Genetic Analysis of Neisseria meningitidis Sequence Type 7 Serogroup X Originating from Serogroup A.
ABSTRACT: Neisseria meningitidis causes meningococcal disease, often resulting in fulminant meningitis, sepsis, and death. Vaccination programs have been developed to prevent infection of this pathogen, but serogroup replacement is a problem. Capsular switching has been an important survival mechanism for N. meningitidis, allowing the organism to evolve in the present vaccine era. However, related mechanisms have not been completely elucidated. Genetic analysis of capsular switching between diverse serogroups would help further our understanding of this pathogen. In this study, we analyzed the genetic characteristics of the sequence type 7 (ST-7) serogroup X strain that was predicted to arise from ST-7 serogroup A at the genomic level. By comparing the genomic structures and sequences, ST-7 serogroup X was closest to ST-7 serogroup A, whereas eight probable recombination regions, including the capsular gene locus, were identified. This indicated that serogroup X originated from serogroup A by recombination leading to capsular switching. The recombination involved approximately 8,540 bp from the end of the ctrC gene to the middle of the galE gene. There were more recombination regions and strain-specific single-nucleotide polymorphisms in serogroup X than in serogroup A genomes. However, no specific gene was found for each serogroup except those in the capsule gene locus.
Project description:Neisseria meningitidis is a leading cause of septicemia and meningitis worldwide. N. meningitidis capsular polysaccharides have been classified into 13 distinct serogroups which are defined by antibody reactivity and structural analysis, and the capsule plays an important role in virulence. Serogroups A, B, C, W135, and Y have been reported to be clinically important. Several newly identified serogroup C isolates belonging to the unique sequence type 7 (ST-7) were identified in China. Since most ST-7 isolates from China belonged to serogroup A, the newly identified ST-7 serogroup C strains were proposed to have arisen from those belonging to ST-7 serogroup A. In this study, six ST-7 serogroup C and three ST-7 serogroup A isolates were analyzed by pulsed-field gel electrophoresis to confirm their sequence type. In order to clarify the genetic basis of capsular switching between ST-7 serogroup A and C strains, the whole capsular gene clusters and surrounding genes of the two representative ST-7 strains belonging to serogroups A and C, respectively, were sequenced and compared. Potential recombination sites were analyzed using the RDP3 beta software, and recombination-related regions in two other ST-7 serogroup A and five ST-7 serogroup C strains were also sequenced and compared to the representative ST-7 serogroup A and C strain sequences.
Project description:Neisseria meningitidis is an important cause of meningococcal disease globally. Sequence type (ST)-11 clonal complex (cc11) is a hypervirulent meningococcal lineage historically associated with serogroup C capsule and is believed to have acquired the W capsule through a C to W capsular switching event. We studied the sequence of capsule gene cluster (cps) and adjoining genomic regions of 524 invasive W cc11 strains isolated globally. We identified recombination breakpoints corresponding to two distinct recombination events within W cc11: A 8.4-kb recombinant region likely acquired from W cc22 including the sialic acid/glycosyl-transferase gene, csw resulted in a C?W change in capsular phenotype and a 13.7-kb recombinant segment likely acquired from Y cc23 lineage includes 4.5 kb of cps genes and 8.2 kb downstream of the cps cluster resulting in allelic changes in capsule translocation genes. A vast majority of W cc11 strains (497/524, 94.8%) retain both recombination events as evidenced by sharing identical or very closely related capsular allelic profiles. These data suggest that the W cc11 capsular switch involved two separate recombination events and that current global W cc11 meningococcal disease is caused by strains bearing this mosaic capsular switch.
Project description:Neisseria meningitidis is a leading bacterial cause of sepsis and meningitis globally with dynamic strain distribution over time. Beginning with an epidemic among Hajj pilgrims in 2000, serogroup W (W) sequence type (ST) 11 emerged as a leading cause of epidemic meningitis in the African 'meningitis belt' and endemic cases in South America, Europe, Middle East and China. Previous genotyping studies were unable to reliably discriminate sporadic W ST-11 strains in circulation since 1970 from the Hajj outbreak strain (Hajj clone). It is also unclear what proportion of more recent W ST-11 disease clusters are caused by direct descendants of the Hajj clone. Whole genome sequences of 270 meningococcal strains isolated from patients with invasive meningococcal disease globally from 1970 to 2013 were compared using whole genome phylogenetic and major antigen-encoding gene sequence analyses. We found that all W ST-11 strains were descendants of an ancestral strain that had undergone unique capsular switching events. The Hajj clone and its descendants were distinct from other W ST-11 strains in that they shared a common antigen gene profile and had undergone recombination involving virulence genes encoding factor H binding protein, nitric oxide reductase, and nitrite reductase. These data demonstrate that recent acquisition of a distinct antigen-encoding gene profile and variations in meningococcal virulence genes was associated with the emergence of the Hajj clone. Importantly, W ST-11 strains unrelated to the Hajj outbreak contribute a significant proportion of W ST-11 cases globally. This study helps illuminate genomic factors associated with meningococcal strain emergence and evolution.
