Search

Pharyngeal colonisation by the commensal bacterium Neisseria lactamica inhibits colonisation by Neisseria meningitidis and has an inverse epidemiological association with meningococcal disease. The mechanisms that underpin this relationship are unclear, but could involve the induction of cross-reactive immunity. In this study, we aimed to evaluate whether colonisation with N lactamica induces N lactamica-specific B-cell responses that are cross-reactive with N meningitidis.In this randomised, placebo-controlled, human infection trial at University Hospital Southampton Clinical Research Facility (Southampton, UK), healthy adults aged 18-45 years were randomly assigned (2:1) to receive intranasal inoculation with either 10Of 50 participants assessed for eligibility between Sept 5, 2018, and March 3, 2019, 31 were randomly assigned (n=20 N lactamica, n=11 PBS). Among the 17 participants who were colonised with N lactamica, the median baselines compared with peak post-colonisation N lactamica-specific plasma-cell frequencies (per 10Natural immunity to N meningitidis after colonisation with N lactamica might be due to cross-reactive adaptive responses. Exploitation of this microbial mechanism with a genetically modified live vector could protect against N meningitidis colonisation and disease.Wellcome Trust, Medical Research Council, and NIHR Southampton Biomedical Research Centre.

Neisseria lactamica is a nonpathogenic commensal of the human upper respiratory tract that has been associated with protection against N. meningitidis colonization and disease. We have previously utilized the N. lactamica controlled human infection model to investigate the protective effect of N. lactamica colonization on N. meningitidis colonization, the nature of cross-reactive immune responses mounted toward N. meningitidis following N. lactamica colonization, and the microevolution of N. lactamica over a 5-month colonization period. More recently, we have assessed the possibility of utilizing genetically modified strains of N. lactamica to enable use of the commensal as a vehicle for prolonged exposure of the nasopharynx of humans to antigens of interest, expressed in carried organisms. A controlled infection with N. lactamica expressing the meningococcal antigen NadA has been executed and the results demonstrate that this strategy is effective at generating immune responses to the target antigen. Throughout this chapter, we outline in a step-by-step manner the methodologies utilized when performing controlled human infection with N. lactamica including procedures relating to: (1) the dilution of N. lactamica stock vials to derive intranasal inocula, (2) the delivery of intranasal inocula to human volunteers, (3) the determination of N. lactamica colonization status following intranasal inoculation using oropharyngeal swabbing and nasal wash sampling, (4) the microbiological procedures utilized to identify N. lactamica colonization among study volunteers, and (5) the identification of N. lactamica colonies as strain Y92-1009 using polymerase chain reaction.

Background Penicillin and ciprofloxacin are important for invasive meningococcal disease (IMD) management and prevention. IMD cases caused by penicillin- and ciprofloxacin-resistant Neisseria meningitidis containing a ROB-1 β-lactamase gene (blaROB-1) and a mutated DNA gyrase gene (gyrA) have been recently reported in the United States. Methods We examined 2097 meningococcal genomes collected through US population-based surveillance from January 2011 to February 2020 to identify IMD cases caused by strains with blaROB-1- or gyrA-mediated resistance. Antimicrobial resistance was confirmed phenotypically. The US isolate genomes were compared to non-US isolate genomes containing blaROB-1. Interspecies transfer of ciprofloxacin resistance was assessed by comparing gyrA among Neisseria species. Results Eleven penicillin- and ciprofloxacin-resistant isolates were identified after December 2018; all were serogroup Y, sequence type 3587, clonal complex (CC) 23, and contained blaROB-1 and a T91I-containing gyrA allele. An additional 22 penicillin-resistant, blaROB-1- containing US isolates with wild-type gyrA were identified from 2013 to 2020. All 33 blaROB-1-containing isolates formed a single clade, along with 12 blaROB-1-containing isolates from 6 other countries. Two-thirds of blaROB-1-containing US isolates were from Hispanic individuals. Twelve additional ciprofloxacin-resistant isolates with gyrA T91 mutations were identified. Ciprofloxacin-resistant isolates belonged to 6 CCs and contained 10 unique gyrA alleles; 7 were similar or identical to alleles from Neisseria lactamica or Neisseria gonorrhoeae. Conclusions Recent IMD cases caused by a dual resistant serogroup Y suggest changing antimicrobial resistance patterns in the United States. The emerging dual resistance is due to acquisition of ciprofloxacin resistance by β-lactamase–containing N. meningitidis. Routine antimicrobial resistance surveillance will effectively monitor resistance changes and spread.

