Networks and groups within the genus Neisseria: analysis of argF, recA, rho, and 16S rRNA sequences from human Neisseria species.

To understand the pattern of nucleotide sequence variation among bacteria that frequently exchange chromosomal genes, we analyzed sequences of the recA, argF, and rho genes, as well as part of the small-subunit (16S) rRNA gene, from about 50 isolates of human commensal Neisseria species and the pathogenic N. meningitidis and N. gonorrhoeae. Almost all isolates of these species could be assigned to five phylogenetic groups that are found for all genes examined and generally are supported by high bootstrap values. In contrast, the phylogenetic relationships among groups varied according to the gene analyzed with notable incongruences involving N. cinerea and N. lactamica. Further analysis using split decomposition showed that for each gene, including 16S rRNA, the patterns of sequence divergence within N. meningitidis and closely related species were inconsistent with a bifurcating treelike phylogeny and better represented by an interconnected network. These data indicate that the human commensal Neisseria species can be separated into discrete groups of related species but that the relationships both within and among these groups, including those reconstructed using 16S rRNA, have been distorted by interspecies recombination events.     

Characterisation of the lpdA gene from Neisseria meningitidis by polymerase chain reaction, restriction fragment length polymorphism and sequencing

P64k protein from Neisseria meningitidis is well recognised in sera from individuals convalescent from meningococcal disease or vaccinated with the Cuban antimeningococcal vaccine VA-MENGOC-BC. The presence of the protein in more than 80 meningococcal strains has also been verified. It is immunogenic in animal models and the antibodies elicited show bactericidal activity against meningococci. To further investigate at the molecular level whether lpdA, the gene coding for P64k protein, is conserved among different N. meningitidis strains, a total of 20 strains isolated from different geographic areas were differentiated on the basis of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the lpdA gene and restriction endonuclease digestion with HpaII. Although a total of five different PCR-RFLP patterns were present, nucleotide sequence determination showed that identity levels were as high as 93-99% among the N. meningitidis strains analysed.     

The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis.

Multilocus sequence typing (MLST) is a recently developed nucleotide sequence-based method for the definitive assignment of isolates within bacterial populations to specific clones. MLST uses the same principles as multilocus enzyme electrophoresis and provides data that can be used to investigate aspects of the population genetics and evolution of bacterial species. We used an MLST data set consisting of the sequences of approximately 450-bp fragments from seven housekeeping loci from a large strain collection of Neisseria meningitidis to estimate the relative impact of recombination compared with point mutation in the diversification of N. meningitidis clonal complexes. 126 meningococcal isolates were assigned to 10 clonal complexes, 9 of which contained minor clonal variants. The allelic variation within each complex was classified as a recombinational exchange or a putative point mutation through a comparison of the sequences of each variant allele with that of the allele typically found in the clonal complex. The nine clonal complexes contained a total of 23 allelic variants, and analysis of the sequences of these variant alleles revealed that a single nucleotide site in a meningococcal housekeeping gene is at least 80-fold more likely to change as a result of recombination than as a result of mutation. This value is estimated to be 10-50-fold for Escherichia coli and approximately 50-fold for Streptococcus pneumoniae.     

Sulfonamide resistance in Neisseria meningitidis as defined by site-directed mutagenesis could have its origin in other species.

Sulfonamide resistance in Neisseria meningitidis is mediated by altered forms of the chromosomal gene for the drug target enzyme dihydropteroate synthase. Sulfonamides have been used for decades both for prophylaxis and the treatment of meningococcal disease, and resistance is common. Two types of resistance determinants have been identified, and regions important for drug insusceptibility to the corresponding enzyme have been defined by site-directed mutagenesis. Both types of resistance traits have spread among strains of N. meningitidis of different serogroups and serotypes, and the large differences at the nucleotide level in a comparison of the resistance genes with the dhps genes of susceptible meningococci indicate the origin of one or maybe both types in other Neisseria species. One sulfonamide-sensitive strain of N. meningitidis was found to have a mosaic dhps gene with a central part identical to the corresponding part of a gonococcal strain. This observation supports the idea of an interspecies transfer of genetic material in Neisseria species as a mechanism for the development of chromosomally mediated resistance.     

