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1.  Opacity-Associated Adhesin Repertoire in Hyperinvasive Neisseria meningitidis†  
Infection and Immunity  2006;74(9):5085-5094.
The opacity (Opa) proteins mediate a variety of interactions between the bacterium Neisseria meningitidis and its human host. These interactions are thought to be of central importance in both the asymptomatic colonization of the nasopharynx and the sporadic occurrence of meningococcal disease. The receptor specificities of a limited number of Opa protein variants have been explored, but the high level of amino acid sequence diversity among variants has complicated the assignment of specific roles to individual Opa variants or combinations of variants. In addition, the distribution of Opa protein variants among diverse meningococci, information that is potentially informative for studies of Opa function, is poorly understood. A systematic survey of the genetic diversity in the four opa gene loci in each of 77 meningococcal isolates was undertaken. These isolates were representative of the seven hyperinvasive meningococcal clonal complexes that caused the majority of meningococcal disease over the last 50 years. Consistent with previous studies, a high level of sequence diversity was observed among the opa genes and the proteins that they encoded; however, particular sets of Opa protein variants were consistently associated with each of the clonal complexes over time periods often spanning decades and during global spread. These observations were consistent with the postulate that particular combinations of Opa proteins confer fitness advantages to individual clonal complexes and have implications for studies of Opa function and the inclusion of Opa proteins in novel meningococcal vaccines.
doi:10.1128/IAI.00293-06
PMCID: PMC1594835  PMID: 16926400
2.  Clonal Waves of Neisseria Colonisation and Disease in the African Meningitis Belt: Eight- Year Longitudinal Study in Northern Ghana 
PLoS Medicine  2007;4(3):e101.
Background
The Kassena-Nankana District of northern Ghana lies in the African “meningitis belt” where epidemics of meningococcal meningitis have been reoccurring every eight to 12 years for the last 100 years. The dynamics of meningococcal colonisation and disease are incompletely understood, and hence we embarked on a long-term study to determine how levels of colonisation with different bacterial serogroups change over time, and how the patterns of disease relate to such changes.
Methods and Findings
Between February 1998 and November 2005, pharyngeal carriage of Neisseria meningitidis in the Kassena-Nankana District was studied by twice-yearly colonisation surveys. Meningococcal disease was monitored throughout the eight-year study period, and patient isolates were compared to the colonisation isolates. The overall meningococcal colonisation rate of the study population was 6.0%. All culture-confirmed patient isolates and the majority of carriage isolates were associated with three sequential waves of colonisation with encapsulated (A ST5, X ST751, and A ST7) meningococci. Compared to industrialised countries, the colonising meningococcal population was less constant in genotype composition over time and was genetically less diverse during the peaks of the colonisation waves, and a smaller proportion of the isolates was nonserogroupable. We observed a broad age range in the healthy carriers, resembling that of meningitis patients during large disease epidemics.
Conclusions
The observed lack of a temporally stable and genetically diverse resident pharyngeal flora of meningococci might contribute to the susceptibility to meningococcal disease epidemics of residents in the African meningitis belt. Because capsular conjugate vaccines are known to impact meningococcal carriage, effects on herd immunity and potential serogroup replacement should be monitored following the introduction of such vaccines.
An analysis of pharyngeal carriage of meningococci in one district of Ghana examined the features of the isolates that might contribute to the susceptibility to meningococcal epidemics in the African meningitis belt.
Editors' Summary
Background.
Bacterial meningitis is a rare but often fatal infection of the meninges—the thin membrane around the brain and the spinal cord. It can be caused by several types of bacteria, but meningococcal meningitis, which is caused by Neisseria meningitidis, is the most common form of bacterial meningitis in children and the second most common form in adults. About 10% of healthy people have N. meningitidis growing in their nose and throat; the bacteria are spread by exposure to infected respiratory secretions. In these “carriers,” the immune system keeps the bug in check but sometimes this surveillance fails, N. meningitidis enters the bloodstream and travels to the brain, where it infects the meninges and causes inflammation. The symptoms of meningococcal meningitis are sudden fever, headache, and a stiff neck and, even if strong antibiotics are given quickly, 10%–15% of patients die.
Why Was This Study Done?
Outbreaks of meningococcal meningitis occur all over the world, but the highest burden of disease is in the African meningitis belt, which stretches across sub-Saharan Africa from Senegal to Ethiopia. Here, localized epidemics of meningococcal meningitis occur every eight to 12 years during the dry season. Control of these epidemics relies on their early detection followed by mass immunization. This approach can be hard to implement in countries with limited resources, but the introduction of other control measures (for example, routine childhood immunization) requires an understanding of how the spread of different strains of N. meningitides through the community causes periodic epidemics. In this study, the researchers have studied the long-term dynamics of colonization by N. meningitidis and the occurrence of meningococcal meningitis in one region of the African meningitis belt.
What Did the Researchers Do and Find?