Project description:The (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate meningococcal capsule of serogroup A Neisseria meningitidis is biochemically distinct from the sialic acid-containing capsules produced by other disease-associated meningococcal serogroups (e.g., B, C, Y, and W-135). We defined the genetic cassette responsible for expression of the serogroup A capsule. The cassette comprised a 4,701-bp nucleotide sequence located between the outer membrane capsule transporter gene, ctrA, and galE, encoding the UDP-glucose-4-epimerase. Four open reading frames (ORFs) not found in the genomes of the other meningococcal serogroups were identified. The first serogroup A ORF was separated from ctrA by a 218-bp intergenic region. Reverse transcriptase (RT) PCR and primer extension studies of serogroup A mRNA showed that all four ORFs were cotranscribed in the opposite orientation to ctrA and that transcription of the ORFs was initiated from the intergenic region by a sigma-70-type promoter that overlapped the ctrA promoter. The first ORF exhibited 58% amino acid identity with the UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) 2-epimerase of Escherichia coli, which is responsible for the conversion of UDP-GlcNAc into UDP-N-acetyl-D-mannosamine. Polar or nonpolar mutagenesis of each of the ORFs resulted in an abrogation of serogroup A capsule production as determined by colony immunoblots and enzyme-linked immunosorbent assay. Replacement of the serogroup A biosynthetic gene cassette with a serogroup B cassette by transformation resulted in capsule switching from a serogroup A capsule to a serogroup B capsule. These data indicate that assembly of the serogroup A capsule likely begins with monomeric UDP-GlcNAc and requires proteins encoded by three other genes found in the serogroup A N. meningitidis-specific operon located between ctrA and galE.
Project description:Three group B Neisseria meningitidis isolates, recovered from meningococcal disease cases in Canada and typed as B:2c:P1.5, were characterized. Multilocus sequence typing showed that all three isolates were related because of an identical sequence type (ST) 573. Isolates typed as 2c:P1.5 are common in serogroup Y meningococci but rare in isolates from serogroups B or C. Although no serogroup Y isolates have been typed as ST-573, eight isolates showed five to six housekeeping gene alleles that were identical to that of ST-573. This suggested that the B:2c:P1.5 isolates may have originated from serogroup Y organisms, possibly by capsule switching.
Project description:Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate (M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the M16917 genome sequence, which led to the introduction of an ?2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.
Project description:Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.
Project description:Neisseria meningitidis serogroup W135 has been associated with global outbreaks since the 2000 Hajj. Considering that N. meningitidis serogroup W135 is the third most prevalent serogroup isolated in Brazil in the last 10 years, and the possibility that the Hajj-related N. meningitidis serogroup W135 clone has been causing disease in Brazil, the present study characterized invasive N. meningitidis serogroup W135 isolates recovered in Brazil from 1990 to 2005.The isolates were characterized by serotyping, PorA and PorB VR typing, FetA and 16S rRNA typing, multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE).Based on MLST, 73% of the isolates were clustered in one major clone of ST-11 complex/ET37 complex. Strains of this clone had the same STs, serotypes and PorA VR types as found in Hajj-related N. meningitidis serogroup W135 clone. One of these strains had the Hajj-2000 outbreak strain genotype, including 16S rRNA gene sequence 31 and 84% relatedness by PFGE.Taken together, these data suggest that the Hajj-related N. meningitidis serogroup W135 clone is present in Brazil but has not yet caused a substantial number of infections. Given the emergence of N. meningitidis serogroup W135 globally and the unpredictability of meningococcal disease epidemiology, continued surveillance for this invasive N. meningitidis serogroup W135 clone is needed for control and prevention strategies.
Project description:BACKGROUND:The aim of this study is to describe the molecular epidemiology of Neisseria meningitidis invasive disease before the introduction of serogroup C conjugate vaccine in Amazonas State in 2010. METHODS:Meningococcal disease reported cases were investigated in Amazonas State during the period 2000-2010. N. meningitidis isolates (n?=?196) recovered from patients were genotyped by multilocus sequence typing (MLST) and sequencing of porB, porA, fetA, fHbp and penA. Antimicrobial susceptibility was determined using E-test. RESULTS:In the study period, 948 cases were reported; the incidence was 2.8 for the entire state and 4.8 per 100,000 in the capital of Manaus. Most meningococcal disease was caused by N. meningitidis belonging to ST-32 (72%; 141/196) or ST-103 (21%; 41/196) clonal complexes. Capsular switching (B?C) was suggested within clonal complex (cc) 32. There were 6 (3%; 6/196) strains with intermediate susceptibility to penicillin and a single strain was resistant to rifampicin. Since 2007, serogroup C strains belonging to the cc103 have predominated and case-fatality has increased. CONCLUSION:We demonstrate a high rate of meningococcal disease in Amazonas State, where, like other parts of Brazil, serogroup C replaced serogroup B during 2000s. These data serve as a baseline to measure impact of serogroup C conjugate vaccine introduction in 2010. This study emphasizes the need for enhanced surveillance to monitor changes in meningococcal disease trends following the introduction of meningococcal vaccines.
Project description:The bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus.