The epidemiology of meningococcal disease varies by geography and time. Whole-genome sequencing of Neisseria meningitidis serogroup X isolates from sub-Saharan Africa and Europe showed that serogroup X emergence in sub-Saharan Africa resulted from expansion of particular variants within clonal complex 181. Virulence of these isolates in experimental mouse models was high.

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-alpha is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-alpha receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-alpha. 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.

Invasive disease caused by Neisseria meningitidis serogroup W (MenW) has historically had a low incidence in Sweden, with an average incidence of 0.03 case/100,000 population from 1995 to 2014. In recent years, a significant increase in the incidence of MenW has been noted in Sweden, to an average incidence of 0.15 case/100,000 population in 2015 to 2016. In 2017 (1 January to 30 June), 33% of invasive meningococcal disease cases (7/21 cases) were caused by MenW. In the present study, all invasive MenW isolates from Sweden collected in 1995 to June 2017 (n = 86) were subjected to whole-genome sequencing to determine the population structure and to compare isolates from Sweden with historical and international cases. The increase of MenW in Sweden was determined to be due to isolates belonging to the South American sublineage of MenW clonal complex 11, namely, the novel U.K. 2013 lineage. This lineage was introduced in Sweden in 2013 and has since been the dominant lineage of MenW.

Neisseria meningitidis serogroups W and Y are the most common serogroups causing invasive meningococcal disease in Sweden. The majority of cases are caused by the serogroup W UK 2013 strain of clonal complex (cc) 11, and subtype 1 of the serogroup Y, YI strain of cc23. In this study, virulence factors of several lineages within cc11 and cc23 were investigated in transgenic BALB/c mice expressing human transferrin. Transgenic mice were infected intraperitoneally with serogroup W and Y isolates. Levels of bacteria and the proinflammatory cytokine CXCL1 were determined in blood collected 3 h and 24 h post-infection. Apoptosis was investigated in immune cells from peritoneal washes of infected mice. Adhesion and induction of apoptosis in human epithelial cells were also scored.

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis emerged in Europe during the 2000s. Draft genomes of serogroup Y isolates in Sweden revealed that although the population structure of these isolates was similar to other serogroup Y isolates internationally, a distinct strain (YI) and more specifically a sublineage (1) of this strain was responsible for the increase of serogroup Y IMD in Sweden. We performed single molecule real-time (SMRT) sequencing on eight serogroup Y isolates from different sublineages to unravel the genetic and epigenetic factors delineating them, in order to understand the serogroup Y emergence. Extensive comparisons between the serogroup Y sublineages of all coding sequences, complex genomic regions, intergenic regions, and methylation motifs revealed small point mutations in genes mainly encoding hypothetical and metabolic proteins, and non-synonymous variants in genes involved in adhesion, iron acquisition, and endotoxin production. The methylation motif CACNNNNNTAC was only found in isolates of sublineage 2. Only seven genes were putatively differentially expressed, and another two genes encoding hypothetical proteins were only present in sublineage 2. These data suggest that the serogroup Y IMD increase in Sweden was most probably due to small changes in genes important for colonization and transmission.