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis (NmY) is rare in China; recently, an invasive NmY isolate, Nm512, was discovered in Shanghai with decreased susceptibility to penicillin (Pen(NS)). Here, we investigated the epidemiology of NmY isolates in Shanghai and explored the potential commensal Neisseria lactamica donor of the Pen(NS) NmY isolate. A total of 491 N. meningitidis and 724 commensal Neisseria spp. isolates were collected. Eleven NmY isolates were discovered from IMD (n = 1) and carriers (n = 10), including two Pen(NS) isolates with five-key-mutation-harboring (F504L-A510V-I515V-H541N-I566V) penA genes. Five of the eight ST-175 complex (CC175) isolates had a genotype [Y:P1.5-1,2-2:F5-8:ST-175(CC175)] identical to that of the predominant invasive clone found in South Africa. Only one invasive NmY CC23 isolate (Nm512) was discovered; this isolate carried a novel Pen(NS) penA832 allele, which was identified in commensal N. lactamica isolates locally. Recombination analysis and transformation of the penA allele highlighted that N. meningitidis Nm512 may acquire resistance from its commensal donor; this was supported by the similar distribution of transformation-required DNA uptake sequence variants and the highly cognate receptor ComP between N. meningitidis and N. lactamica. In 2,309 NmY CC23 genomes from the PubMLST database, isolates with key-mutation-harboring penA genes comprised 12% and have been increasing since the 1990s, accompanied by recruitment of the bla(ROB-1) and/or quinolone resistance allele. Moreover, penA22 was predominant among genomes without key mutations in penA. These results strongly suggest that Nm512 is a descendant of the penA22-harboring CC23 isolate from Europe and acquired its penicillin resistance locally from commensal N. lactamica species by natural transformation.

Infant upper respiratory microbiota are derived partly from the maternal respiratory tract, and certain microbiota are associated with altered risk of infections and respiratory disease.20 healthy pregnant women will receive nasal inoculation withThis study has been approved by the London Central Research Ethics Committee (21/PR/0373). Findings will be published in peer-reviewed open-access journals as soon as possible.NCT04784845.

Neisseria spp. possess four genogroups of filamentous prophages, termed Nf1 to 4. A filamentous bacteriophage from the Nf1 genogroup termed meningococcal disease-associated phage (MDA φ) is associated with clonal complexes of Neisseria meningitidis that cause invasive meningococcal disease. Recently, we recovered an isolate of Neisseria gonorrhoeae (ExNg63) from a rare case of gonococcal meningitis, and found that it possessed a region with 90% similarity to Nf1 prophages, specifically, the meningococcal MDA φ. This led to the hypothesis that the Nf1 prophage may be more widely distributed amongst the genus Neisseria. An analysis of 92 reference genomes revealed the presence of intact Nf1 prophages in the commensal species, Neisseria lactamica and Neisseria cinerea in addition to the pathogen N. gonorrhoeae. In N. gonorrhoeae, Nf1 prophages had a restricted distribution but were present in all representatives of MLST ST1918. Of the 160 phage integration sites identified, only one common insertion site was found between one isolate of N. gonorrhoeae and N. meningitidis. There was an absence of any obvious conservation of the receptor for prophage entry, PilE, suggesting that the phage may have been obtained by natural transformation. An examination of the restriction modification systems and mutated mismatch repair systems with prophage presence suggested that there was no obvious preference for these hosts. A timed phylogeny inferred that N. meningitidis was the donor of the Nf1 prophages in N. lactamica and N. gonorrhoeae. Further work is required to determine whether Nf1 prophages are active and can act as accessory colonization factors in these species.

The human nasopharynx contains a stable microbial ecosystem of commensal and potentially pathogenic bacteria, which can elicit protective primary and secondary immune responses. Experimental intranasal infection of human adults with the commensal Neisseria lactamica produced safe, sustained pharyngeal colonization. This has potential utility as a vehicle for sustained release of antigen to the human mucosa, but commensals in general are thought to be immunologically tolerated. Here, we show that engineered N. lactamica, chromosomally transformed to express a heterologous vaccine antigen, safely induces systemic, antigen-specific immune responses during carriage in humans. When the N. lactamica expressing the meningococcal antigen Neisseria Adhesin A (NadA) was inoculated intranasally into human volunteers, all colonized participants carried the bacteria asymptomatically for at least 28 days, with most (86%) still carrying the bacteria at 90 days. Compared to an otherwise isogenic but phenotypically wild-type strain, colonization with NadA-expressing N. lactamica generated NadA-specific immunoglobulin G (IgG)- and IgA-secreting plasma cells within 14 days of colonization and NadA-specific IgG memory B cells within 28 days of colonization. NadA-specific IgG memory B cells were detected in peripheral blood of colonized participants for at least 90 days. Over the same period, there was seroconversion against NadA and generation of serum bactericidal antibody activity against a NadA-expressing meningococcus. The controlled infection was safe, and there was no transmission to adult bedroom sharers during the 90-day period. Genetically modified N. lactamica could therefore be used to generate beneficial immune responses to heterologous antigens during sustained pharyngeal carriage.

