Neisseria genes required for persistence identified via in vivo screening of a transposon mutant library.

The mechanisms used by human adapted commensal Neisseria to shape and maintain a niche in their host are poorly defined. These organisms are common members of the mucosal microbiota and share many putative host interaction factors with Neisseria meningitidis and Neisseria gonorrhoeae. Evaluating the role of these shared factors during host carriage may provide insight into bacterial mechanisms driving both commensalism and asymptomatic infection across the genus. We identified host interaction factors required for niche development and maintenance through in vivo screening of a transposon mutant library of Neisseria musculi, a commensal of wild-caught mice which persistently and asymptomatically colonizes the oral cavity and gut of CAST/EiJ and A/J mice. Approximately 500 candidate genes involved in long-term host interaction were identified. These included homologs of putative N. meningitidis and N. gonorrhoeae virulence factors which have been shown to modulate host interactions in vitro. Importantly, many candidate genes have no assigned function, illustrating how much remains to be learned about Neisseria persistence. Many genes of unknown function are conserved in human adapted Neisseria species; they are likely to provide a gateway for understanding the mechanisms allowing pathogenic and commensal Neisseria to establish and maintain a niche in their natural hosts. Validation of a subset of candidate genes confirmed a role for a polysaccharide capsule in N. musculi persistence but not colonization. Our findings highlight the potential utility of the Neisseria musculi-mouse model as a tool for studying the pathogenic Neisseria; our work represents a first step towards the identification of novel host interaction factors conserved across the genus.

The first pediatric case of mild encephalitis/encephalopathy with a reversible splenial lesion (MERS) associated with Neisseria meningitidis.

BACKGROUND: Mild encephalitis/encephalopathy with a reversible splenial lesion (MERS) is a clinic-radiological syndrome characterized by neurological deficiencies and reversible magnetic resonance imaging findings in the splenium of the corpus callosum. Usually, it has a good prognosis, and patients recover without any sequelae. A viral infection has caused most MERS cases, and bacterial agents have rarely been reported as a cause of MERS. CASE REPORT: A 5-month-old male was admitted to the hospital with fever, poor feeding, decreased activity and groaning. He had focal seizures and required mechanical ventilation. A lumbar puncture was performed, and nucleic acid amplification tests (NAATs) of cerebral spinal fluid was positive for non-typeable serogroup of Neisseria meningitidis. Cranial magnetic resonance imaging (MRI) performed on day 3 of admission showed T2 hyperintensity and diffusion restriction at the splenium of corpus callosum (SCC). Diffusion restriction in the occipital horn of the left ventricular compatible with empyema was also obvious. According to the findings on cranial MRI, this clinical course was diagnosed as MERS associated with meningitis. His fever resolved in one week, cefotaxime was discontinued after 14 days, and the patient completely recovered. A follow-up MRI performed after three weeks of admission showed complete resolution of the signal intensity changes in the SCC and the occipital horn of the left lateral ventricle. CONCLUSION: To the best of our knowledge, this is the first report of MERS associated with Neisseria meningitidis in children. Bacterial agents as a cause of MERS should be kept in mind, and we should avoid unnecessary treatment strategies due to the good prognosis of MERS in children.

Capsule-dependent impact of MAPK signalling on host cell invasion and immune response during infection of the choroid plexus epithelium by Neisseria meningitidis.