Neisseria meningitidis can be a human commensal in the upper respiratory tract but is also capable of causing invasive diseases such as meningococcal meningitis and septicaemia. No specific genetic markers have been detected to distinguish carriage from disease isolates. The aim here was to find genetic traits that could be linked to phenotypic outcomes associated with carriage versus invasive N. meningitidis disease through a bacterial genome-wide association study (GWAS). In this study, invasive N. meningitidis isolates collected in Sweden (n=103) and carriage isolates collected at Örebro University, Sweden (n=213) 2018-2019 were analysed. The GWAS analysis, treeWAS, was applied to single-nucleotide polymorphisms (SNPs), genes and k-mers. One gene and one non-synonymous SNP were associated with invasive disease and seven genes and one non-synonymous SNP were associated with carriage isolates. The gene associated with invasive disease encodes a phage transposase (NEIS1048), and the associated invasive SNP glmU S373C encodes the enzyme N-acetylglucosamine 1-phosphate (GlcNAC 1-P) uridyltransferase. Of the genes associated with carriage isolates, a gene variant of porB encoding PorB class 3, the genes pilE/pilS and tspB have known functions. The SNP associated with carriage was fkbp D33N, encoding a FK506-binding protein (FKBP). K-mers from PilS, tbpB and tspB were found to be associated with carriage, while k-mers from mtrD and tbpA were associated with invasiveness. In the genes fkbp, glmU, PilC and pilE, k-mers were found that were associated with both carriage and invasive isolates, indicating that specific variations within these genes could play a role in invasiveness. The data presented here highlight genetic traits that are significantly associated with invasive or carriage N. meningitidis across the species population. These traits could prove essential to our understanding of the pathogenicity of N. meningitidis and could help to identify future vaccine targets.

Lytic transglycosylases (LT) are enzymes involved in peptidoglycan (PG) remodeling. However, their contribution to cell-wall-modifying complexes and their potential as antimicrobial drug targets remains unclear. Here, we determined a high-resolution structure of the LT, an outer membrane lipoprotein from Neisseria species with a disordered active site helix (alpha helix 30). We show that deletion of the conserved alpha-helix 30 interferes with the integrity of the cell wall, disrupts cell division, cell separation, and impairs the fitness of the human pathogen Neisseria meningitidis during infection. Additionally, deletion of alpha-helix 30 results in hyperacetylated PG, suggesting this LtgA variant affects the function of the PG de-O-acetylase (Ape 1). Our study revealed that Ape 1 requires LtgA for optimal function, demonstrating that LTs can modulate the activity of their protein-binding partner. We show that targeting specific domains in LTs can be lethal, which opens the possibility that LTs are useful drug-targets.

Invasive meningococcal disease (IMD) caused by Neisseria meningitidis serogroup Y has increased in Europe, especially in Scandinavia. In Sweden, serogroup Y is now the dominating serogroup, and in 2012, the serogroup Y disease incidence was 0.46/100,000 population. We previously showed that a strain type belonging to sequence type 23 was responsible for the increased prevalence of this serogroup in Sweden. The objective of this study was to investigate the serogroup Y emergence by whole-genome sequencing and compare the meningococcal population structure of Swedish invasive serogroup Y strains to those of other countries with different IMD incidence. Whole-genome sequencing was performed on invasive serogroup Y isolates from 1995 to 2012 in Sweden (n = 186). These isolates were compared to a collection of serogroup Y isolates from England, Wales, and Northern Ireland from 2010 to 2012 (n = 143), which had relatively low serogroup Y incidence, and two isolates obtained in 1999 in the United States, where serogroup Y remains one of the major causes of IMD. The meningococcal population structures were similar in the investigated regions; however, different strain types were prevalent in each geographic region. A number of genes known or hypothesized to have an impact on meningococcal virulence were shown to be associated with different strain types and subtypes. The reasons for the IMD increase are multifactorial and are influenced by increased virulence, host adaptive immunity, and transmission. Future genome-wide association studies are needed to reveal additional genes associated with serogroup Y meningococcal disease, and this work would benefit from a complete serogroup Y meningococcal reference genome.

In 2015, Niger reported the largest epidemic of Neisseria meningitidis serogroup C (NmC) meningitis in sub-Saharan Africa. The NmC epidemic coincided with serogroup W (NmW) cases during the epidemic season, resulting in a total of 9,367 meningococcal cases through June 2015. To clarify the phylogenetic association, genetic evolution, and antibiotic determinants of the meningococcal strains in Niger, we sequenced the genomes of 102 isolates from this epidemic, comprising 81 NmC and 21 NmW isolates. The genomes of 82 isolates were completed, and all 102 were included in the analysis. All NmC isolates had sequence type 10217, which caused the outbreaks in Nigeria during 2013-2014 and for which a clonal complex has not yet been defined. The NmC isolates from Niger were substantially different from other NmC isolates collected globally. All NmW isolates belonged to clonal complex 11 and were closely related to the isolates causing recent outbreaks in Africa.

Since 2015, the incidence of invasive meningococcal disease (IMD) caused by serogroup W (MenW) has increased in Sweden, due to the introduction of the 2013 strain belonging to clonal complex 11. The aim of this study was to describe the clinical presentation of MenW infections, in particular the 2013 strain, including genetic associations. Medical records of confirmed MenW IMD cases in Sweden during the years 1995-2019 (n = 113) were retrospectively reviewed and the clinical data analysed according to strain. Of all MenW patients, bacteraemia without the focus of infection was seen in 44%, bacteraemic pneumonia in 26%, meningitis in 13% and epiglottitis in 8%, gastrointestinal symptoms in 48% and 4% presented with petechiae. Phylogenetic analysis was used for possible links between genetic relationship and clinical picture. The 2013 strain infections, particularly in one cluster, were associated with more severe disease compared with other MenW infections. The patients with 2013 strain infections (n = 68) were older (52 years vs. 25 years for other strains), presented more often with diarrhoea as an atypical presentation (P = 0.045) and were more frequently admitted for intensive care (P = 0.032). There is a risk that the atypical clinical presentation of MenW infections, with predominantly gastrointestinal or respiratory symptoms rather than neck stiffness or petechiae, may lead to delay in life-saving treatment.

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.

A rising incidence of meningococcal serogroup W disease has been evident in many countries worldwide. Serogroup W isolates belonging to the sequence type (ST)-11 clonal complex have been associated with atypical symptoms and increased case fatality rates. The continued expansion of this clonal complex in the later part of the 2010s has been largely due to a shift from the so-called original UK strain to the 2013 strain. Here we used single-molecule real-time (SMRT) sequencing to determine the methylomes of the two major serogroup W strains belonging to ST-11 clonal complex. Five methylated motifs were identified in this study, and three of the motifs, namely 5'-GATC-3', 5'-GAAGG-3', 5'-GCGCGC-3', were found in all 13 isolates investigated. The results showed no strain-specific motifs or difference in active restriction modification systems between the two strains. Two phase variable methylases were identified and the enrichment or depletion of the methylation motifs generated by these methylases varied between the two strains. Results from this work give further insight into the low diversity of methylomes in highly related strains and encourage further research to decipher the role of regions with under- or overrepresented methylation motifs.

Meningococcal disease occurs as sporadic cases in developed countries, with the occasional emergence of new clones of Neisseria meningitidis. Here, we report the genome sequence of N. meningitidis strain LNP27256, an isolate of sequence type 11 linked to a recent outbreak among men who have sex with men in Europe.

Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis, which are typically transmitted via respiratory droplets, are leading causes of invasive diseases, including bacteraemic pneumonia and meningitis, and of secondary infections subsequent to post-viral respiratory disease. The aim of this study was to investigate the incidence of invasive disease due to these pathogens during the early months of the COVID-19 pandemic.

Historically, the major cause of meningococcal epidemics in the meningitis belt of sub-Saharan Africa has been Neisseria meningitidis serogroup A (NmA), but the incidence has been substantially reduced since the introduction of a serogroup A conjugate vaccine starting in 2010. We performed whole-genome sequencing on isolates collected post-2010 to assess their phylogenetic relationships and inter-country transmission.