Rationale: The respiratory microbiota is increasingly being appreciated as an important mediator in the susceptibility to childhood respiratory tract infections (RTIs). Pathogens are presumed to originate from the nasopharyngeal ecosystem. Objectives: To investigate the association between early-life respiratory microbiota and development of childhood RTIs. Methods: In a prospective birth cohort (Microbiome Utrecht Infant Study: MUIS), we characterized the oral microbiota longitudinally from birth until six months of age of 112 infants (9 regular samples/subject) and compared them with nasopharyngeal microbiota using 16S-rRNA-based sequencing. We also characterized oral and nasopharynx samples during RTI episodes in the first half year of life. Measurements and Main Results: Oral microbiota were driven mostly by feeding type, followed by age, mode of delivery and season of sampling. In contrast to our previously published associations between nasopharyngeal microbiota development and susceptibility to RTIs, oral microbiota development was not directly associated with susceptibility to RTI development. However, we did observe an influx of oral taxa, such as Neisseria lactamica, Streptococcus, Prevotella nanceiensis, Fusobacterium and Janthinobacterium lividum, in the nasopharyngeal microbiota prior to and during RTIs, which was accompanied by reduced presence and abundance of Corynebacterium, Dolosigranulum and Moraxella spp. Moreover, this phenomenon was accompanied by reduced niche differentiation indicating loss of ecological topography preceding confirmed RTIs. This loss of ecological topography was further augmented by start of daycare, and linked to consecutive development of symptomatic infections.Conclusions: Together, our results link the loss of topography to subsequent development of RTI episodes. This may lead to new insights for prevention of RTIs and antibiotic utilization in childhood.

The human nasopharynx is colonized by commensal bacteria and pathobionts, which comprise a complex microbial ecosystem capable of generating primary and secondary immune responses. Experimental intranasal infection of human adults with the commensal Neisseria lactamica results in safe, sustained colonization. Herein is described a novel technology to chromosomally transform N. lactamica with heterologous antigen, for the purpose of safe delivery to the mucosal surface and the generation of an antigen-specific immune response. N. lactamica was transformed to express the meningococcal vaccine antigen Neisseria Adhesin A (NadA) and was inoculated intranasally into humans at a dose of 105 colony-forming units. NadA-expressing N. lactamica colonized these individuals and was carried asymptomatically for 3 months. Colonization with NadA-expressing N. lactamica generated NadA-specific IgG-secreting plasma cells within 14 days of colonization and both NadA-specific IgG and NadA-specific IgG memory B cells within 28 days of colonization. NadA-specific IgG memory B cells circulate in the bloodstream of colonized participants for at least 90 days. Genetically transformed N. lactamica has the potential to be a safe bacterial vehicle to generate beneficial immune responses to a wide range of heterologous antigens during sustained pharyngeal carriage.

Innate immune cells such as monocytes and macrophages are activated in response to microbial and other challenges and mount an inflammatory defensive response. Exposed cells develop the so-called innate memory, which allows them to react differently to a subsequent challenge, aiming at better protection. In this study, using human primary monocytes in vitro, we have assessed the memory-inducing capacity of two antigenic molecules of Schistosoma mansoni in soluble form compared to the same molecules coupled to outer membrane vesicles of Neisseria lactamica. The results show that particulate challenges are much more efficient than soluble molecules in inducing innate memory, which is measured as the production of inflammatory and anti-inflammatory cytokines (TNFα, IL-6, IL-10). Controls run with LPS from Klebsiella pneumoniae compared to the whole bacteria show that while LPS alone has strong memory-inducing capacity, the entire bacteria are more efficient. These data suggest that microbial antigens that are unable to induce innate immune activation can nevertheless participate in innate activation and memory when in a particulate form, which is a notion that supports the use of nanoparticulate antigens in vaccination strategies for achieving adjuvant-like effects of innate activation as well as priming for improved reactivity to future challenges.

Asymptomatic carriers are a likely source of transmission of Neisseria meningitidis to close contacts who are placed at a higher risk for invasive meningococcal disease (IMD). Although N. meningitidis ciprofloxacin-resistance is rare, there have been an increase in the reports of resistant isolates mainly in patients diagnosed with IMD, and little is known about the N. meningitidis ciprofloxacin-resistance in the carrier populations. We performed a pharyngeal carriage study during a 2017 military setting outbreak in Peru, caused by a ciprofloxacin-resistant N. meningitidis B. The isolates analysed came from two hospitalized cases and six asymptomatic carriers. Whole-genome sequence-based analysis was performed and showed that strains carrying the Thr91Ile mutation, in the gene encoding for subunit A of DNA gyrase (gyrA), were responsible for the fluoroquinolone resistance (MICs ≥0.256 µg ml−1) and were closely related to highly virulent strains from France, Norway and the UK. Phylogenetic analysis of the gyrA gene revealed that likely these Peruvian isolates acquired resistance through horizontal gene transfer from Neisseria lactamica . Our study provides evidence for the emergence and propagation of ciprofloxacin-resistant N. meningitidis B from asymptomatic carriers, and recommends the introduction of serogroup B vaccines for high-risk populations.

Chlamydia trachomatis is the most frequently detected sexually transmitted bacterial pathogen in the world. Attempts to control these infections with screening programs and antibiotics have failed and, therefore, a vaccine is the best approach to control this epidemic. The Chlamydia major outer membrane protein (MOMP) is the most protective subunit vaccine so far tested. Protection induced by MOMP is, in part, dependent on its tertiary structure. We have previously described new recombinant antigens composed of the Neisseria lactamica PorB engineered to express the variable domains (VD) from Chlamydia muridarum MOMP. Here we tested antigens containing each individual MOMP VD and different VD combinations. Following immunization, mice were challenged intranasally with C. muridarum. Our results show that three constructs, PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4, elicited high serum IgG titers in vivo, significant IFN-γ levels upon T cells re-stimulation in vitro, and evidence of protective immunity in vivo. PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4 immunized mice lost less body weight, had lighter lungs, and decreased numbers of inclusion forming units (IFUs) in lungs than other PorB/VD construct tested and mock PBS-immunized mice. These results suggest that this approach may be a promising alternative to the use of MOMP in a Chlamydia vaccine.