BACKGROUND: The Gram-negative bacterium Neisseria meningitidis (Nm) can cause meningitis in humans, but the host signalling pathways manipulated by Nm during central nervous system (CNS) entry are not completely understood. METHODS: We investigate the role of the mitogen-activated protein kinases (MAPK) Erk1/2 and p38 in an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB) based on human epithelial choroid plexus (CP) papilloma (HIBCPP) cells during infection with Nm serogroup B (NmB) and serogroup C (NmC) strains. A transcriptome analysis of HIBCPP cells following infection with Nm by massive analysis of cDNA ends (MACE) was done to further characterize the cellular response to infection of the barrier. RESULTS: Interestingly, whereas NmB and NmC wild type strains required active Erk1/2 and p38 pathways for infection, invasion by capsule-deficient mutants was independent of Erk1/2 and, in case of the NmB strain, of p38 activity. The transcriptome analysis of HIBCPP cells following infection with Nm demonstrated specific regulation of genes involved in the immune response dependent on Erk1/2 signalling. Gene ontology (GO) analysis confirmed loss of MAPK signalling after Erk1/2 inhibition and revealed an additional reduction of cellular responses including NFκB and JAK-STAT signalling. Interestingly, GO terms related to TNF signalling and production of IL6 were lost specifically following Erk1/2 inhibition during infection with wild type Nm, which correlated with the reduced infection rates by the wild type in absence of Erk1/2 signalling. CONCLUSION: Our data point towards a role of MAPK signalling during infection of the CP epithelium by Nm, which is strongly influenced by capsule expression, and affects infection rates as well as the host cell response.

Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis.

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5' region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.

Modelling evolutionary pathways for commensalism and hypervirulence in Neisseria meningitidis.

Neisseria meningitidis, the meningococcus, resides exclusively in humans and causes invasive meningococcal disease (IMD). The population of N. meningitidis is structured into stable clonal complexes by limited horizontal recombination in this naturally transformable species. N. meningitidis is an opportunistic pathogen, with some clonal complexes, such as cc53, effectively acting as commensal colonizers, while other genetic lineages, such as cc11, are rarely colonizers but are over-represented in IMD and are termed hypervirulent. This study examined theoretical evolutionary pathways for pathogenic and commensal lineages by examining the prevalence of horizontally acquired genomic islands (GIs) and loss-of-function (LOF) mutations. Using a collection of 4850 genomes from the BIGSdb database, we identified 82 GIs in the pan-genome of 11 lineages (10 hypervirulent and one commensal lineage). A new computational tool, Phaser, was used to identify frameshift mutations, which were examined for statistically significant association with genetic lineage. Phaser identified a total of 144 frameshift loci of which 105 were shown to have a statistically significant non-random distribution in phase status. The 82 GIs, but not the LOF loci, were associated with genetic lineage and invasiveness using the disease carriage ratio metric. These observations have been integrated into a new model that infers the early events of the evolution of the human adapted meningococcus. These pathways are enriched for GIs that are involved in modulating attachment to the host, growth rate, iron uptake and toxin expression which are proposed to increase competition within the meningococcal population for the limited environmental niche of the human nasopharynx. We surmise that competition for the host mucosal surface with the nasopharyngeal microbiome has led to the selection of isolates with traits that enable access to cell types (non-phagocytic and phagocytic) in the submucosal tissues leading to an increased risk for IMD.

Invasion of the choroid plexus epithelium by Neisseria meningitidis is differently mediated by Arp2/3 signaling and possibly by dynamin dependent on the presence of the capsule.

Neisseria meningitis (Nm) is a human-specific bacterial pathogen that can cause sepsis and meningitis. To cause meningitis Nm must enter the central nervous system (CNS) across one of the barriers between the blood and the brain. We have previously shown that a capsule-depleted Serogroup B strain of Nm displays enhanced invasion into human choroid plexus (CP) epithelial papilloma (HIBCPP) cells, which represent an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB). Still, the processes involved during CNS invasion by Nm, especially the role of host cell actin cytoskeleton remodeling, are not investigated in detail. Here, we demonstrate that invasion into CP epithelial cells by encapsulated and capsule-depleted Nm is mediated by distinct host cell pathways. Whereas a Serogroup B wild-type strain enters HIBCPP cells by a possibly dynamin-independent, but actin related protein 2/3 (Arp2/3)-dependent mechanism, invasion by a capsule-depleted mutant is reduced by the dynamin inhibitor dynasore and Arp2/3-independent. Both wild-type and mutant bacteria require Src kinase activity for entry into HIBCPP cells. Our data show that Nm can employ different mechanisms for invasion into the CP epithelium dependent on the presence of a capsule.

In-silico three dimensional structure prediction of important Neisseria meningitidis proteins.

