Meningococcal Genome Research Articles

AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis

Oxidative stress is a principal cause of DNA damage, and mechanisms to repair this damage are among the most highly conserved of biological processes. Oxidative stress is also used by phagocytes to attack bacterial pathogens in defence of the host. We have identified and characterised two apurinic/apyrimidinic (AP) endonuclease paralogues in the human pathogen Neisseria meningitidis. The presence of multiple versions of DNA repair enzymes in a single organism is usually thought to reflect redundancy in activities that are essential for cellular viability. We demonstrate here that these two AP endonuclease paralogues have distinct activities in DNA repair: one is a typical Neisserial AP endonuclease (NApe), whereas the other is a specialised 3'-phosphodiesterase Neisserial exonuclease (NExo). The lack of AP endonuclease activity of NExo is shown to be attributable to the presence of a histidine side chain, blocking the abasic ribose-binding site. Both enzymes are necessary for survival of N. meningitidis under oxidative stress and during bloodstream infection. The novel functional pairing of NExo and NApe is widespread among bacteria and appears to have evolved independently on several occasions.

DNA repair profiles of disease-associated isolates ofNeisseria meningitidis

Neisseria meningitidis, or the meningococcus, is the source of significant morbidity and mortality in humans worldwide. Even though mutability has been linked to the occurrence of outbreaks of epidemic disease, meningococcal DNA repair pathways are poorly delineated. For the first time, a collection of meningococcal disease-associated isolates has been demonstrated to express constitutively the DNA glycosylases MutY and Fpg in vivo. DNA sequence analysis showed considerable variability in the deduced amino acid sequences of MutS and Fpg, while MutY and RecA were highly conserved. Interestingly, multi-locus sequence typing demonstrated a putative link between the pattern of amino acid substitutions and levels of spontaneous mutagenicity in meningococcal strains. These results provide a basis for further studies aimed at resolving the genotype/phenotype relationships of meningococcal genome variability and mutator activity.

Microarray analyses of meningococcal genome composition and gene regulation: a review of the recent literature: Table 1

The development of microarrays for genome comparison and transcriptional profiling along with the public availability of several meningococcal genome sequences has promoted studies elucidating (i) intraspecific and interspecific genomic differences of members of the genus Neisseria, and (ii) the transcriptional response of meningococci to a variety of environmental stresses such as heat shock, iron starvation, serum treatment, and contact with eukaryotic cells. Furthermore, microarray-based finetyping of meningococci is in development. It will remain a difficult, but important, goal to identify sets of genes determining the virulence potential of hypervirulent meningococcal lineages in comparison with apathogenic ones. The recent identification of the meningococcal disease-associated island through the application of microarray analyses has been a step towards this aim. Transcriptional profiling of meningococci has brought about the compilation of large datasets, which also provide information about several regulons. Meningococcal microarray analysis has established a basis for studies clarifying the function of previously unknown genes, and has supported the identification of interesting vaccine candidates. However, harmonization of protocols and tools, as well as central databases are needed to foster the comparability of studies and the integration of knowledge.

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

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.

Meningococcal genome dynamics

Neisseria meningitidis (the meningococcus) is an important commensal, pathogen and model organism that faces up to the environment in its exclusive human host with a small but hyperdynamic genome. Compared with Escherichia coli, several DNA-repair genes are absent in N. meningitidis, whereas the gene products of others interact differently. Instead of responding to external stimuli, the meningococcus spontaneously produces a plethora of genetic variants. The frequent genomic alterations and polymorphisms have profound consequences for the interaction of this microorganism with its host, impacting structural and antigenic changes in crucial surface components that are relevant for adherence and invasion as well as antibiotic resistance and vaccine development.

Unusual Genetic Organization of a Functional Type I Protein Secretion System in Neisseria meningitidis

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.