Neisseria meningitidis and Neisseria gonorrhoeae, two highly related species that might have emerged from a common commensal ancestor, constitute major human threats. Vaccines are available to prevent N. meningitidis infection, whereas there are only a limited number of antibiotics available for N. gonorrhoeae. Unfortunately, some strains of these species are rapidly evolving and capable of escaping human interventions., Neisseria meningitidis and Neisseria gonorrhoeae, two highly related species that might have emerged from a common commensal ancestor, constitute major human threats. Vaccines are available to prevent N. meningitidis infection, whereas there are only a limited number of antibiotics available for N. gonorrhoeae. Unfortunately, some strains of these species are rapidly evolving and capable of escaping human interventions. Thus, it is now urgent to develop new avenues to fight these bacteria. This study reports that a boron-based salt, sodium tetraphenylborate (NaBPh4), displays high bactericidal activity and remarkable specificity against N. meningitidis and N. gonorrhoeae. Other closely related commensal species such as Neisseria lactamica, which is found in the normal flora of healthy individuals, were found to be less affected even at 5-fold higher doses of NaBPh4. This specificity was further observed when much lower sensitivity was found for more distant Neisseriaceae species (such as Neisseria elongata or Kingella oralis) and completely unrelated species. Significant boron uptake by N. meningitidis cells was observed after incubation with 5 μM NaBPh4, as measured by inductively coupled plasma mass spectrometry, suggesting that this drug candidate’s target(s) could be located intracellularly or within the cell envelope. Furthermore, mutants with slightly decreased susceptibility displayed alterations in genes coding for cell envelope elements, which reduced their virulence in an animal model of infection. Finally, a single dose of NaBPh4 resulted in a significant reduction in bacterial burden in a mouse model of N. meningitidis bacteremia. Although numerous boron-containing species were previously reported for their complex biological activities, the observation of this narrow selectivity is unprecedented and of potential importance from a therapeutic standpoint.

Leading CRISPR-Cas technologies employ Cas9 and Cas12 enzymes that generate RNA-guided dsDNA breaks. Yet, the most abundant microbial adaptive immune systems, Type I CRISPRs, are under-exploited for eukaryotic applications. Here, we report the adoption of a minimal CRISPR-Cas3 from Neisseria lactamica (Nla) type I-C system to create targeted large deletions in the human genome. RNP delivery of its processive Cas3 nuclease and target recognition complex Cascade can confer ∼95% editing efficiency. Unexpectedly, NlaCascade assembly in bacteria requires internal translation of a hidden component Cas11 from within the cas8 gene. Furthermore, expressing a separately encoded NlaCas11 is the key to enable plasmid- and mRNA-based editing in human cells. Finally, we demonstrate that supplying cas11 is a universal strategy to systematically implement divergent I-C, I-D, and I-B CRISPR-Cas3 editors with compact sizes, distinct PAM preferences, and guide orthogonality. These findings greatly expand our ability to engineer long-range genome edits.

Neisseria lactamica is a nonpathogenic commensal bacterium that is potentially associated with the development of natural immunity against N. meningitidis. However, the genetic variation present in natural populations of N. lactamica has not been fully investigated. To better understand its epidemiology and genetic variation, we studied N. lactamica carriage in 1200 students aged 11-19 years old in Salvador, Brazil. The carriage prevalence was 4.5% (54/1200), with no statistical difference among sex and age, although we observed a trend towards higher carriage prevalence among 11-year-old individuals. Whole genome sequence analysis revealed a high genetic diversity among the isolates, with the presence of 32 different STs, 28 (87.5%) of which were new. A total of 21/50 (42%) isolates belonged to three different clonal complexes. While none of the isolates contained nadA or fHpb alleles, we detected 21 FetA variants, 20 NhbA variants and two variants of PorB. The data provide detailed information on circulating N. lactamica isolates in adolescents in Brazil and are complementary to studies in other countries.

Although invasive meningococcal disease is rare in Japan (0.028 cases per 100,000 population), its incidence is 10 times greater in many other countries. Colonization is a prerequisite for invasive meningococcal disease. However, no study in Japan has involved specifically analyzing the carriage rate of Neisseria meningitidis in children. During 5 months in 2015, the respiratory tract specimens of patients who presented to 3 hospitals with respiratory symptoms were cultured. The bacteria were identified in selective media using a meningococcal detection kit and the serogroup was identified using polymerase chain reaction analysis. In 389 patients aged ≤15 years with respiratory symptoms, the N. meningitidis isolation rate was 0.26% (1/389). The serogroup of the only child who tested positive was Y. In this study, we detected a low meningococcal isolation rate in pediatric patients. Due to increasing globalization, the risk of invasive meningococcal disease is likely increasing in Japan. Accordingly, invasive meningococcal diseases should be continuously monitored in Japan. Future large-scale studies should assess meningococcal isolation rates and corresponding serogroups.