Pathogenic bacteria Neisseria meningitidis cause serious infection i.e. meningitis (infection of the brain) worldwide. Among five pathogenic serogroups, serogroup B causes life threatening illness as there is no effective vaccine available due to its poor immunogenicity. A total of 73 genes in N. meningitidis genome have identified that were proved to be essential for meningococcal disease and were considered as crucial drug targets. We targeted five of those proteins, which are known to involve in amino acid biosynthesis, for homology-based three dimensional structure determinations by MODELLER (v9.19) and evaluated the models by PROSA and PROCHECK programs. Detailed structural analyses of NMB0358, NMB0943, NMB1446, NMB1577 and NMB1814 proteins were carried out during the present research. Based on a high degree of sequence conservation between target and template protein sequences, excellent models were built. The overall three dimensional architectures as well as topologies of all the proteins were quite similar with that of the templates. Active site residues of all the homology models were quite conserved with respect to their respective templates indicating similar catalytic mechanisms in these orthologues. Here, we are reporting, for the first time, detailed three dimensional folds of N. meningitidis pathogenic factors involved in a crucial cellular metabolic pathway. Moreover, the three dimensional structural information of these important drug targets would be utilized in computer-aided drug designing in future.

Genome-wide methylome analysis of two strains belonging to the hypervirulent Neisseria meningitidis serogroup W ST-11 clonal complex.

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.

Peritonitis primària per N. meningitidis serogrup B / Peritonitis primaria por N. Meningitidis serogrupo B / Primary peritonitis by serogroup B N. Meningitidis

INTRODUCCIÓ: Neisseria meningitidis és un dels principals causants de sèpsia I meningitis adquirida a la comunitat en pacients pediàtrics. Se'n descriuen presentacions atípiques, com la peritonitis primària. Presentem aquest cas per la seva excepcionalitat: una patologia infreqüent en edat pediàtrica causada per un germen poc habitual. CAS CLÍNIC: Es presenta un lactant de 4 mesos que acut al servei d'Urgències amb els seus pares per febre I lesions cutànies disseminades de 24 hores d'evolució, amb vòmits I diarrea. Davant la sospita de sèpsia, s'inicien maniobres de reanimació inicial, amb càrrega de volum I antibioteràpia, prèvia extracció d'hemocultiu. Tot I la millora clínica inicial, persisteix la palpació dolorosa a l'hipogastri. Es valora juntament amb Cirurgia I es fa una tomografia computada abdominal que mostra hipercaptació de nanses d'intestí prim I líquid intraabdominal compatible amb procés inflamatori peritoneal. Es fa una laparoscòpia exploradora I s'obté abundant líquid purulent; es revisen íntegrament budell I colon sense observar lesions. Es manté a dieta absoluta durant 48 hores després de la cirurgia, amb progressió alimentària posterior sense incidències. S'obtenen resultats microbiològics: hemocultiu positiu per N. meningitidis B I reacció en cadena de la polimerasa N. meningitidis positiva en sang I en líquids cefalorraquidi I peritoneal. COMENTARIS: La peritonitis primària en pacients pediàtrics sans és una entitat de baixa incidència. Entre els gèrmens descrits en la literatura és molt poc freqüent trobar N. meningitidis. És necessari pensar en aquesta entitat davant d'un pacient amb malaltia invasiva per N. meningitidis I exploració abdominal patològica. La base del tractament són la cirurgia I l'antibioteràpia endovenosa