Comparative genomic view of mechanisms of variation in Neisseria meningitidis through sequencing and analysis of a third meningococcal genome from serogroup C strain FAM18

Comparative genomic view of mechanisms of variation in Neisseria meningitidis through sequencing and analysis of a third meningococcal genome from serogroup C strain FAM18

Modulation of the mtrCDE‐encoded efflux pump gene complex of Neisseria meningitidis due to a Correia element insertion sequence

The mtr (multiple transferable resistance) gene complex in Neisseria gonorrhoeae encodes an energy-dependent efflux pump system that is responsible for export of anti-bacterial hydrophobic agents. Expression of the mtrCDE operon in gonococci is negatively regulated by the MtrR protein. Hydrophobic agent resistance mediated by the mtr system is also inducible, which results from an AraC-like protein termed MtrA. In this work, we identified and characterized a pump similar to the gonococcal mtr system in various strains of Neisseria meningitidis. Unlike the situation with gonococci, the mtr system in meningococci is not subject to the MtrR or MtrA regulatory schemes. An analysis of the promoter region of the mtrCDE operon in a panel of meningococcal strains revealed the presence of one or two classes of insertion sequence elements. A 155-159 bp insertion sequence element known as the Correia element, previously identified elsewhere in the gonococcal and meningococcal genomes, was present in the mtrCDE promoter region of all meningococcal strains tested. In addition to the Correia element, a minority of strains had a tandemly linked, intact copy of IS1301. As described previously, a binding site for the integration host factor (IHF) was present at the centre of the Correia element upstream of mtrCDE genes. IHF was found to bind specifically to this site and deletion of the IHF binding site enhanced mtrC transcription. We also identified a post-transcriptional regulation of the mtrCDE transcript by cleavage in the inverted repeat of the Correia element, as previously described by Mazzone et al. [Gene278: 211-222 (2001)] and De Gregorio et al. [Biochim Biophys Acta 1576: 39-44 (2002)]for other Correia element. We conclude that the mtr efflux system in meningococci is subject to transcriptional regulation by IHF and post-transcriptional regulation by cleavage in the inverted repeat of the Correia element.

Natural transformation and phase variation modulation in Neisseria meningitidis.

Neisseria meningitidis has evolved the ability to control the expression-state of numerous genes by phase variation. It has been proposed that the process aids this human pathogen in coping with the diversity of microenvironments and host immune systems. Therefore, increased frequencies of phase variation may augment the organism's adaptability and virulence. In this study, we found that DNA derived from various neisserial co-colonizers of the human nasopharynx increased N. meningitidis switching frequencies, indicating that heterologous neisserial DNA modulates phase variation in a transformation-dependent manner. In order to determine whether the effect of heterologous DNA was specific to the Hb receptor, HmbR, we constructed a Universal Rates of Switching cassette (UROS). With this cassette, we demonstrated that heterologous DNA positively affects phase variation throughout the meningococcal genome, as UROS phase variation frequencies were also increased in the presence of neisserial DNA. Overexpressing components of the neisserial mismatch repair system partially alleviated DNA-induced changes in phase variation frequencies, thus implicating mismatch repair titration as a cause of these transformation-dependent increases in switching. The DNA-dependent effect on phase variation was transient and may serve as a mechanism for meningococcal genetic variability that avoids the fitness costs encountered by global mutators.

Meningococcal vaccines.

Meningococcal disease is one of the most feared and serious infections in the young and its prevention by vaccination is an important goal. The high degree of antigenic variability of the organism makes the meningococcus a challenging target for vaccine prevention. Meningococcal polysaccharide vaccines against serogroup A and C are efficacious and have been widely used, often in combination with serogroup Y and W135 components. Their relative lack of immunogenicity in young children and infants can be overcome by conjugation to a protein carrier. The effectiveness of serogroup C glycoconjugate vaccines in children of all ages has been demonstrated and they have now been introduced into routine vaccination schedules. Conjugate vaccines against other serogroups, including A, Y, and W135 will soon be available and it is hoped they may emulate this success. Prevention of serogroup B disease has proven more elusive. Several serogroup B vaccines based on outer membrane vesicles have been shown to be immunogenic and reasonably effective in adults and older children, but the protection offered by them is chiefly strain-specific. Multivalent recombinant PorA vaccines have been developed to broaden the protective effect, but no efficacy data are available as yet. Intensive efforts have been directed at other outer membrane protein vaccine candidates and lipopolysaccharide, and some of these have been shown to offer protection in experimental animal models. Nonpathogenic Neisseriae spp. such as Neisseria lactamica are also possible vaccine candidates. Previously unknown proteins have been identified from in silico analysis of the meningococcal genome and their vaccine potential explored. However, none of these has yet been presented as the 'universal' protective antigen and work in this field continues to be held back by our limited knowledge concerning the mechanisms of natural protection against serogroup B meningococci.