Purpose of review Nonpathogenic commensal Neisseria are rarely considered in the clinical setting despite evidence that they can cause invasive opportunistic infections. In contrast, they may offer protection against pathogenic Neisseria, and such relationships are being actively explored in experimental studies. Recent findings Recent case reports are presented of invasive infection caused by nonpathogenic Neisseria in patients on novel biologic therapies. On the other hand, Neisseria lactamica, a nonpathogenic commensal, has been shown in human challenge studies to inhibit colonization by Neisseria meningitidis. Experimental mouse models have also explored the inhibitory effects of nonpathogenic Neisseria on Neisseria gonnhoreae infection. Cutting-edge advances in metagenomics and microbiomics are being used to understand the mechanisms underpinning these effects. Summary Clinicians should have increased awareness of nonpathogenic Neisseria. First, as new immunomodulating therapies become licenced, the interactions that maintain balance between commensals and their human hosts may be altered. Second, these bacteria are showing promise in their capacity to exclude pathogenic Neisseria species from their anatomical niches.

IntroductionNeisseria lactamicais a commensal organism found in the human nasopharynx and is closely related to the pathogenN. meningitidis(meningococcus). Carriage ofN. lactamicais associated with reduced meningococcal carriage and disease. We summarise an ethically approved protocol for an experimental human challenge study using a genetically modified strain ofN. lactamicathat expresses the meningococcal antigen NadA. We aim to develop a model to study the role of specific bacterial antigens in nasopharyngeal carriage and immunity, to evaluate vaccines for their efficacy in preventing colonisation and to provide a proof of principle for the development of bacterial medicines.Methods and analysisHealthy adult volunteers aged 18–45 years will receive an intranasal inoculation of either the NadA containing strain ofN. lactamicaor a genetically modified, but wild-type equivalent control strain. These challenge volunteers will be admitted for 4.5 days observation following inoculation and will then be discharged with strict infection control rules. Bedroom contacts of the challenge volunteers will also be enrolled as contact volunteers. Safety, colonisation, shedding, transmission and immunogenicity will be assessed over 90 days after which carriage will be terminated with antibiotic eradication therapy.Ethics and disseminationThis study has been approved by the Department for Environment, Food and Rural Affairs and South Central Oxford A Research Ethics Committee (reference: 18/SC/0133). Findings will be published in peer-reviewed open-access journals as soon as possible.Trial registration numberNCT03630250; Pre-results.

Journal of molecular biology 431(11), 2082 - 2094 (2019). doi:10.1016/j.jmb.2019.04.010, Specificity engineering is challenging and particularly difficult for enzymes that have the catalytic machinery and specificity determinants in close proximity. Restriction endonucleases have been used as a paradigm for protein engineering, but successful cases are rare. Here, we present the results of a directed evolution approach to the engineering of a dimeric, blunt end cutting restriction enzyme NlaIV (GGN/NCC). Based on the remote similarity to EcoRV endonuclease, regions for random mutagenesis and in vitro evolution were chosen. The obtained variants cleaved target sites with an up to 100-fold kcat/KM preference for AT or TA (GGW/WCC) over GC or CG (GGS/SCC) in the central dinucleotide step, compared to the only ~ 17-fold preference of the wild-type enzyme. To understand the basis of the increased specificity, we determined the crystal structure of NlaIV. Despite the presence of DNA in the crystallization mix, the enzyme crystallized in the free form. We therefore constructed a computational model of the NlaIV–DNA complex. According to the model, the mutagenesis of the regions that were in the proximity of DNA did not lead to the desired specificity change, which was instead conveyed in an indirect manner by substitutions in the more distant regions, Published by Elsevier, Amsterdam [u.a.]

The genus Neisseria includes three major species of importance to human health and disease ( Neisseria gonorrhoeae , Neisseria meningitidis , and Neisseria lactamica ) that express broad-spectrum O -linked protein glycosylation (Pgl) systems. The potential for related Pgl systems in other species in the genus, however, remains to be determined. Using a strain of Neisseria elongata subsp. glycolytica , a unique tetrasaccharide glycoform consisting of di- N -acetylbacillosamine and glucose as the first two sugars followed by a rare sugar whose mass spectrometric fragmentation profile was most consistent with di- N -acetyl hexuronic acid and a N -acetylhexosamine at the nonreducing end has been identified. Based on established mechanisms for UDP-di- N -acetyl hexuronic acid biosynthesis found in other microbes, we searched for genes encoding related pathway components in the N. elongata subsp. glycolytica genome. Here, we detail the identification of such genes and the ensuing glycosylation phenotypes engendered by their inactivation. While the findings extend the conservative nature of microbial UDP-di- N -acetyl hexuronic acid biosynthesis, mutant glycosylation phenotypes reveal unique, relaxed specificities of the glycosyltransferases and oligosaccharyltransferases to incorporate pathway intermediate UDP-sugars into mature glycoforms. IMPORTANCE Broad-spectrum protein glycosylation (Pgl) systems are well recognized in bacteria and archaea. Knowledge of how these systems relate structurally, biochemically, and evolutionarily to one another and to others associated with microbial surface glycoconjugate expression is still incomplete. Here, we detail reverse genetic efforts toward characterization of protein glycosylation mutants of N. elongata subsp. glycolytica that define the biosynthesis of a conserved but relatively rare UDP-sugar precursor. The results show both a significant degree of intra- and transkingdom conservation in the utilization of UDP-di- N -acetyl-glucuronic acid and singular properties related to the relaxed specificities of the N. elongata subsp. glycolytica system

