the inner core.     

Characterization of DsbD in Neisseria meningitidis

Proper periplasmic disulfide bond formation is important for folding and stability of many secreted and membrane proteins, and is catalysed by three DsbA oxidoreductases in Neisseria meningitidis. DsbD provides reducing power to DsbC that shuffles incorrect disulfide bond in misfolded proteins as well as to the periplasmic enzymes that reduce apo-cytochrome c (CcsX) or repair oxidative protein damages (MrsAB). The expression of dsbD, but not other dsb genes, is positively regulated by the MisR/S two-component system. Quantitative real-time PCR analyses showed significantly reduced dsbD expression in all misR/S mutants, which was rescued by genetic complementation. The direct and specific interaction of MisR with the upstream region of the dsbD promoter was demonstrated by electrophoretic mobility shift assay, and the MisR binding sequences were mapped. Further, the expression of dsbD was found to be induced by dithiothrietol (DTT), through the MisR/S regulatory system. Surprisingly, we revealed that inactivation of dsbD can only be achieved in a strain carrying an ectopically located dsbD, in the dsbA1A2 double mutant or in the dsbA1A2A3 triple mutant, thus DsbD is indispensable for DsbA-catalysed oxidative protein folding in N. meningitidis. The defects of the meningococcal dsbA1A2 mutant in transformation and resistance to oxidative stress were more severe in the absence of dsbD.     

Susceptibility of Neisseria meningitidis Strains from the Civilian Population to Sulfadiazine, Penicillin, and Rifampin

The minimal inhibitory concentration (MIC) values of sulfadiazine, penicillin, and rifampin for meningococcal strains isolated from civilians during 1970 were compared. The strains were isolated from various sources and geographical areas and represented several serogroups. The ranges of MIC values were as follows: 0.05 to 20 mg/100 ml for sulfadiazine, 0.01 to 0.4 mug/ml for penicillin, and 0.01 to 0.8 mug/ml for rifampin. There was no significant relationship between MIC values of sensitive or resistant sulfadiazine strains and the MIC values to the other two antimicrobial agents. Comparisons of sulfadiazine MIC values with inhibition zones around sulfathiazole discs showed excellent correlation, provided the strains were separated into sensitive and resistant groups on the basis of growth at 1 mg/100 ml. Regression curves for penicillin and rifampin sensitivity showed homologous sensitive populations with the strains studied.     

Construction of Opa-Positive and Opa-Negative Strains of Neisseria meningitidis to Evaluate a Novel Meningococcal Vaccine

Neisseria meningitidis is a major global pathogen causing invasive disease with a mortality of 5–10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opacity-associated adhesin (Opa) proteins are major meningococcal outer membrane proteins, which have shown recent promise as a potential novel vaccine. Immunisation of mice with different Opa variants elicited high levels of meningococcal-specific bactericidal antibodies, demonstrating proof in principle for this approach. Opa proteins are critical in meningococcal pathogenesis, mediating bacterial adherence to host cells, and modulating human cellular immunity via interactions with T cells and neutrophils, although there are conflicting data regarding their effects on CD4⁺ T cells. We constructed Opa-positive and Opa-negative meningococcal strains to allow further evaluation of Opa as a vaccine component. All four opa genes from N. meningitidis strain H44/76 were sequentially disrupted to construct all possible combinations of N. meningitidis strains deficient in one, two, three, or all four opa genes. The transformations demonstrated that homologous recombination of exogenous DNA into the meningococcal chromosome can occur with as little as 80 bp, and that minor sequence differences are permissible. Anti-Opa bactericidal antibody responses following immunisation of mice with recombinant Opa were specific to the Opa variant used in immunisation. No immunomodulatory effects were observed when Opa was contained within meningococcal outer membrane vesicles (OMVs), compared to Opa-negative OMVs. These observations support the incorporation of Opa in meningococcal vaccines.     

Carriage of Neisseria meningitidis and Neisseria lactamica in northern Greece

In response to an increase in the number of cases of invasive meningococcal disease (IMD) in northern regions of Greece, a survey was carried out to determine if there was an increase in carriage of Neisseria meningitidis, particularly in areas where there have been increases in immigrant populations from neighbouring countries. The second objective was to determine if there was an increase in the serogroup C:2a:P1.5,2 a phenotype associated with recent outbreaks or changes in antibiotic sensitivities. As carriage of Neisseria lactamica is associated with development of natural immunity to IMD, the third objective was to determine the carriage rate of N. lactamica in this population. Among 3167 individuals tested, meningococci were isolated from 334 (10.5%). Compared with our previous studies, the proportion of meningococcal carriers was significantly increased among children in secondary education (11.3%) (chi2=9.67, P<0.005) and military recruits (37.4%) (chi2=21.11, P<0.000). Only 5/334 (1.5%) isolates expressed the phenotype associated with the increase in IMD in Greece. N. lactamica was isolated from 146/3167 (4.6%) participants. It was isolated from 71/987 (7.2%) children attending primary or nursery schools; however, the highest proportion of carriers (11.3%) was found in the boarding school for young Albanian men. In the 21-59-year age range, the majority of N. lactamica isolates (22/25, 88%) were from women, probably due to closer or more prolonged contact with children in the primary school age range. Smoking was significantly associated with isolation of meningococci from men but not from women. Penicillin-insensitive strains (25/334, 7.5%) were identified in all four regions examined; the majority (14/25, 56%) were obtained from military personnel. We conclude that there was a higher proportion of carriers in the population of northern Greece; however, the increase in carriage rate was not associated with the influx of immigrants from neighbouring countries, and there was not a higher incidence of the C:2a:P1.5,2 strain responsible for increased disease activity in Greece in either the immigrant or local populations.     

Characterization of the opa (class 5) gene family of Neisseria meningitidis

Class 5 outer membrane proteins of Neisseria meningitidis show both phase- and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used lambda gt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.     

Iron Transport Systems in Neisseria meningitidis

Acquisition of iron and iron complexes has long been recognized as a major determinant in the pathogenesis of Neisseria meningitidis. In this review, high-affinity iron uptake systems, which allow meningococci to utilize the human host proteins transferrin, lactoferrin, hemoglobin, and haptoglobin-hemoglobin as sources of essential iron, are described. Classic features of bacterial iron transport systems, such as regulation by the iron-responsive repressor Fur and TonB-dependent transport activity, are discussed, as well as more specific features of meningococcal iron transport. Our current understanding of how N. meningitidis acquires iron from the human host and the vaccine potentials of various components of these iron transport systems are also reviewed.     

Nitric oxide metabolism in Neisseria meningitidis

Neisseria meningitidis, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease. The genome of N. meningitidis includes the genes aniA and norB, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively. These gene products should allow the bacterium to denitrify nitrite to nitrous oxide. We show that N. meningitidis can support growth microaerobically by the denitrification of nitrite via NO and that norB is required for anaerobic growth with nitrite. NorB and, to a lesser extent, the cycP gene product cytochrome c' are able to counteract toxicity due to exogenously added NO. Expression of these genes by N. meningitidis during colonization and disease may confer protection against exogenous or endogenous nitrosative stress.