Meningococcal vaccines

The successful introduction of a protein–polysaccharide conjugate vaccine against serogroup C meningococci into the UK infant immunization schedule, in combination with a catch-up campaign for individuals less than 18 years of age, has seen virtually all group C disease eliminated in childhood. From being a devastating disease with a very high mortality, the possibility of eradicating invasive meningococcal disease now seems eminently feasible. Since similar technology is likely to facilitate prevention of disease caused be serogroup A, Y and W135 meningococci, the major hurdle in achieving the goal of eradication is development of a safe and immunogenic vaccine against serogroup B infections. Outer membrane vesicle vaccines remain in development and further trials are anticipated. Through the recent availability of the meningococcal genome sequence, many new vaccine candidates are being identified and there is increasing optimism that a solution to the problem can be found.

Prospects offered by genome studies for combating meningococcal disease by vaccination.

Meningococcal disease was first recognised and Neisseria meningitidis isolated as the causative agent over 100 years ago, but despite more than a century of research, attempts to eliminate this distressing illness have so far been thwarted. The main problem lies in the fact that N. meningitidis usually exists as a harmless commensal inhabitant of the human nasopharynx, the pathogenic state being the exception rather than the norm. As man is its only host, the meningococcus is uniquely adapted to this ecological niche and has evolved an array of mechanisms for evading clearance by the human immune response. Progress has been made in combating the disease by developing vaccines that target specific pathogenic serogroups of meningococci. However, a fully comprehensive vaccine that protects against all pathogenic strains is still just beyond reach. The publication of the genome sequences of two meningococcal strains, one each from serogroups A and B and the imminent completion of a third illustrates the extent of the problems to be overcome, namely the vast array of genetic mechanisms for the generation of meningococcal diversity. Fortunately, genome studies also provide new hope for solutions to these problems in the potential for a greater understanding of meningococcal pathogenesis and possibilities for the identification of new vaccine candidates. This review describes some of the approaches that are currently being used to exploit the information from meningococcal genome sequences and seeks to identify future prospects for combating meningococcal disease.

Neisserial TonB-dependent outer-membrane proteins: detection, regulation and distribution of three putative candidates identified from the genome sequences.

Computer searches were carried out of the gonococcal and meningococcal genome databases for previously unknown members of the TonB-dependent family (Tdf) of outer-membrane receptor proteins. Seven putative non-contiguous genes were found and three of these (identified in gonococcal strain FA1090) were chosen for further study. Consensus motif analysis of the peptide sequences was consistent with the three genes encoding TonB-dependent receptors. In view of the five previously characterized TonB-dependent proteins of pathogenic neisseriae, the putative genes were labelled tdfF, tdfG and tdfH. TdfF had homology with the siderophore receptors FpvA of Pseudomonas aeruginosa and FhuE of Escherichia coli, whereas TdfG and TdfH had homology with the haemophore receptor HasR of Serratia marcescens. The aim of this project was to characterize these proteins and determine their expression, regulation, distribution and surface exposure. Strain surveys of iron-stressed commensal and pathogenic neisseriae revealed that TdfF is unlikely to be expressed, TdfG is expressed by gonococci only and that TdfH is expressed by both meningococci and gonococci. Expression of TdfH was unaffected by iron availability. Susceptibility of TdfH to cleavage by proteases in live gonococci was consistent with surface exposure of this protein. TdfH may function as a TonB-dependent receptor for a non-iron nutrient source. Furthermore, TdfH is worthy of future investigation as a potential meningococcal vaccine candidate as it is a highly conserved, widely distributed and surface-exposed outer-membrane protein.

Generation and characterization of a PhoP homologue mutant of Neisseria meningitidis.