Background Neisseria meningitidis are common colonizers of the human nasopharynx. In some circumstances, N. meningitidis becomes an opportunistic pathogen that invades tissues and causes meningitis. While a vaccine against a number of serogroups has been in effective use for many years, a vaccine against N. meningitidis group B has not yet been universally adopted. Bacterial heat shock protein complex (HSPC) vaccines comprise bacterial HSPs, purified with their chaperoned protein cargo. HSPC vaccines use the intrinsic adjuvant activity of their HSP, thought to act via Toll-like receptors (TLR), to induce an immune response against their cargo antigens. This study evaluated HSPC vaccines from N. meningitidis and the closely related commensal N. lactamica . Results The protein composition of N. lactamica and N. meningitidis HSPCs were similar. Using human HEK293 cells we found that both HSPCs can induce an innate immune response via activation of TLR2. However, stimulation of TLR2 or TLR4 deficient murine splenocytes revealed that HSPCs can activate an innate immune response via multiple receptors. Vaccination of wildtype mice with the Neisseria HSPC induced a strong antibody response and a Th1-restricted T helper response. However, vaccination of mice deficient in the major TLR adaptor protein, MyD88, revealed that while the Th1 response to Neisseria HSPC requires MyD88, these vaccines unexpectedly induced an antigen-specific antibody response via a MyD88-independent mechanism. Conclusions N. lactamica and N. meningitidis HSPC vaccines both have potential utility for immunising against neisserial meningitis without the requirement for an exogenous adjuvant. The mode of action of these vaccines is highly complex, with HSPCs inducing immune responses via both MyD88-dependent and -independent mechanisms. In particular, these HSPC vaccines induced an antibody response without detectable T cell help.

Although acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is a manageable disease for many, it is still a source of significant morbidity and economic hardship for many others. The predominant mode of transmission of HIV/AIDS is sexual intercourse, and measures to reduce transmission are needed. Previously, we showed that caerin 1 antimicrobial peptides (AMPs) originally derived from Australian amphibians inhibited in vitro transmission of HIV at relatively low concentrations and had low toxicity for T cells and an endocervical cell line. The use of AMPs as part of microbicidal formulations would expose the vaginal microbiome to these agents and cause potential harm to protective lactobacilli. Here, we tested the effects of caerin 1 peptides and their analogs on the viability of two species of common vaginal lactobacilli (Lactobacillus rhamnosus and Lactobacillus crispatus). Several candidate peptides had limited toxicity for the lactobacilli at a range of concentrations that would inhibit HIV. Three AMPs were also tested for their ability to inhibit growth of Neisseria lactamica, a close relative of the sexually transmissible Neisseria gonorrhoeae. Neisseria lactamica was significantly more sensitive to the AMPs than the lactobacilli. Thus, several candidate AMPs have the capacity to inhibit HIV and possible N. gonorrhoeae transmission at concentrations that are significantly less harmful to the resident lactobacilli.

Neisseria lactamica is a harmless coloniser of the infant respiratory tract, and has a mutually-excluding relationship with the pathogen Neisseria meningitidis. Here we report controlled human infection with genomically-defined N. lactamica and subsequent bacterial microevolution during 26 weeks of colonisation. We find that most mutations that occur during nasopharyngeal carriage are transient indels within repetitive tracts of putative phase-variable loci associated with host-microbe interactions (pgl and lgt) and iron acquisition (fetA promotor and hpuA). Recurrent polymorphisms occurred in genes associated with energy metabolism (nuoN, rssA) and the CRISPR-associated cas1. A gene encoding a large hypothetical protein was often mutated in 27% of the subjects. In volunteers who were naturally co-colonised with meningococci, recombination altered allelic identity in N. lactamica to resemble meningococcal alleles, including loci associated with metabolism, outer membrane proteins and immune response activators. Our results suggest that phase variable genes are often mutated during carriage-associated microevolution., Carriage of Neisseria lactamica, a harmless coloniser of the human respiratory tract, is inversely correlated with Neisseria meningitidis infection. Here, Pandey et al. provide insights into micro-evolutionary processes in N. lactamica during controlled infection of healthy volunteers.