The researchers took throat swabs twice a year from people living in rural northern Ghana for eight years. They tested each swab for N. meningitidis and determined the serogroup of the bacteria they found. Bacterial serogroups differ only in terms of the antigens (molecules recognized by the immune system) that they express; N. meningitidis is classified into 13 serogroups based on the sugars that coat its surface. The researchers also used DNA sequencing to group the bacterial isolates into genoclouds—genetically closely related groups of meningococci represented by a sequence type (ST) number. Finally, they monitored meningococcal disease throughout the study and determined the serogroup and genocloud of patient isolates. Their results show colonization of 6% of the study population by N. meningitidis and reveal three consecutive waves of colonization and disease characterized by the presence of a serogroup A ST5 genocloud, a serogroup X ST751 genocloud, and finally a serogroup A ST7 genocloud. Colonizing bacteria isolated in this study in Ghana, the researchers report, changed their genotype more frequently but were less genetically diverse than those isolated in industrialized countries. In addition, the commonest serogroups of N. meningitidis in carriers in Ghana were disease-causing serogroups, whereas in industrialized countries these serogroups are rarely seen in carriers. However, non-groupable bacteria (bacteria that lack surface sugars), although common in industrialized countries, were rare in Ghana.
What Do These Findings Mean?
These findings begin to explain why epidemics of meningococcal meningitis are common in the African meningitis belt. Because there isn't a stable, genetically diverse population of N. meningitidis in carriers, the immune systems of people living here may not be optimally prepared to deal with new bacterial clones that arrive in the region, and this lack of immunity could result in frequent epidemics. However, because the researchers took relatively few samples every six months from one small area of the meningitis belt, the genetic diversity of N. meningitidis over the whole region might be considerably greater than that colonizing the study population. Nevertheless, the description of successive waves of meningococci colonization in Ghana has important implications for the proposed introduction of childhood vaccination against meninogococcal disease in the African meningitis belt. If this vaccination program goes ahead, warn the researchers, it will be essential to monitor which strains of N. meningitidis are colonizing the population and to have emergency plans ready to deal with any emerging disease-causing serogroups that are not covered by the vaccine.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040101.
The Web sites of the institutions at which this research was performed, the Swiss Tropical Institute and the Navrongo Health Research Centre, provide more information about the programs
The World Health Organization provides information on meningococcal disease, including the African meningitis belt (in English, Spanish, Chinese, Russian, and Arabic)
Information on meningitis and vaccines and their potential use in Africa is available from the Meningitis Vaccine Project (in English and French)
Medline Plus has encyclopedia pages on meningococcal meningitis
The US Centers for Disease Control and Prevention provides information on meningococcal disease (in English and Spanish)
doi:10.1371/journal.pmed.0040101
PMCID: PMC1831736  PMID: 17388665
3.  Differential Modulation of TNF-α–Induced Apoptosis by Neisseria meningitidis 
PLoS Pathogens  2009;5(5):e1000405.
Infections by Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell–bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods (FITC-Annexin V staining followed by FACS analysis, caspase assays and DNA fragmentation). Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-α is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-α receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-α. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx.
Author Summary
Acquisition of Neisseria meningitidis often leads to asymptomatic colonization (carriage) and rarely results in invasive disease associated with tissue injury. The reasons that make disease-associated isolates (pathogenic isolates) but not asymptomatic carriage isolates able to invade the host to establish disease are not understood. Isolates belonging to the ST-11 clonal complex are most frequently associated with the disease and rarely found in carriers. These hyper-invasive isolates may be able to induce cytopathic effects in target cells. We aimed to investigate the cytopathic effect of meningococcal isolates on epithelial cells using both ST-11 pathogenic isolates and carriage isolates. We showed that cytopathic effects were strongly associated with pathogenic isolates and infected cells exhibited features of apoptosis. This effect is mainly mediated by bacterial endotoxin (lipooligosaccharide) and involved an autocrine signaling mechanism of secreted TNF-α through its receptor TNF-RI. In contrast, carriage isolates down-regulate TNF-RI on the surface of infected cells by increasing TNF-RI shedding into the medium. We suggest that chelating secreted TNF-α protects cells from apoptosis. Our results unravel a differential modulation of TNF-α signaling by meningococcal isolates leading to cell survival or death and would therefore contribute to better understanding of the duality between carriage and invasiveness.
doi:10.1371/journal.ppat.1000405
PMCID: PMC2669886  PMID: 19412525
4.  Variation and molecular evolution of HmbR, the Neisseria meningitidis haemoglobin receptor 
Microbiology  2010;156(Pt 5):1384-1393.
Meningococcal disease caused by serogroup B Neisseria meningitidis remains an important health problem in many parts of the world, and there are currently no comprehensive vaccines. Poor immunogenicity, combined with immunological identity to human sialic acids, have hindered the development of a serogroup B conjugate vaccine, resulting in the development of alternative vaccine candidates, including many outer-membrane protein (OMP)-based formulations. However, the design of protein-based meningococcal vaccines is complicated by the high level of genetic and antigenic diversity of the meningococcus. Knowledge of the extent and structuring of this diversity can have implications for the use of particular proteins as potential vaccine candidates. With this in mind, the diversity of the meningococcal OMP HmbR was investigated among N. meningitidis isolates representative of major hyper-invasive lineages. In common with other meningococcal antigens, the genetic diversity of hmbR resulted from a combination of intraspecies horizontal genetic exchange and de novo mutation. Furthermore, genealogical analysis showed an association of hmbR genes with clonal complexes and the occurrence of two hmbR families, A and B. Three variable regions (VR1–VR3), located in loops 2, 3 and 4, were observed with clonal complex structuring of VR types. A minority of codons (3.9 %), located within putative surface-exposed loop regions of a 2D model, were under diversifying selection, indicating regions of the protein likely to be subject to immune attack.
doi:10.1099/mic.0.036475-0
PMCID: PMC3068627  PMID: 20150237
5.  Sequence, distribution and chromosomal context of class I and class II pilin genes of Neisseria meningitidis identified in whole genome sequences 
BMC Genomics  2014;15:253.
Background
Neisseria meningitidis expresses type four pili (Tfp) which are important for colonisation and virulence. Tfp have been considered as one of the most variable structures on the bacterial surface due to high frequency gene conversion, resulting in amino acid sequence variation of the major pilin subunit (PilE). Meningococci express either a class I or a class II pilE gene and recent work has indicated that class II pilins do not undergo antigenic variation, as class II pilE genes encode conserved pilin subunits. The purpose of this work was to use whole genome sequences to further investigate the frequency and variability of the class II pilE genes in meningococcal isolate collections.
Results
We analysed over 600 publically available whole genome sequences of N. meningitidis isolates to determine the sequence and genomic organization of pilE. We confirmed that meningococcal strains belonging to a limited number of clonal complexes (ccs, namely cc1, cc5, cc8, cc11 and cc174) harbour a class II pilE gene which is conserved in terms of sequence and chromosomal context. We also identified pilS cassettes in all isolates with class II pilE, however, our analysis indicates that these do not serve as donor sequences for pilE/pilS recombination. Furthermore, our work reveals that the class II pilE locus lacks the DNA sequence motifs that enable (G4) or enhance (Sma/Cla repeat) pilin antigenic variation. Finally, through analysis of pilin genes in commensal Neisseria species we found that meningococcal class II pilE genes are closely related to pilE from Neisseria lactamica and Neisseria polysaccharea, suggesting horizontal transfer among these species.
Conclusions
Class II pilins can be defined by their amino acid sequence and genomic context and are present in meningococcal isolates which have persisted and spread globally. The absence of G4 and Sma/Cla sequences adjacent to the class II pilE genes is consistent with the lack of pilin subunit variation in these isolates, although horizontal transfer may generate class II pilin diversity. This study supports the suggestion that high frequency antigenic variation of pilin is not universal in pathogenic Neisseria.
doi:10.1186/1471-2164-15-253
PMCID: PMC4023411  PMID: 24690385
Type four pilus; Neisseria meningitidis; Class I pilin; Class II pilin; Antigenic variation
6.  Potential of Recombinant Opa Proteins as Vaccine Candidates against Hyperinvasive Meningococci ▿ † 
Infection and Immunity  2011;79(7):2810-2818.
Neisseria meningitidis causes half a million cases of septicemia and meningitis globally each year. The opacity (Opa) integral outer membrane proteins from N. meningitidis are polymorphic and highly immunogenic. Particular combinations of Opa proteins are associated with the hyperinvasive meningococcal lineages that have caused the majority of serogroup B and C meningococcal disease in industrialized countries over the last 60 years. For the first time, this genetic structuring of a diverse outer membrane protein family has been used to select a novel combination of representative antigens for immunogenicity testing. Fourteen recombinant Opa variants were produced and used in murine immunizations inducing an increase in specific antimeningococcal total IgG levels. All 14 Opa proteins elicited bactericidal antibodies against at least one hyperinvasive meningococcal isolate, and most isolates from each hyperinvasive lineage were killed by at least one Opa antiserum at a titer of 1:16 or greater. Cross-reactive bactericidal antibody responses were observed among clonal complexes. A theoretical coverage of 90% can be achieved by using a particular combination of 6 Opa proteins against an isolate collection of 227 recent United Kingdom disease cases. This study indicates the potential of Opa proteins to provide broad coverage against multiple meningococcal hyperinvasive lineages.
doi:10.1128/IAI.01338-10
PMCID: PMC3191958  PMID: 21464082
7.  Molecular epidemiology and emergence of worldwide epidemic clones of Neisseria meningitidis in Taiwan 
Background
Meningococcal disease is infrequently found in Taiwan, a country with 23 million people. Between 1996 and 2002, 17 to 81 clinical cases of the disease were reported annually. Reported cases dramatically increased in 2001–2002. Our record shows that only serogroup B and W135 meningococci have been isolated from patients with meningococcal disease until 2000. However, serogroup A, C and Y meningococci were detected for the first time in 2001 and continued to cause disease through 2002. Most of serogroup Y meningococcus infections localized in Central Taiwan in 2001, indicating that a small-scale outbreak of meningococcal disease had occurred. The occurrence of a meningococcal disease outbreak and the emergence of new meningococcal strains are of public health concern.
Methods
Neisseria meningitidis isolates from patients with meningococcal disease from 1996 to 2002 were collected and characterized by serogrouping, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genetic relatedness and clonal relationship between the isolates were analyzed by using the PFGE patterns and the allelic profiles of the sequence types (STs).
Results
Serogroups A, B, C, W135, Y, and non-serogroupable Neisseria meningitidis were, respectively, responsible for 2%, 50%, 2%, 35%, 9%, and 2% of 158 culture-confirmed cases of meningococcal disease in 1996–2002. Among 100 N. meningitidis isolates available for PFGE and MLST analyses, 51 different PFGE patterns and 30 STs were identified with discriminatory indices of 0.95 and 0.87, respectively. Of the 30 STs, 21 were newly identified and of which 19 were found in serogroup B isolates. A total of 40 PFGE patterns were identified in 52 serogroup B isolates with the patterns distributed over several distinct clusters. In contrast, the isolates within each of the serogroups A, C, W135, and Y shared high levels of PFGE pattern similarity. Analysis of the allelic profile of the 30 STs suggested the serogroup B isolates be assigned into 5 clonally related groups/ clonal complexes and 7 unique clones. The ST-41/44 complex/Lineage 3, and the ST-3439 and ST-3200 groups represented 79% of the serogroup B meningococci. In contrast, isolates within serogroups A, serogroup W135 (and C), and serogroup Y, respectively, simply belonged to ST-7, ST-11, and ST-23 clones.
Conclusion
Our data suggested that serogroup B isolates were derived from several distinct lineages, most of which could either be indigenous or were introduced into Taiwan a long time ago. The serogroup A, W135 (and C), and Y isolates, respectively, belonged to the ST-7, ST-11, and ST-23, and the represented clones that are currently the major circulating clones in the world and are introduced into Taiwan more recently. The emergence of serogroup A, C and Y strains contributed partly to the increase in cases of meningococcal disease in 2001–2002.
doi:10.1186/1471-2334-6-25
PMCID: PMC1431541  PMID: 16478548
8.  The Effect of Immune Selection on the Structure of the Meningococcal Opa Protein Repertoire 
PLoS Pathogens  2008;4(3):e1000020.
The opa genes of the Gram negative bacterium Neisseria meningitidis encode Opacity-associated outer membrane proteins whose role is to promote adhesion to the human host tissue during colonisation and invasion. Each meningococcus contains 3–4 opa loci, each of which may be occupied by one of a large number of alleles. We analysed the Opa repertoire structure in a large, well-characterised collection of asymptomatically carried meningococci. Our data show an association between Opa repertoire and meningococcal lineages similar to that observed previously for meningococci isolated from cases of invasive disease. Furthermore, these Opa repertoires exhibit discrete, non-overlapping structure at a population level, and yet low within-repertoire diversity. These data are consistent with the predictions of a mathematical model of strong immune selection upon a system where identical alleles may occupy different loci.
Author Summary
Neisseria meningitidis is a globally important pathogen that causes 2,000–3,000 cases of invasive meningococcal disease annually in the United Kingdom. The meningococcal Opa proteins are important in mediating adhesion to and invasion of human tissues, and are important for evasion of the host immune response. They are encoded by a repertoire of 3–4 genomic loci in each meningococcus and exhibit high levels of sequence diversity. Here we analyzed the Opa repertoires of a large, well-characterised, asymptomatically carried meningococcal isolate collection. We found that the Opa repertoires were specific to individual meningococcal genotypes, similar to that observed in isolates from cases of invasive disease. These repertoires exhibited discrete, non-overlapping structure at a population level, and yet low within-repertoire diversity. These data were consistent with the predictions of a mathematical model of strong immune selection, suggesting that the collective immune response of the host population shapes the antigenic diversity of the meningococcal Opa repertoire. This study provides new insights into Opa-mediated meningococcal pathogenesis and the effect of host population immunity on the biodiversity and population structure of bacterial pathogens. These data may also have implications for the design of new meningococcal vaccines based on surface proteins.
doi:10.1371/journal.ppat.1000020
PMCID: PMC2265424  PMID: 18369470
9.  Carried Meningococci in the Czech Republic: a Diverse Recombining Population 
Journal of Clinical Microbiology  2000;38(12):4492-4498.
Population and evolutionary analyses of pathogenic bacteria are frequently hindered by sampling strategies that concentrate on isolates from patients with invasive disease. This is especially so for the gram-negative diplococcus Neisseria meningitidis, a cause of septicemia and meningitis worldwide. Meningococcal isolate collections almost exclusively comprise organisms originating from patients with invasive meningococcal disease, although this bacterium is a commensal inhabitant of the human nasopharynx and very rarely causes pathological effects. In the present study, molecular biology-based techniques were used to establish the genetic relationships of 156 meningococci isolated from healthy young adults in the Czech Republic during 1993. None of the individuals sampled had known links to patients with invasive disease. Multilocus sequence typing (MLST) showed that the bacterial population was highly diverse, comprising 71 different sequence types (STs) which were assigned to 34 distinct complexes or lineages. Three previously identified hyperinvasive lineages were present: 26 isolates (17%) belonged to the ST-41 complex (lineage 3); 4 (2.6%) belonged to the ST-11 (electrophoretic type [ET-37]) complex, and 1 (0.6%) belonged to the ST-32 (ET-5) complex. The data were consistent with the view that most nucleotide sequence diversity resulted from the reassortment of alleles by horizontal genetic exchange.
PMCID: PMC87626  PMID: 11101585
10.  Two-Partner Secretion Systems of Neisseria meningitidis Associated with Invasive Clonal Complexes▿ †  
Infection and Immunity  2008;76(10):4649-4658.