Neisseria meningitidis is an exclusively human pathogen frequently carried asymptomatically in the nasopharynx but it can also provoke invasive infections such as meningitis and septicemia. N. meningitidis uses a limited range of carbon sources during infection, such as glucose, that is usually transported into bacteria via the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), in which the phosphocarrier protein HPr (encoded by the ptsH gene) plays a central role. Although N. meningitidis possesses an incomplete PTS, HPr was found to be required for its virulence. We explored the role of HPr using bioluminescent wild-type and ΔptsH strains in experimental infection in transgenic mice expressing the human transferrin. The wild-type MC58 strain was recovered at higher levels from the peritoneal cavity and particularly from blood compared to the ΔptsH strain. The ΔptsH strain provoked lower levels of septicemia in mice and was more susceptible to complement-mediated killing than the wild-type strain. We tested whether meningococcal structures impacted complement resistance and observed that only the capsule level was decreased in the ΔptsH mutant. We therefore compared the transcriptomic profiles of wild-type and ΔptsH strains and identified 49 differentially expressed genes. The HPr regulon contains mainly hypothetical proteins (43%) and several membrane-associated proteins that could play a role during host interaction. Some other genes of the HPr regulon are involved in stress response. Indeed, the ΔptsH strain showed increased susceptibility to environmental stress conditions. Our data suggest that HPr plays a pleiotropic role in host-bacteria interactions most likely through the innate immune response that may be responsible for the enhanced clearance of the ΔptsH strain from blood.

Bacterial meningitis is a major global cause of morbidity and mortality. Rapid identification of the aetiological agent of meningitis is essential for clinical and public health management and disease prevention given the wide range of pathogens that cause the clinical syndrome and the availability of vaccines that protect against some, but not all, of these. Since microbiological culture is complex, slow, and often impacted by prior antimicrobial treatment of the patient, molecular diagnostic assays have been developed for bacterial detection. Distinguishing between meningitis caused by Neisseria meningitidis (meningococcus), Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae, and Streptococcus agalactiae and identifying their polysaccharide capsules is especially important. Here, we review methods used in the identification of these bacteria, providing an up-to-date account of available assays, allowing clinicians and diagnostic laboratories to make informed decisions about which assays to use.

Differential modulation of NF-κB during meningococcal infection is critical in innate immune response to meningococcal disease. Non-invasive isolates of Neisseria meningitidis provoke a sustained NF-κB activation in epithelial cells. However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis. We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells. This cleavage was mediated by the secreted 150 kDa meningococcal ST-11 IgA protease carrying nuclear localisation signals (NLS) in its α-peptide moiety that allowed efficient intra-nuclear transport. In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport. This part of iga polymorphism allows non-invasive isolates lacking NLS, unlike hyperinvasive ST-11 isolates of N. meningitides habouring NLS in their α-peptide, to be carried asymptomatically in the human nasopharynx through selective eradication of their ability to induce apoptosis in infected epithelial cells.

Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml ± 0.1), CD80 (LOGEC50 = 4.88 µg/ml ± 0.15) and CD86 (LOGEC50 = 5.36 µg/ml ± 0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7 ± 5.1% cells versus 12 ± 2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed thatPBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP) at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.

The WHO recently endorsed an ambitious plan, "Defeating Meningitis by 2030", that aims to control/eradicate invasive bacterial infection epidemics by 2030. Vaccination is one of the pillars of this road map, with the goal to reduce the number of cases and deaths due to Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae and Streptococcus agalactiae. The risk of developing invasive bacterial infections (IBI) due to these bacterial species includes genetic and acquired factors that favor repeated and/or severe invasive infections. We searched the PubMed database to identify host risk factors that increase the susceptibility to these bacterial species. Here, we describe a number of inherited and acquired risk factors associated with increased susceptibility to invasive bacterial infections. The burden of these factors is expected to increase due to the anticipated decrease in cases in the general population upon the implementation of vaccination strategies. Therefore, detection and exploration of these patients are important as vaccination may differ among subjects with these risk factors and specific strategies for vaccination are required. The aim of this narrative review is to provide information about these factors as well as their impact on vaccination against the four bacterial species. Awareness of risk factors for IBI may facilitate early recognition and treatment of the disease. Preventive measures including vaccination, when available, in individuals with increased risk for IBI may prevent and reduce the number of cases.