The spread of antibiotic resistance within and between different bacterial populations is a major health problem on a global scale. The identification of genetic transformation in genomic data from Neisseria meningitidis, the meningococcus (Mc), and other bacteria is problematic, since similar or even identical alleles may be involved. A particular challenge in naturally transformable bacteria generally is to distinguish between common ancestry and true recombined sites in sampled genome sequences. Furthermore, the identification of recombination following experimental transformation of homologous alleles requires identifiable differences between donor and recipient, which in itself influences the propensity for homologous recombination (HR). This study identifies the distribution of HR events following intraspecies and interspecies Mc transformations of rpoB alleles encoding rifampicin resistance by whole-genome DNA sequencing and single nucleotide variant analysis. The HR events analysed were confined to the genomic region surrounding the single nucleotide genetic marker used for selection. An exponential length distribution of these recombined events was found, ranging from a few nucleotides to about 72 kb stretches. The lengths of imported sequences were on average found to be longer following experimental transformation of the recipient with genomic DNA from an intraspecies versus an interspecies donor (P

We present the high quality, complete genome assembly of Neisseria lactamica Y92–1009 used to manufacture an outer membrane vesicle (OMV)-based vaccine, and a member of the Neisseria genus. The strain is available on request from the Public Health England Meningococcal Reference Unit. This Gram negative, dipplococcoid bacterium is an organism of worldwide clinical interest because human nasopharyngeal carriage is related inversely to the incidence of meningococcal disease, caused by Neisseria meningitidis. The organism sequenced was isolated during a school carriage survey in Northern Ireland in 1992 and has been the subject of a variety of laboratory and clinical studies. Four SMRT cells on a RSII machine by Pacific Biosystems were used to produce a complete, closed genome assembly. Sequence data were obtained for a total of 30,180,391 bases from 2621 reads and assembled using the HGAP algorithm. The assembly was corrected using short reads obtained from an Illumina HiSeq 2000instrument. This resulted in a 2,146,723 bp assembly with approximately 460 fold mean coverage depth and a GC ratio of 52.3%.

In meningococci, reduced penicillin susceptibility is associated with five specific mutations in the transpeptidase region of penicillin binding protein 2 (PBP2). We showed that the same set of mutations was present in 64 of 123 Neisseria lactamica strains obtained from a carriage study (MIC range: 0.125-2.0mg/L). The PBP2 encoding penA alleles in these strains were genetically similar to those found in intermediate resistant meningococci suggesting frequent interspecies genetic exchange. Fifty-six N. lactamica isolates with mostly lower penicillin MICs (range: 0.064-0.38mg/L) exhibited only three of the five mutations. The corresponding penA alleles were unique to N. lactamica and formed a distinct genetic clade. PenA alleles with no mutations on the other hand were unique to meningococci. Under penicillin selective pressure, genetic transformation of N. lactamica penA alleles in meningococci was only possible for alleles encoding five mutations, but not for those encoding three mutations; the transfer resulted in MICs comparable to those of meningococci harboring penA alleles that encoded PBP2 with five mutations, but considerably lower than those of the corresponding N. lactamica donor strains. Due to a transformation barrier the complete N. lactamica penA could not be transformed into N. meningitidis. In summary, penicillin MICs in N. lactamica were associated with the number of mutations in the transpeptidase region of PBP2. Evidence for interspecific genetic transfer was only observed for penA alleles associated with higher MICs, suggesting that alleles encoding only three mutations in the transpeptidase region are biologically not effective in N. meningitidis. Factors other than PBP2 seem to be responsible for the high levels of penicillin resistance in N. lactamica. A reduction of penicillin susceptibility in N. meningitidis by horizontal gene transfer from N. lactamica is unlikely to happen.

The three species Neisseria meningitidis , Neisseria gonorrheae , and Neisseria lactamica are often regarded as highly recombining bacteria. N . meningitidis has been considered a paradigmatic case of the “semiclonal model” or of “epidemic clonality,” demonstrating occasional bouts of clonal propagation in an otherwise recombining species. In this model, occasional clonality generates linkage disequilibrium in the short term. In the long run, however, the effects of clonality are countered by recombination. We show that many data are at odds with this proposal and that N . meningitidis fits the criteria that we have proposed for predominant clonal evolution (PCE). We point out that ( i ) the proposed way to distinguish epidemic clonality from PCE may be faulty and ( ii ) the evidence of deep phylogenies by microarrays and whole-genome sequencing is at odds with the predictions of the semiclonal model. Last, we revisit the species status of N . meningitidis , N . gonorrheae , and N . lactamica in the light of the PCE model.

Background Deprivation is associated with an increased risk of invasive Neisseria meningitidis disease, but little is known about the relationship between deprivation and asymptomatic carriage of N. meningitidis. This analysis was conducted to examine the relationship between meningococcal carriage and deprivation. Methods As part of a rapid meningococcal carriage prevalence study conducted in West Cumbria to investigate an apparent cluster of invasive meningococcal disease, data were collected on lifestyle and social factors, including area-level indicators of socioeconomic status, to identify factors associated with meningococcal carriage. Results In a multivariable log binomial regression model adjusted for age, lower socioeconomic status was significantly associated with higher prevalence of meningococcal carriage. A 1-unit increase in Index of Multiple Deprivation (2010) score was associated with a 1.7% increase in meningococcal carriage prevalence (95% confidence interval 0.3-3.0%). Age was the only significant predictor of carriage of Neisseria lactamica. Conclusions Living in a deprived area is associated with increased carriage of Group B meningococcus. Deprivation is an important factor to consider in the evaluation of the effectiveness and cost-effectiveness of the introduction of new meningococcal B vaccines and the development and implementation of immunization policies. Further work is required to understand whether deprivation has an effect on meningococcal carriage through other factors such as smoking.