The two-partner secretion (TPS) pathway is widespread among gram-negative bacteria and facilitates the secretion of very large and often virulence-related proteins. TPS systems consist of a secreted TpsA protein and a TpsB protein involved in TpsA transport across the outer membrane. Sequenced Neisseria meningitidis genomes contain up to five TpsA- and two TpsB-encoding genes. Here, we investigated the distribution of TPS-related open reading frames in a collection of disease isolates. Three distinct TPS systems were identified among meningococci. System 1 was ubiquitous, while systems 2 and 3 were significantly more prevalent among isolates of hyperinvasive clonal complexes than among isolates of poorly invasive clonal complexes. In laboratory cultures, systems 1 and 2 were expressed. However, several sera from patients recovering from disseminated meningococcal disease recognized the TpsAs of systems 2 and 3, indicating the expression of these systems during infection. Furthermore, we showed that the major secreted TpsAs of systems 1 and 2 depend on their cognate TpsBs for transport across the outer membrane and that the system 1 TpsAs undergo processing. Together, our data indicate that TPS systems may contribute to the virulence of N. meningitidis.
doi:10.1128/IAI.00393-08
PMCID: PMC2546848  PMID: 18678657
11.  The Distribution and ‘In Vivo’ Phase Variation Status of Haemoglobin Receptors in Invasive Meningococcal Serogroup B Disease: Genotypic and Phenotypic Analysis 
PLoS ONE  2013;8(9):e76932.
Two haemoglobin-binding proteins, HmbR and HpuAB, contribute to iron acquisition by Neisseria meningitidis. These receptors are subject to high frequency, reversible switches in gene expression - phase variation (PV) - due to mutations in homopolymeric (poly-G) repeats present in the open reading frame. The distribution and PV state of these receptors was assessed for a representative collection of isolates from invasive meningococcal disease patients of England, Wales and Northern Ireland. Most of the major clonal complexes had only the HmbR receptor whilst the recently expanding ST-275-centred cluster of the ST-269 clonal complex had both receptors. At least one of the receptors was in an ‘ON’ configuration in 76.3% of the isolates, a finding that was largely consistent with phenotypic analyses. As PV status may change during isolation and culture of meningococci, a PCR-based protocol was utilised to confirm the expression status of the receptors within contemporaneously acquired clinical specimens (blood/cerebrospinal fluid) from the respective patients. The expression state was confirmed for all isolate/specimen pairs with <15 tract repeats indicating that the PV status of these receptors is stable during isolation. This study therefore establishes a protocol for determining in vivo PV status to aid in determining the contributions of phase variable genes to invasive meningococcal disease. Furthermore, the results of the study support a putative but non-essential role of the meningococcal haemoglobin receptors as virulence factors whilst further highlighting their vaccine candidacy.
doi:10.1371/journal.pone.0076932
PMCID: PMC3786947  PMID: 24098814
12.  Virulence Evolution of the Human Pathogen Neisseria meningitidis by Recombination in the Core and Accessory Genome 
PLoS ONE  2011;6(4):e18441.
Background
Neisseria meningitidis is a naturally transformable, facultative pathogen colonizing the human nasopharynx. Here, we analyze on a genome-wide level the impact of recombination on gene-complement diversity and virulence evolution in N. meningitidis. We combined comparative genome hybridization using microarrays (mCGH) and multilocus sequence typing (MLST) of 29 meningococcal isolates with computational comparison of a subset of seven meningococcal genome sequences.
Principal Findings
We found that lateral gene transfer of minimal mobile elements as well as prophages are major forces shaping meningococcal population structure. Extensive gene content comparison revealed novel associations of virulence with genetic elements besides the recently discovered meningococcal disease associated (MDA) island. In particular, we identified an association of virulence with a recently described canonical genomic island termed IHT-E and a differential distribution of genes encoding RTX toxin- and two-partner secretion systems among hyperinvasive and non-hyperinvasive lineages. By computationally screening also the core genome for signs of recombination, we provided evidence that about 40% of the meningococcal core genes are affected by recombination primarily within metabolic genes as well as genes involved in DNA replication and repair. By comparison with the results of previous mCGH studies, our data indicated that genetic structuring as revealed by mCGH is stable over time and highly similar for isolates from different geographic origins.
Conclusions
Recombination comprising lateral transfer of entire genes as well as homologous intragenic recombination has a profound impact on meningococcal population structure and genome composition. Our data support the hypothesis that meningococcal virulence is polygenic in nature and that differences in metabolism might contribute to virulence.
doi:10.1371/journal.pone.0018441
PMCID: PMC3082526  PMID: 21541312
13.  exl, an Exchangeable Genetic Island in Neisseria meningitidis 
Infection and Immunity  2001;69(3):1687-1696.
The genetic structure and evolution of a novel exchangeable meningococcal genomic island was defined for the important human pathogen Neisseria meningitidis. In 125 meningococcal strains tested, one of three unrelated nucleotide sequences, designated exl (exchangeable locus), was found between a gene required for heme utilization, hemO, and col, encoding a putative Escherichia coli collagenase homologue. The 5′ boundary of each exl cassette was the stop codon of hemO, whereas the 3′ boundary was delineated by a 33-bp repeat containing neisserial uptake sequences located downstream of col. One of the three alternative exl cassettes contained the meningococcal hemoglobin receptor gene, hmbR (exl3). In other meningococcal strains, hmbR was absent from the genome and was replaced by either a nucleotide sequence containing a novel open reading frame, exl2, or a cassette containing exl3. The proteins encoded by exl2 and exl3 had no significant amino acid homology to HmbR but contained six motifs that are also present in the lipoprotein components of the lactoferrin (LbpB), transferrin (TbpB), and hemoglobin-haptoglobin (HpuA) uptake systems. To determine the evolutionary relationships among meningococci carrying hmbR, exl2, or exl3, isolates representing 92 electrophoretic types were examined. hmbR was found throughout the population structure of N. meningitidis (genetic distance, >0.425), whereas exl2 and exl3 were found in clonal groups at genetic distances of <0.2. The commensal neisserial species were identified as reservoirs for all of the exl cassettes found in meningococci. The structure of these cassettes and their correlation with clonal groups emphasize the extensive gene pool and frequent horizontal DNA transfer events that contribute to the evolution and virulence of N. meningitidis.
doi:10.1128/IAI.69.3.1687-1696.2001
PMCID: PMC98073  PMID: 11179344
14.  Distribution of Surface Protein Variants among Hyperinvasive Meningococci: Implications for Vaccine Design  
Infection and Immunity  2004;72(10):5955-5962.
The bacterium Neisseria meningitidis is a major cause of meningitis and septicemia worldwide. Outer membrane proteins (OMPs) are candidates in the search for comprehensive meningococcal vaccines; however, the formulation of OMP vaccines is complicated by antigenic diversity, which is generated by high levels of genetic reassortment and strong positive selection in the meningococcal antigen genes. The genetic and antigenic diversity of three OMPs (FetA, PorA, and PorB) among a global collection of meningococcal isolates representative of the major hyperinvasive clonal complexes was determined. There was evidence for antigenic structuring among the three OMPs that could not be explained purely by descent. These observations violated the predictions of the clonal and epidemic clonal models of population structure but were in concordance with models of strain structure which propose that host immunity selects for nonoverlapping antigen combinations. The patterns of antigenic variant combinations suggested that an OMP-based vaccine with as few as six PorA and five FetA variant sequences could generate homologous immune responses against all 78 isolates examined.
doi:10.1128/IAI.72.10.5955-5962.2004
PMCID: PMC517544  PMID: 15385499
15.  Population Structure and Capsular Switching of Invasive Neisseria meningitidis Isolates in the Pre-Meningococcal Conjugate Vaccine Era, United States, 2000–2005 
The Journal of infectious diseases  2010;201(8):1208-1224.
Background
A quadrivalent meningococcal conjugate vaccine (MCV4) was licensed in the United States in 2005; no serogroup B vaccine is available. Neisseria meningitidis changes its capsular phenotype through capsular switching, which has implications for vaccines that do not protect against all serogroups.
Methods
Meningococcal isolates from 10 Active Bacterial Core Surveillance sites from 2000–2005 were analyzed so that changes following MCV4 licensure can be identified. Isolates were characterized by multilocus sequence typing (MLST) and outer membrane protein gene sequencing. Isolates expressing capsular polysaccharide different from that associated with the MLST lineage were considered to demonstrate capsule switching.
Results
Among 1,160 isolates, the most common genetic lineages were the ST-23, ST-32, ST-11, and ST-41/44 clonal complexes. Of serogroup B and Y isolates, 8 (1.5%) and 3 (0.9%), respectively, demonstrated capsular switching, compared to 36 (12.9%) for serogroup C (p <0.0001); most serogroup C switches were from virulent serogroup B and/or serogroup Y lineages.
Conclusions
A limited number of genetic lineages caused the majority of invasive meningococcal infections. A substantial proportion of isolates had evidence of capsular switching. The high prevalence of capsular switching requires surveillance to detect changes in meningococcal population structure that may impact effectiveness of meningococcal vaccines.
doi:10.1086/651505
PMCID: PMC2838939  PMID: 20199241
Neisseria meningitidis; meningococcus, meningococcal; population structure; multilocus sequence typing; DNA sequencing; outer membrane proteins; fetA; porA; porB; molecular epidemiology; capsular switching
16.  Distribution of transferrin binding protein B gene (tbpB) variants among Neisseria species 
BMC Microbiology  2008;8:66.
Background
Transferrin binding protein B (tbpB), an outer membrane lipoprotein, is required for the acquisition of iron from human transferrin. Two tbpB families have been documented in Neisseria meningitidis: an isotype I tbpB gene of 1.8 kb and an isotype II tbpB gene of 2.1 kb, the former expressed by meningococci in the disease-associated ST-11 clonal complex and the latter found among meningococci belonging to the hyper-invasive clonal complexes including ST-8, ST-18, ST-32, ST-41/44 as well as N. gonorrhoeae isolates. The origin of the isotype I tbpB gene is unknown, however several features in common with non-pathogenic Neisseria and the ST-11 clonal complex N. meningitidis isolate FAM18 have been documented leading to the hypothesis that the isotype I tbpB gene may also be shared between non-pathogenic Neisseria and ST-11 meningococci. As a result, the diversity of the tbpB gene was investigated in a defined collection of Neisseria species.
Results
Two families of isotype I tbpB were identified: family A containing conserved genes belonging to ST-11 meningococci, N. polysaccharea and N. lactamica isolates and family B including more diverse isotype I tbpB genes from N. sicca, N. mucosa, N. flava, N. subflava as well as N. cinerea, N. flavescens and N. polysaccharea isolates. Three isotype II tbpB families were identified with: family C containing diverse tbpB genes belonging to N. polysaccharea, N. lactamica, N. gonorrhoeae and N. meningitidis isolates, family D including another subset of isotype II tbpB genes from N. lactamica isolates and family E solely composed of N. gonorrhoeae tbpB genes.
Conclusion
This study reveals another instance of similarity between meningococci of the ST-11 clonal complex and non-pathogenic Neisseria with the origin of the isotype I tbpB gene resulting from a horizontal genetic transfer event occurring between these two populations.
doi:10.1186/1471-2180-8-66
PMCID: PMC2386816  PMID: 18430216
17.  Genetic Diversity and Levels of Expression of Factor H Binding Protein among Carriage Isolates of Neisseria meningitidis 
PLoS ONE  2014;9(9):e107240.
The prevention of meningococcal disease may be improved by recombinant vaccines such as 4CMenB and rLP2086 that target the factor H binding protein (fHbp), an immunogenic surface component of Neisseria meningitidis present as one of three variants. Whether such vaccines decrease carriage of invasive isolates and thus induce herd immunity is unknown. We analyzed the genetic diversity and levels of expression of fHbp among 268 carriage strains and compare them to those of 467 invasive strains. fhbp gene sequencing showed higher proportions of variants 2 and 3 among carriage isolates (p<0.0001). Carriage isolates expressed lower levels of fHbp (p<0.01) but that remain high enough to predict targeting by antibodies against fHbp particularly in group B isolates belonging to the frequent hypervirulent clonal complexes in Europe and North America (cc32, cc41/44, cc269). This suggests that fHbp targeting meningococcal vaccines might reduce, at least in part, the acquisition of some hyperinvasive isolates.
doi:10.1371/journal.pone.0107240
PMCID: PMC4172500  PMID: 25247300
18.  Invasive Serogroup B Neisseria meningitidis in Québec, Canada, 2003 to 2010: Persistence of the ST-269 Clone Since It First Emerged in 2003 
Journal of Clinical Microbiology  2012;50(5):1545-1551.
In the era after the introduction of the meningococcal serogroup C conjugate vaccine, from 1 January 2003 to 31 December 2010, serogroup B meningococci were the major cause of invasive meningococcal disease in the province of Québec, Canada, being responsible for 72% of all meningococcal disease cases. Of the 334 invasive serogroup B Neisseria meningitidis strains analyzed, 53.9% belonged to the ST-269 clonal complex (CC). Since it first emerged in 2003, the percentage of invasive serogroup B isolates that belonged to the ST-269 CC had increased from 35% in 2003 to 76% in 2010. Among the 180 meningococci in the ST-269 CC, 91.7% belonged to a single ST (ST-269). The most common PorA genotypes identified in the ST-269 CC were (i) VR1 19-1, VR2 15-11, VR3 36 (84%) and (ii) VR1 18-7, VR2 9, VR3 35-1 (9%). Cases of invasive disease due to the ST-269 CC were commonly found in those aged 11 to 19 years (30.5%) and 20 to 40 years (25.5%). Meningococci of the ST-269 CC were uncommon in other Canadian provinces. In contrast to the ST-269 CC, invasive serogroup B meningococci that belonged to the ST-41/44 CC were much more diverse genetically. However, one ST (ST-571), which is uncommon in the United States, accounted for 35% of all cases due to this CC. The current finding suggests that the ST-269 clone may indeed represent an emerging hypervirulent clone of meningococci.
doi:10.1128/JCM.06835-11
PMCID: PMC3347115  PMID: 22337990
19.  Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells 
PLoS Pathogens  2014;10(6):e1004160.
The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains.
Author Summary
Neisseria meningitidis, an obligate human pathogen, is a causative agent of septicemia and meningitis worldwide. Meningococcal infection manifests in a variety of forms, including meningitis, meningococcemia with meningitis or meningococcemia without obvious meningitis. The interaction of N. meningitidis with human cells lining the blood vessels of the blood-cerebrospinal fluid barrier is a prerequisite for the development of meningitis. As a major pathogenicity factor, the meningococcal outer membrane protein Opc enhances bacterial entry into brain endothelial cells, however, mechanisms underlying trapping of receptors and signaling molecules following this interaction remained elusive. We now show that Opc-expressing meningococci activate acid sphingomyelinase (ASM) in brain endothelial cells, which hydrolyses sphingomyelin to cause ceramide release and formation of extended ceramide-enriched membrane platforms wherein ErbB2, an important receptor involved in bacterial uptake, clusters. Mechanistically, ASM activation relied on binding of N. meningitidis to its attachment receptor, HSPG, followed by activation of PC-PLC. Meningococcal isolates of the ST-11 clonal complex, which are reported to be more likely to cause severe sepsis, but rarely meningitis, barely invaded brain endothelial cells and revealed a highly restricted ability to induce ASM and ceramide release. Thus, our results unravel a differential activation of the ASM/ceramide system by the species N. meningitidis determining its invasiveness into brain endothelial cells.
doi:10.1371/journal.ppat.1004160
PMCID: PMC4055770  PMID: 24945304
20.  Meningococcal carriage and disease—Population biology and evolution 
Vaccine  2009;27(4):B64-B70.