Two-component regulatory systems are important regulators of virulence genes in a number of bacteria. Genes encoding a two-component regulator system, with homology to the phoP/phoQ system in salmonella, were identified in the meningococcal genome. Allele replacement was used to generate a meningococcal knock-out mutant of the regulator component of this system, and its phenotype was examined. The mutant displayed many differences in protein profiles compared with wild type, consistent with it being a gene-regulatory mutation. Many of the growth characteristics of the mutant were similar to those of phoP mutants of salmonella: it was unable to grow at low concentrations of magnesium and was sensitive to defensins and other environmental stresses. Magnesium-regulated differences in protein expression were abrogated in the mutant, indicating that the meningococcal PhoP/PhoQ system may, as in salmonella, respond to changes in environmental magnesium levels. These results are consistent with the PhoP homologue playing a similar role in the meningococcus to PhoP in salmonella and suggest that it may similarly be involved in the regulation of virulence genes in response to environmental stimuli in the meningococcus. In support of this conclusion, we found the mutant grew was unable to grow in mouse serum and was attenuated in its ability to traverse through a layer of human epithelial cells. Identification of those genes regulated by the meningococcal PhoP may provide a route towards the identification of virulence genes in the meningococcus.

Genomics of Neisseria meningitidis

Abstract An important feature of disease caused by Neisseria meningitidis is the propensity to invade the meninges. Much progress has been made in our understanding of how this pathogen circumvents the physical properties of this cellular barrier. This review will address the new possibilities offered by the recent availability of meningococcal genome sequences.

Mutagenesis of Neisseria meningitidis by in vitro transposition of Himar1 mariner.

Now that the meningococcal genome sequence has been completed, the lack of a suitable method for saturation mutagenesis remains a major obstacle to the unraveling of the pathogenic propensity of Neisseria meningitidis. Here, we demonstrate that in vitro Himar1 mariner transposition on chromosomal or PCR-amplified meningococcal DNA, which is subsequently reintroduced into N. meningitidis by natural transformation, is an extremely efficient mutagenesis method. Southern blot analysis, sequencing the Himar1 insertion point in numerous transposition mutants, and a limited screening of the mutant libraries for clones impaired in maltose catabolism confirmed that Himar1 transposed randomly in N. meningitidis. Taken together, these data demonstrate that Himar1 in vitro transposition can lead to the exhaustive mutagenesis of N. meningitidis, allowing for the first time a genomic-scale mutational analysis of this important human pathogen.

Microbial genomics

Hot on the heels of the Neisseria meningitidis serogroup B genome sequence comes publication of a second complete meningococcal genome sequence, this time from a serogroup A strain, Z2491. Although not common in the UK, serogroup A strains are responsible for most meningococal disease and death on a global basis. Julian Parkhill and others at the Sanger Centre, with collaborators from Oxford and Berlin, thanks to funding from the Wellcome Trust have recently described the 2.18-Mb genome sequence 1 Parkhill J. et al. Complete DNA sequence of a serogroup A strain of Neisseria menigitidis Z2491. Nature. 2000; 404: 502-506 Crossref PubMed Scopus (614) Google Scholar . Two notable discoveries were the presence of many hundreds of repetitive elements and of at least 60 coding regions (nearly 5% in total) with a lower than average G+C content (i.e. probably representing recently acquired DNA). Detailed analysis of the genome suggested three mechanisms of repeat-mediated antigenic variation: on/off switching by slipped-strand mispairing; intragenomic recombination leading to the use of different carboxy termini for surface-exposed proteins; and intergenomic gene conversion of surface-protein-encoding genes associated with large arrays of repeats, mediated by the internalization of related DNA through a DNA uptake sequence. Work is already underway at the Sanger Centre on a third meningococcal genome sequence, of a serogroup C strain. The genome sequences and associated information are accessible through the Sanger web site: (http://www.sanger.ac.uk/ Projects/N_meningitidis/).