INTRODUCCIÓN: Neisseria meningitidis es una de las principales causas de sepsis y meningitis adquirida en la comunidad en pacientes pediátricos. Se describen presentaciones atípicas entre las cuales consta de forma excepcional la peritonitis primaria. Este caso ha sido seleccionado por su excepcionalidad, pues se trata de una patología poco frecuente en edad pediátrica y causada por un germen poco habitual. CASO CLÍNICO: Se presenta a un lactante de 4 meses que acude al servicio de Urgencias con sus padres por fiebre y lesiones cutáneas diseminadas de 24 horas de evolución, junto con vómitos y diarrea. Ante la sospecha de sepsis se realizan maniobras de reanimación inicial con carga de volumen y antibioterapia, con previa toma de hemocultivo. A pesar de la mejoría clínica inicial, persiste palpación dolorosa en hipogastrio. Se valora junto con Cirugía y se realiza tomografía computarizada abdominal que muestra hipercaptación de asas de intestino delgado, asociado a líquido intra-abdominal compatible con proceso inflamatorio peritoneal. Se realiza laparoscopia exploradora y se obtiene abundante líquido purulento y esfacelos; se revisan de forma íntegra intestino y colon sin observar lesiones. Se mantiene a dieta absoluta durante 48 horas tras la cirugía, con progresión alimentaria posterior y sin incidencias. Se obtienen resultados microbiológicos: hemocultivo positivo para N. meningitidis B sensible y reacción en cadena de la polimerasa N. meningitidis B positiva en sangre y en líquidos cefalorraquídeo y peritoneal. COMENTARIOS: La peritonitis primaria en pacientes pediátricos sanos es una entidad de baja incidencia. Entre los gérmenes descritos en la literatura es muy poco frecuente encontrar N. meningitidis. Es necesario pensar en esta entidad ante un paciente con infección invasiva por N. meningitidis con exploración abdominal patológica. La base de su tratamiento son la cirugía y la antibioterapia endovenosa

INTRODUCTION: Neisseria meningitidis is one of the main causes of community acquired sepsis and meningitis in children. Atypical presentations have been described, one of them is primary peritonitis. CASE REPORT: We present a 4-month-old infant admitted to the emergency department with fever and disseminated petechia of 24 hours of evolution along with vomiting and diarrhea. With the diagnosis of sepsis, resuscitation maneuvers were started with intravenous fluids and antibiotics after a blood culture was obtained. In spite of the initial clinical improvement, painful palpation persisted in hypogastrium. The patient was assessed together with surgery and an abdominal computed tomography was performed, which showed a small intestine loop enlargement, associated with intraabdominal fluid that suggested peritonitis. Exploratory laparoscopy was performed, obtaining abundant purulent fluid and necrotic and inflammatory tissue. The entire bowel and colon were inspected and no lesions were found. The patient remained fasting for 48 hours after surgery. Enteral nutrition was started afterwards with no complications. N. menigitidis B was isolated in blood culture and polymerase chain reaction positive for N. meningitidis B was positive in blood and in cerebrospinal and peritoneal fluids. COMMENTS: Primary peritonitis in healthy pediatric patients is rare. N. meningitidis is very rare causative pathogen. Primary peritonitis should be considered when having a patient with invasive infection by N. meningitidis with abnormal abdominal examination. Treatment includes surgery and intravenous antibiotics

Type IV pilus retraction enables sustained bacteremia and plays a key role in the outcome of meningococcal sepsis in a humanized mouse model.

Neisseria meningitidis (the meningococcus) remains a major cause of bacterial meningitis and fatal sepsis. This commensal bacterium of the human nasopharynx can cause invasive diseases when it leaves its niche and reaches the bloodstream. Blood-borne meningococci have the ability to adhere to human endothelial cells and rapidly colonize microvessels. This crucial step enables dissemination into tissues and promotes deregulated inflammation and coagulation, leading to extensive necrotic purpura in the most severe cases. Adhesion to blood vessels relies on type IV pili (TFP). These long filamentous structures are highly dynamic as they can rapidly elongate and retract by the antagonistic action of two ATPases, PilF and PilT. However, the consequences of TFP dynamics on the pathophysiology and the outcome of meningococcal sepsis in vivo have been poorly studied. Here, we show that human graft microvessels are replicative niches for meningococci, that seed the bloodstream and promote sustained bacteremia and lethality in a humanized mouse model. Intriguingly, although pilus-retraction deficient N. meningitidis strain (ΔpilT) efficiently colonizes human graft tissue, this mutant did not promote sustained bacteremia nor induce mouse lethality. This effect was not due to a decreased inflammatory response, nor defects in bacterial clearance by the innate immune system. Rather, TFP-retraction was necessary to promote the release of TFP-dependent contacts between bacteria and, in turn, the detachment from colonized microvessels. The resulting sustained bacteremia was directly correlated with lethality. Altogether, these results demonstrate that pilus retraction plays a key role in the occurrence and outcome of meningococcal sepsis by supporting sustained bacteremia. These findings open new perspectives on the role of circulating bacteria in the pathological alterations leading to lethal sepsis.