Meningococcal disease occurs worldwide with incidence rates varying from 1 to 1000 cases per 100,000. The causative organism, Neisseria meningitidis, is an obligate commensal of humans, which normally colonizes the mucosa of the upper respiratory tract without causing invasive disease, a phenomenon known as carriage. Studies using molecular methods have demonstrated the extensive genetic diversity of meningocococci isolated from carriers, in contrast to a limited number of genetic types, known as the hyperinvasive lineages, associated with invasive disease. Population and evolutionary models that invoke positive selection can be used to resolve the apparent paradox of virulent lineages persisting during the global spread of a non-clonal and normally commensal bacterium. The application of insights gained from studies of meningococcal population biology and evolution is important in understanding the spread of disease, as well as in vaccine development and implementation, especially with regard to the challenge of producing comprehensive vaccines based on sub-capsular antigens and measuring their effectiveness.
doi:10.1016/j.vaccine.2009.04.061
PMCID: PMC2719693  PMID: 19464092
Meningococcal disease; Carriage; Population structure
21.  Implications of Differential Age Distribution of Disease-Associated Meningococcal Lineages for Vaccine Development 
New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects <1 year of age experienced lower risk of sequence type 11 (ST-11) cc, ST-32 cc, and ST-269 cc disease and higher risk of disease due to unassigned STs, 1- to 4-year-olds experienced lower risk of ST-11 cc and ST-32 cc disease, 5- to 14-year-olds were less likely to experience ST-11 cc and ST-269 cc disease, and ≥25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups.
doi:10.1128/CVI.00133-14
PMCID: PMC4054250  PMID: 24695776
22.  Evolutionary and genomic insights into meningococcal biology 
Future microbiology  2012;7(7):873-885.
Summary
Epidemic disease caused by Neisseria meningitidis, the meningococcus, has been recognised for two centuries, but remains incompletely controlled and understood. There have been dramatic reductions in serogroup A and C meningococcal disease following the introduction of protein-polysaccharide conjugate vaccines but there is currently no comprehensive vaccine against serogroup B meningococci. Genetic analyses of meningococcal populations have provided many insights into the biology, evolution, and pathogenesis of this important pathogen. The meningococcus, and its close relative the gonococcus, are the only pathogenic members of the genus Neisseria, and the invasive propensity of meningococci varies widely, with around a dozen ‘hyper invasive lineages’ responsible for most disease. Despite this, attempts to identify a ‘pathogenome’, a subset of genes associated with the invasive phenotypes have failed; however, genome-wide studies of representative meningococcal isolates using high throughput sequencing are beginning to provide detail on the relationship of invasive phenotype and genotype in this fascinating organism and how this relationship has evolved.
doi:10.2217/fmb.12.62
PMCID: PMC3492750  PMID: 22827308
Neisseria meningitidis; population genomics; pathogenomics; molecular evolution; molecular epidemiology
23.  Multicenter Study for Defining the Breakpoint for Rifampin Resistance in Neisseria meningitidis by rpoB Sequencing▿  
Identification of clinical isolates of Neisseria meningitidis that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number (n = 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of ≥0.25 mg/liter (n = 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of <0.25 mg/liter were also examined (n = 231). Typing of isolates was performed, and a 660-bp DNA fragment of the rpoB gene was sequenced. Sequences differing by at least one nucleotide were defined as unique rpoB alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of >1 mg/liter possessed rpoB alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of ≤1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of ≤1 mg/liter. The rpoB allele sequence and isolate data have been incorporated into the PubMLST Neisseria database (http://pubmlst.org/neisseria/). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates.
doi:10.1128/AAC.00315-10
PMCID: PMC2934967  PMID: 20606072
24.  The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation 
Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than Dam presence.
doi:10.3389/fmicb.2014.00712
PMCID: PMC4269198  PMID: 25566225
dam replacing gene; Dam activity; Neisseria gonorrhoeae; adhesion; biofilm
25.  Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence 
Microbiology  2011;157(Pt 5):1446-1456.
Neisseria meningitidis can utilize haem, haemoglobin and haemoglobin–haptoglobin complexes as sources of iron via two TonB-dependent phase variable haemoglobin receptors, HmbR and HpuAB. HmbR is over-represented in disease isolates, suggesting a link between haemoglobin acquisition and meningococcal disease. This study compared the distribution of HpuAB and phase variation (PV) status of both receptors in disease and carriage isolates. Meningococcal disease (n = 214) and carriage (n = 305) isolates representative of multiple clonal complexes (CCs) were investigated for the distribution, polyG tract lengths and ON/OFF status of both haemoglobin receptors, and for the deletion mechanism for HpuAB. Strains with both receptors or only hmbR were present at similar frequencies among meningococcal disease isolates as compared with carriage isolates. However, >90 % of isolates from the three CCs CC5, CC8 and CC11 with the highest disease to carriage ratios contained both receptors. Strains with an hpuAB-only phenotype were under-represented among disease isolates, suggesting selection against this receptor during systemic disease, possibly due to the receptor having a high level of immunogenicity or being inefficient in acquisition of iron during systemic spread. Absence of hpuAB resulted from either complete deletion or replacement by an insertion element. In an examination of PV status, one or both receptors were found in an ON state in 91 % of disease and 71 % of carriage isolates. We suggest that expression of a haemoglobin receptor, either HmbR or HpuAB, is of major importance for systemic spread of meningococci, and that the presence of both receptors contributes to virulence in some strains.
doi:10.1099/mic.0.046946-0
PMCID: PMC3352162  PMID: 21310784

Results 1-25 (1035323)