Genetic diversity of the iron-binding protein (Fbp) gene of the pathogenic and commensal Neisseria

Abstract The pathogenic Neisseria and most commensal Neisseria species produce an iron-binding protein (Fbp) when grown under iron-limited conditions. In the current study, we confirmed the presence of Fbp, as well as DNA sequences homologous to the gonococcal fbp , in strains of N. gonorrhoeae, N. meningitidis, N. cinerea, N. lactamica, N. subflava, N. kochii and N. polysaccharea . The fbp genes from these strains were amplified by the polymerase chain reaction, digested with Stu I or Rsa I, and the restriction patterns examined. The patterns for the gonococcal and meningococcal fbp were virtually identical; however, variations were observed in the fbp sequences of the commensal Neisseria species. N. flavescens, N. mucosa, N. sicca, N. ovis and Branhamella catarrhalis , did not produce Fbp as detected by sodium dodecyl sulfate-polyacrylamide gel electropheris and reactivity with an Fbp specific monoclonal antibody, nor did they hybridize to an fbp -specific DNA probe.     

The influence of mutation, recombination, population history, and selection on patterns of genetic diversity in Neisseria meningitidis

Patterns of genetic diversity within populations of human pathogens, shaped by the ecology of host-microbe interactions, contain important information about the epidemiological history of infectious disease. Exploiting this information, however, requires a systematic approach that distinguishes the genetic signal generated by epidemiological processes from the effects of other forces, such as recombination, mutation, and population history. Here, a variety of quantitative techniques were employed to investigate multilocus sequence information from isolate collections of Neisseria meningitidis, a major cause of meningitis and septicemia world wide. This allowed quantitative evaluation of alternative explanations for the observed population structure. A coalescent-based approach was employed to estimate the rate of mutation, the rate of recombination, and the size distribution of recombination fragments from samples from disease-associated and carried meningococci obtained in the Czech Republic in 1993 and a global collection of disease-associated isolates collected globally from 1937 to 1996. The parameter estimates were used to reject a model in which genetic structure arose by chance in small populations, and analysis of molecular variation showed that geographically restricted gene flow was unlikely to be the cause of the genetic structure. The genetic differentiation between disease and carriage isolate collections indicated that, whereas certain genotypes were overrepresented among the disease-isolate collections (the "hyperinvasive" lineages), disease-associated and carried meningococci exhibited remarkably little differentiation at the level of individual nucleotide polymorphisms. In combination, these results indicated the repeated action of natural selection on meningococcal populations, possibly arising from the coevolutionary dynamic of host-pathogen interactions.     

Genetic subgroups of Neisseria meningitidis serogroup A isolated from patients with disseminated forms of meningococcal infection in Moscow, 1969-2006

Results of microbiological monitoring for serogroup A Neisseria meningitidis circulated in Moscow from 2002 to 2006 are presented. Using multilocus sequence-typing, molecular and epidemiologic characteristics of 32 cultures isolated from cerebro-spinal fluid of patients with generalized forms of meningococcal infection. Typed isolates belonged to 4 sequence types: CT-3349 (detected in 24 cultures), CT-2 (detected in 5 cultures), CT-75 (detected in 2 cultures), and CT-5803 (detected in 1 culture). All sequence types (except CT-5803) were detected in Moscow in previous years. Using Internet database (http://pubmlst.org/neisseria) they were genetically characterized and compared with data on serogroup A meningococci circulated in Moscow before 2002., meningococci belonging to epidemically dangerous genetic subgroup III were not detected between characterized strains. Typed isolates were distributed between subgroups VI and X, which are typical for the area under surveillance. Genetic changes in Moscow population of Neisseria meningitidis serogroup A, which manifested by shift of dominating genetic subgroup after 2002-2003, were analyzed.     

Rapid laboratory detection of meningococcal disease outbreaks caused by serogroup C Neisseria meningitidis

Molecular subtyping is of significant importance to the recognition of outbreaks of meningococcal disease caused by serogroup C Neisseria meningitidis. We describe the application of multilocus variable number tandem repeat analysis (MLVA) for the molecular subtyping of N. meningitidis and compare its performance to that of pulsed-field gel electrophoresis (PFGE). For MLVA, a multiplex PCR assay targeting five variable number tandem repeat regions was developed and evaluated using a panel of sporadic and outbreak-associated serogroup C N. meningitidis isolates. MLVA was highly reproducible and provided results within 6 h. Overall, the discriminatory power of MLVA was equivalent to that of PFGE. The utilization of MLVA for subtyping N. meningitidis isolates provides a rapid and safer alternative to PFGE for identifying outbreaks of meningococcal disease. As such, it may provide public health officials with timely information that may minimize the spread of outbreak-related cases through prophylaxis.     