Neisseria meningitidis IgA1-specific serine protease exhibits novel cleavage activity against IgG3.

Neisseria meningitidis (meningococcus) is a common bacterial colonizer of the human nasopharynx but can occasionally cause very severe systemic infections with rapid onset. Meningococci are able to degrade IgA encountered during colonization of mucosal membranes using their IgA1-specific serine protease. During systemic infection, specific IgG can induce complement-mediated lysis of the bacterium. However, meningococcal immune evasion mechanisms in thwarting IgG remain undescribed. In this study, we report for the first time that the meningococcal IgA1-specific serine protease is able to degrade IgG3 in addition to IgA. The IgG3 heavy chain is specifically cleaved in the lower hinge region thereby separating the antigen binding part from its effector binding part. Through molecular characterization, we demonstrate that meningococcal IgA1-specific serine protease of cleavage type 1 degrades both IgG3 and IgA, whereas cleavage type 2 only degrades IgA. Epidemiological analysis of 7581 clinical meningococcal isolates shows a significant higher proportion of cleavage type 1 among isolates from invasive cases compared to carrier cases, regardless of serogroup. Notably, serogroup W cc11 which is an increasing cause of invasive meningococcal disease globally harbors almost exclusively cleavage type 1 protease. Our study also shows an increasing prevalence of meningococcal isolates encoding IgA1P cleavage type 1 compared to cleavage type 2 during the observed decade (2010-2019). Altogether, our work describes a novel mechanism of IgG3 degradation by meningococci and its association to invasive meningococcal disease.

Genomic analysis of the meningococcal ST-4821 complex-Western clade, potential sexual transmission and predicted antibiotic susceptibility and vaccine coverage.

INTRODUCTION: The ST-4821 complex (cc4821) is a leading cause of serogroup C and serogroup B invasive meningococcal disease in China where diverse strains in two phylogenetic groups (groups 1 and 2) have acquired fluoroquinolone resistance. cc4821 was recently prevalent among carriage isolates in men who have sex with men in New York City (USA). Genome-level population studies have thus far been limited to Chinese isolates. The aim of the present study was to build upon these with an extended panel of international cc4821 isolates. METHODS: Genomes of isolates from Asia (1972 to 2017), Europe (2011 to 2018), North America (2007), and South America (2014) were sequenced or obtained from the PubMLST Neisseria database. Core genome comparisons were performed in PubMLST. RESULTS: Four lineages were identified. Western isolates formed a distinct, mainly serogroup B sublineage with alleles associated with fluoroquinolone susceptibility (MIC <0.03 mg/L) and reduced penicillin susceptibility (MIC 0.094 to 1 mg/L). A third of these were from anogenital sites in men who have sex with men and had unique denitrification gene alleles. Generally 4CMenB vaccine strain coverage was reliant on strain-specific NHBA peptides. DISCUSSION: The previously identified cc4821 group 2 was resolved into three separate lineages. Clustering of western isolates was surprising given the overall diversity of cc4821. Possible association of this cluster with the anogenital niche is worthy of monitoring given concerns surrounding antibiotic resistance and potential subcapsular vaccine escape.

Construction of a complete set of Neisseria meningitidis mutants and its use for the phenotypic profiling of this human pathogen.

The bacterium Neisseria meningitidis causes life-threatening meningitis and sepsis. Here, we construct a complete collection of defined mutants in protein-coding genes of this organism, identifying all genes that are essential under laboratory conditions. The collection, named NeMeSys 2.0, consists of individual mutants in 1584 non-essential genes. We identify 391 essential genes, which are associated with basic functions such as expression and preservation of genome information, cell membrane structure and function, and metabolism. We use this collection to shed light on the functions of diverse genes, including a gene encoding a member of a previously unrecognised class of histidinol-phosphatases; a set of 20 genes required for type IV pili function; and several conditionally essential genes encoding antitoxins and/or immunity proteins. We expect that NeMeSys 2.0 will facilitate the phenotypic profiling of a major human bacterial pathogen.

Gene Polymorphisms of TLR4 and TLR9 and Haemophilus influenzae Meningitis in Angolan Children.

Bacterial meningitis (BM) is a severe disease caused by various bacterial pathogens. Toll-like receptors (TLRs) protect humans from invading pathogens. In this study, we determined whether single nucleotide polymorphisms (SNPs) of TLR4 and TLR9 are associated with susceptibility to and outcome of BM in Angolan children. Samples were taken from 241 patients and 265 age-matched ethnic controls. The SNPs TLR4 rs4986790 (896A > G) and TLR9 rs187084 (-1486T > C) were determined by high-resolution melting analysis (HRMA). The frequency of variant genotypes in TLR4 was significantly higher in patients with Haemophilus influenzae meningitis than controls (odds ratio (OR), 2.5; 95% confidence interval (CI), 1.2-5.4; p = 0.021), whereas the frequency of variant genotypes in TLR9 was significantly lower in patients with H. influenzae meningitis than controls (OR, 0.4; 95% CI, 0.2-0.9; p = 0.036). No such differences were found with other causative pathogens, such as Streptococcus pneumoniae and Neisseria meningitidis. At the time of discharge, patients with meningitis caused by Gram-negative bacteria who were carriers of variant TLR4 genotypes had a higher risk of ataxia (OR, 12.91; 95% CI, 1.52-109.80; p = 0.019) and other neurological sequelae (OR, 11.85; 95% CI, 1.07-131.49; p = 0.044) than those with the wild-type TLR4 genotype. Our study suggests an association between H. influenzae meningitis and genetic variation between TLR4 and TLR9 in Angolan children.

Neisseria meningitidis Urethritis Outbreak Isolates Express a Novel Factor H Binding Protein Variant That Is a Potential Target of Group B-Directed Meningococcal (MenB) Vaccines.

Factor H binding protein (FHbp) is an important Neisseria meningitidis virulence factor that binds a negative regulator of the alternative complement pathway, human factor H (FH). Binding of FH increases meningococcal resistance to complement-mediated killing. FHbp also is reported to prevent interaction of the antimicrobial peptide (AMP) LL-37 with the meningococcal surface and meningococcal killing. FHbp is a target of two licensed group B-directed meningococcal (MenB) vaccines. We found a new FHbp variant, peptide allele identification no. 896 (ID 896), was highly expressed by an emerging meningococcal pathotype, the nonencapsulated urethritis clade (US_NmUC). This clade has been responsible for outbreaks of urethritis in multiple U.S. cities since 2015, other mucosal infections, and cases of invasive meningococcal disease. FHbp ID 896 is a member of the variant group 1 (subfamily B), bound protective anti-FHbp monoclonal antibodies, bound high levels of human FH, and enhanced the resistance of the clade to complement-mediated killing in low levels of human complement likely present at human mucosal surfaces. Interestingly, expression of FHbp ID 896 resulted in augmented killing of the clade by LL-37. FHbp ID 896 of the clade was recognized by antibodies elicited by FHbp in MenB vaccines.

Molecular characterization of Neisseria meningitidis isolates recovered from patients with invasive meningococcal disease in Colombia from 2013 to 2016.

BACKGROUND: Neisseria meningitidis is a significant cause of morbidity and mortality worldwide. Meningococcal isolates have a highly dynamic population structure and can be phenotypically and genetically differentiated into serogroups and clonal complexes. The aim of this study was to describe the phenotypic and genotypic characteristics of invasive isolates recovered in Colombia from 2013 to 2016. METHODOLOGY: A total of 193 invasive isolates were analyzed. Phenotypic and genotypic characteristics were determined by serotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. RESULTS: Based on the results, meningococcal serogroups C, B and Y were responsible for 47.9%, 41.7%, and 9.4% of cases, respectively, and the distribution of serogroups B and C changed over time. Fifteen clonal groups and 14 clonal complexes (cc) were identified by PFGE and genome sequencing. The main clonal group included serogroup B isolates with sequence type (ST)-9493 and its four single-locus variants, which has only been identified in Colombian isolates. The clonal population structure demonstrates that the isolates in this study mainly belong to four clonal complexes: ST-11 cc, ST-32 cc, ST-35 cc and ST-41/44 cc. Thirty-eight penA alleles were identified, but no correlation between MICs and specific sequences was observed. CONCLUSION: This study shows that most meningococcal isolates recovered from patients with invasive meningococcal disease in Colombia are strains associated with distinct globally disseminated hyperinvasive clones.

Meningococcal core and accessory phasomes vary by clonal complex.

Neisseria meningitidis is a Gram-negative human commensal pathogen, with extensive phenotypic plasticity afforded by phase-variable (PV) gene expression. Phase variation is a stochastic switch in gene expression from an ON to an OFF state, mediated by localized hypermutation of simple sequence repeats (SSRs). Circulating N. meningitidis clones vary in propensity to cause disease, with some clonal complexes (ccs) classified as hypervirulent and others as carriage-associated. We examined the PV gene repertoires, or phasome, of these lineages in order to determine whether phase variation contributes to disease propensity. We analysed 3328 genomes representative of nine circulating meningococcal ccs with PhasomeIt, a tool that identifies PV genes by the presence of SSRs and homologous gene clusters. The presence, absence and functions of all identified PV gene clusters were confirmed by annotation or blast searches within the Neisseria PubMLST database. While no significant differences were detected in the number of PV genes or the core, conserved phasome content between hypervirulent and carriage lineages, individual ccs exhibited major variations in PV gene numbers. Phylogenetic clusters produced by phasome or core genome analyses were similar, indicating co-evolution of PV genes with the core genome. While conservation of PV clusters is high, with 76 % present in all meningococcal isolates, maintenance of an SSR is variable, ranging from conserved in all isolates to present only in a single cc, indicating differing evolutionary trajectories for each lineage. Diverse functional groups of PV genes were present across the meningococcal lineages; however, the majority directly or indirectly influence bacterial surface antigens and could impact on future vaccine development. Finally, we observe that meningococci have open pan phasomes, indicating ongoing evolution of PV gene content and a significant potential for adaptive changes in this clinically relevant genus.

Difference in virulence between Neisseria meningitidis serogroups W and Y in transgenic mice.

BACKGROUND: 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. RESULTS: The levels of bacteraemia, CXCL1, and apoptosis were higher in serogroup W infected mice than in serogroup Y infected mice. Serogroup W isolates also induced higher levels of apoptosis and adhesion in human epithelial cells. No significant differences were observed between different lineages within cc11 and cc23. CONCLUSIONS: N. meningitidis Serogroup W displayed a higher virulence in vivo in transgenic mice, compared to serogroup Y. This was reflected by higher bacteremia, proinflammatory activity, and ability to induce apoptosis in mouse immune cells and human epithelial cells.

Commensal Neisseria cinerea impairs Neisseria meningitidis microcolony development and reduces pathogen colonisation of epithelial cells.

It is increasingly being recognised that the interplay between commensal and pathogenic bacteria can dictate the outcome of infection. Consequently, there is a need to understand how commensals interact with their human host and influence pathogen behaviour at epithelial surfaces. Neisseria meningitidis, a leading cause of sepsis and meningitis, exclusively colonises the human nasopharynx and shares this niche with several other Neisseria species, including the commensal Neisseria cinerea. Here, we demonstrate that during adhesion to human epithelial cells N. cinerea co-localises with molecules that are also recruited by the meningococcus, and show that, similar to N. meningitidis, N. cinerea forms dynamic microcolonies on the cell surface in a Type four pilus (Tfp) dependent manner. Finally, we demonstrate that N. cinerea colocalises with N. meningitidis on the epithelial cell surface, limits the size and motility of meningococcal microcolonies, and impairs the effective colonisation of epithelial cells by the pathogen. Our data establish that commensal Neisseria can mimic and affect the behaviour of a pathogen on epithelial cell surfaces.