Swapping genes

Haemophilus and Neisseria spp. are common, normal inhabitants of the human upper respiratory tract but can occasionally cause life-threatening infections. Many species of both of these genera are naturally competent for DNA transformation, a capacity that allows them to take up fragments of DNA from the environment and incorporate this DNA into their chromosomes. The sequence required for uptake has been defined for Haemophilus influenzae (HmUS) as a 29-bp consensus sequence with a conserved 9-bp core sequence. This sequence is quite different from the 10-bp sequence for Neisseria meningitidis uptake (nUS). During the sequence analysis of the superoxide dismutase gene (sodC) from N. meningitidis, Kroll et al.[ 1 Kroll J.S. et al. Natural genetic exchange between Haemophilus and Neisseria: intergeneric transfer of chromosomal genes between major human pathogens. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12381-12385 Crossref PubMed Scopus (123) Google Scholar ]noted an inverted repeated sequence characteristic of a rho-independent terminator. Interestingly, this inverted repeat sequence included an HmUS. Protein sequence comparison of SodC from N. meningitidis and H. influenzae showed striking similarities, while the adjacent upstream N. meningitidis gene product showed a similarity to the homologous H. influenzae gene product more in keeping with its phylogenetic similarity. These findings suggest that the N. meningitidis sodC gene was obtained by horizontal gene transfer from Haemophilus. By scanning the partial meningococcal genome for homology to the HmUS, two additional loci with similarities to regions from H. influenzae were identified. One of these loci contained the HmUS within a protein coding region; this sequence is apparently not critical for this protein's activity, as it is not present in the corresponding Escherichia coli protein. By analysing the complete H. influenzae genome for potential nUS, DNA from a region near one of these sites was shown to have similarity to the DNA from the same region in N. meningitidis. These sequence comparisons suggest that transfer of DNA from H. influenzae to N. meningitidis has occurred. The potential for DNA transfer between these species during the colonization of the nasopharynx might lead to the acquisition of virulence or other factors that could result in a more pathogenic bacteria. An example of such a horizontal gene transfer may be found in H. influenzae biogroup aegyptius, the cause of septicaemia associated with the disease Brazilian purpuric fever, which acquired a highly virulent `meningococcal' phenotype.

Molecular analysis of the biosynthesis pathway of the α‐2,8 polysialic acid capsule by Neisseria meningitidls serogroup B

The genes encoding all enzymes necessary for capsular polysaccharide biosynthesis in Neisseria meningitidis B are located on a 5 kb DNA fragment within the chromosomal cps gene cluster. Nucleotide sequence analysis revealed four open reading frames (ORFs), which can encode proteins with molecular masses of 41.4 kDa, 24.9 kDa, 38.3 kDa, and 54.4 kDa, respectively. These ORFs constitute a transcriptional unit as demonstrated by Northern blots. Primer extension analysis revealed that the transcriptional start site is preceded by a nucleotide sequence with homologies to the sigma 70 consensus promoter sequence of Escherichia coli. Functional analysis of the proteins encoded by the ORFs indicated that ORF2 encodes the CMP-NeuNAc synthetase, ORF3 encodes the NeuNAc condensing enzyme, and ORF4 encodes the alpha-2,8 polysialyltransferase. ORF1 encodes an enzyme, which provides a precursor molecule for synthesis of monomeric NeuNAc. In E. coli the subcloned ORFs 2-4 were able to synthesize a high-molecular-weight alpha-2,8 polysialic acid. In contrast, inactivation of ORF1 in the meningococcal genome resulted in a complete loss of capsule production. A regulatory enzyme, the CMP-NeuNAc hydrolase, which cleaves CMP-NeuNAc to CMP and NeuNAc, was not found as a part of the capsular polysaccharide biosynthesis gene operon or within the cps gene cluster.

Identification of a UK outbreak strain of Neisseria meningitidis with a DNA probe

To investigate the epidemiology of meningococcal disease, a specific DNA probe pUS210 (carrying insert DNA which is repeated in the meningococcal genome) was isolated. The ability of this probe to hydridise with multiple polymorphic fragments in Southern blots wasexploited to examine genetic relations within strains. Two geographically distinct foci of prolonged meningococcal disease (Gloucester and Plymouth, UK) are due to a clonal population of virulent strains that are distinct from those found elsewhere in the UK.