The effect of the nature of the strain on the character of the production of iron-dependent proteins by meningococci]

The comparative study of three Neisseria meningitidis strains (15, 125, 2394) was carried out by the method of electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate and by the method of immunoblotting. The intensive expression of 8 iron-regulated proteins (IRP) was shown to occur in iron-deficient culture medium. The major IRP with a molecular weight of 35 kD was expressed by all above-mentioned N. meningitidis strains under the conditions of iron deficiency and cross-reacted with 10 mouse and rabbit antisera to N. meningitidis of different groups, i.e. it was common to all Neisseria species. The antigenic activity of various IRP essentially differed with respect to antisera of animals and sera of patients with meningococcal infection.     

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.     

Proteome analysis of Neisseria meningitidis serogroup A

Neisseria meningitidis is an encapsulated Gram-negative bacterium responsible for significant morbidity and mortality worldwide. Meningococci are opportunistic pathogens, carried in the nasopharynx of approximately 10% of asymptomatic adults. Occasionally they enter the bloodstream to cause septicaemia and meningitis. Meningococci are classified into serogroups on the basis of polysaccharide capsule diversity, and serogroup A strains have caused major epidemics mainly in the developing world. Here we describe a two-dimensional gel electrophoresis protein map of the serogroup A strain Z4970, a clinical isolate classified as ancestral to several pandemic waves. To our knowledge this is the first systematically annotated proteomic map for N. meningitidis. Total protein samples from bacteria grown on GC-agar were electrophoretically separated and protein species were identified by matrix-assisted laser desorption/ionization time of flight spectrometry. We identified the products of 273 genes, covering several functional classes, including 94 proteins so far considered as hypothetical. We also describe several protein species encoded by genes reported by DNA microarray studies as being regulated in physiological conditions which are relevant to natural meningococcal pathogenicity. Since menA differs from other serogroups by having a fairly stable clonal population structure (i.e. with a low degree of variability), we envisaged comparative mapping as a useful tool for microevolution studies, in conjunction with established genotyping methods. As a proof of principle, we performed a comparative analysis on the B subunit of the meningococcal transferrin receptor, a vaccine candidate encoded by the tbpB gene, and a known marker of population diversity in meningococci. The results show that TbpB spot pattern variation observed in the maps of nine clinical isolates from diverse epidemic spreads, fits previous analyses based on allelic variations of the tbpB gene.     

Genetic interactions of DNA repair pathways in the pathogen Neisseria meningitidis

The current increase in the incidence and severity of infectious diseases mandates improved understanding of the basic biology and DNA repair profiles of virulent microbes. In our studies of the major pathogen and model organism Neisseria meningitidis, we constructed a panel of mutants inactivating genes involved in base excision repair, mismatch repair, nucleotide excision repair (NER), translesion synthesis, and recombinational repair pathways. The highest spontaneous mutation frequency among the N. meningitidis single mutants was found in the MutY-deficient strain as opposed to mutS mutants in Escherichia coli, indicating a role for meningococcal MutY in antibiotic resistance development. Recombinational repair was recognized as a major pathway counteracting methyl methanesulfonate-induced alkylation damage in the N. meningitidis. In contrast to what has been shown in other species, meningococcal NER did not contribute significantly to repair of alkylation-induced DNA damage, and meningococcal recombinational repair may thus be one of the main pathways for removal of abasic (apurinic/apyrimidinic) sites and strand breaks in DNA. Conversely, NER was identified as the main meningococcal defense pathway against UV-induced DNA damage. N. meningitidis RecA single mutants exhibited only a moderate decrease in survival after UV exposure as opposed to E. coli recA strains, which are extremely UV sensitive, possibly reflecting the lack of a meningococcal SOS response. In conclusion, distinct differences between N. meningitidis and established DNA repair characteristics in E. coli and other species were identified.     

Transposition of Tn916 to different sites in the chromosome of Neisseria meningitidis: a genetic tool for meningococcal mutagenesis

The difficulty in obtaining mutants in pathogenic Neisseria has limited the ability to genetically define determinants responsible for virulence as well as the ability to generate a genetic map. We show that the 16.5kb conjugative transposon Tn916 can be introduced into Neisseria meningitidis on the suicide vectors pAM120 and pAM170. After introduction, Tn916 transposed to different sites in the chromosome of recipient meningococci, apparently at random, and was stably incorporated. Following its integration into the meningococcal chromosome, Tn916 did not appear to readily express its conjugative and transpositional functions. However, chromosomal DNA from Tn916-carrying meningococci could be used to transform other meningococcal strains to tetracycline resistance. These studies indicate that Tn916 may be an important tool for genetic analysis of N. meningitidis.     

Multilocus sequencing--a new method of genotyping bacteria and first results of its use

Comparative characterization (molecular typing) of isolates within a bacterial species is one of the major problems in microbiology and epidemiology. However, it is rather difficult to correlate data obtained in various laboratories, because traditional, including molecular, methods employed in typing pathogenic microorganisms cannot be standardized. In 1998, Maiden et al. proposed multilocus sequence typing (MLST); through which alleles of several housekeeping genes are directly assessed by nucleotide sequencing, each unique allele combination determining a sequence type of a strain. The advantages of this approach are that the culturing of pathogenic microorganisms is avoided, as their gene fragments are amplified directly from biological samples, and that the sequencing data are unambiguous, easy to standardize, and electronically portable. The latter makes it possible to generate an expandable global database for each species at an Internet site, in order to use it for the purposes of genotyping pathogenic bacteria (and other infectious agents). MLST protocols have been elaborated for Neisseria meningitidis, Streptococcus pneumoniae, and Helicobacter pylori; those for Streptococcus pyogenes, Staphylococcus aureus, and Haemophilus influenzae are now being developed. Basic principles and the first results of MLST have been reviewed, including data on the distribution and microevolution of N. meningitidis clones causing epidemic meningococcal infection, the relative recombination and mutation rates in the N. meningitidis genome, the identification of antibiotic-resistant S. pneumoniae clones causing severe generalized infection, the grouping of H. pylori isolates from various geographic regions, etc.     

Analysis in Neisseria meningitidis and other Neisseria species of genes homologous to the FKBP immunophilin family

The immunophilin family of FK506-binding proteins (FKBPs), involved in eukaryotic protein folding and cell regulation, have recently been found to have prokaryotic homologues. Genes with sequences homologous to those encoding human FKBPs were examined in Neisseria species. An FKBP DNA sequence was present, as shown by the polymerase chain reaction and Southern blotting experiments, in the chromosome of Neisseria meningitidis (14 strains) and in all 11 different commensal Neisseria spp. studied, but was not found in Neisseria gonorrhoeae (11 strains tested) or in Moraxella catarrhalis. The nucleotide and predicted protein sequences of the FKBP-encoding domain from five of the meningococcal strains were highly conserved (e.g. ≥97% homologous). The meningococcal nucleotide sequence was ≥93% homologous and the consensus meningococcal protein sequence was ≥97% homologous to FKBP sequences found in seven different commensal Neisseria spp. The meningococcal nucleotide and predicted protein sequences were ≥59% homologous to the conserved C-terminus of the human FKBP gene family. The FKBP nucleotide sequence was present as a single copy in the chromosome of commensal Neisseria spp. and in most strains of N. meningitidis. The FKBP gene was linked to the silent pilin locus, pilS, in class II-piliated meningococcal strains. In meningococcal strains expressing class I pili, the FKBP gene was linked to one of several pilS loci but not the pilE locus present in these strains. FKBP genes found in commensal Neisseria spp. were not linked to known pilin loci.     

Epidemiological evaluation of Neisseria meningitidis serogroup B by pulsed-field gel electrophoresis

Abstract Genomic DNA from 25 strains of serogroup B Neisseria meningitidis was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Spe I. N. meningitidis genomic DNA displayed considerable diversity. The diversity we observed among these strains was stable and included isolates from an outbreak that were phenotypically identical. This confirms the value of macrorestriction profiling and PFGE in providing epidemiologically stable strain markers for typing meningococci.     

The role of fibronectin in the pathogenesis of meningococcal infection

We have characterized an interaction of 20 strains of Neisseria meningitidis serogroups A, B, C, 29E, W-135 and Z with immobilized fibronectin of human plasma. The adhesion of meningococci to fibronectin was determined by the extent of piliated cells and did not depend on the meningococcal serogroup. Binding of non-piliated or weakly piliated strains (2-5% of piliated cells in the stock) was sufficiently greater than those piliated (8-10%), where the adhesion to fibronectin was not at all observed. The examination of two well-piliated strains showed that the loss of pili resulted in the increase of bacterial adhesion to fibronectin. Constants of association and dissociation of piliated and non-piliated strains to fibronectin were calculated. The role of meningococci-fibronectin interaction in the pathogenesis of meningococcal infection is discussed.