We present the high quality, complete genome assembly of Neisseria lactamica Y92–1009 used to manufacture an outer membrane vesicle (OMV)-based vaccine, and a member of the Neisseria genus. The strain is available on request from the Public Health England Meningococcal Reference Unit. This Gram negative, dipplococcoid bacterium is an organism of worldwide clinical interest because human nasopharyngeal carriage is related inversely to the incidence of meningococcal disease, caused by Neisseria meningitidis. The organism sequenced was isolated during a school carriage survey in Northern Ireland in 1992 and has been the subject of a variety of laboratory and clinical studies. Four SMRT cells on a RSII machine by Pacific Biosystems were used to produce a complete, closed genome assembly. Sequence data were obtained for a total of 30,180,391 bases from 2621 reads and assembled using the HGAP algorithm. The assembly was corrected using short reads obtained from an Illumina HiSeq 2000instrument. This resulted in a 2,146,723 bp assembly with approximately 460 fold mean coverage depth and a GC ratio of 52.3%.

Neisseria meningitidis (Nm) and Neisseria lactamica (Nl) are commensal bacteria that live in the human nasopharynx, where they form microcolonies. In contrast to Nl, Nm occasionally causes blood and/or meningitis infection with often fatal consequences. Here, we studied interactions between neisserial strains during biofilm formation. Fluorescent strains were engineered and analyzed for growth in single- and dual-strain biofilms with confocal laser-scanning microscopy. Different strains of diverse Neisseria species formed microcolonies of different sizes and morphologies. Pair-wise combinations of two invasive Nm strains and one Nm carrier isolate showed that these strains can coexist in spite of the fact that they produce toxins to combat congeners. This lack of competition was even observed when the biofilms were formed under nutrient limitation and can be explained by the observation that the separate microcolonies within mixed biofilms are mostly lineage specific. However, these microcolonies showed different levels of interaction. The coexistence of two strains was also observed in mixed biofilms of Nm and Nl strains. Inactivation of the autotransporter NalP, which prevents the release of the heparin-binding antigen NHBA and the α-peptide of IgA protease from the cell surface, and/or the production of autotransporter AutA increased interactions between microcolonies, as evidenced by close contacts between microcolonies on the substratum. Qualitative and quantitative analysis revealed an altered spatial distribution of each strain in mixed biofilms with consequences for the biomass, biofilm architecture and bacterial viability depending on the synthesis of NalP and AutA, the expression of which is prone to phase variation. Being in a consortium resulted in some cases in commensalism and cooperative behavior, which promoted attachment to the substratum or increased survival, possibly as result of the shared use of the biofilm matrix. We hypothesize that Nm strains can cooperate during host colonization, but, possibly, the different capacities of the microcolonies of each strain to resist the host's defenses limits the long-term coexistence of strains in the host.

The poor immunogenicity of the meningococcal serogroup B (MenB) capsule has led to the development of vaccines targeting subcapsular antigens, in particular the immunodominant and diverse outer membrane porin, PorA. These vaccines are largely strain specific; however, they offer limited protection against the diverse MenB-associated diseases observed in many industrialized nations. To broaden the scope of its protection, the multicomponent vaccine (4CMenB) incorporates a PorA-containing outer membrane vesicle (OMV) alongside relatively conserved recombinant protein components, including factor H-binding protein (fHbp), Neisseria adhesin A (NadA), and neisserial heparin-binding antigen (NHBA). The expression of PorA is unique to meningococci ( Neisseria meningitidis ); however, many subcapsular antigens are shared with nonpathogenic members of the genus Neisseria that also inhabit the nasopharynx. These organisms may elicit cross-protective immunity against meningococci and/or occupy a niche that might otherwise accommodate pathogens. The potential for 4CMenB responses to impact such species (and vice versa) was investigated by determining the genetic distribution of the primary 4CMenB antigens among diverse members of the common childhood commensal, Neisseria lactamica . All the isolates possessed nhba but were devoid of fhbp and nadA . The nhba alleles were mainly distinct from but closely related to those observed among a representative panel of invasive MenB isolates from the same broad geographic region. We made similar findings for the immunogenic typing antigen, FetA, which constitutes a major part of the 4CMenB OMV. Thus, 4CMenB vaccine responses may impact or be impacted by nasopharyngeal carriage of commensal neisseriae. This highlights an area for further research and surveillance should the vaccine be routinely implemented.

Colonization of the nasopharynx by non-pathogenic Neisseria species, including N. lactamica, has been suggested to lead to the acquisition of natural immunity against Neisseria meningitidis in young children. The aim of this study was to identify a model complex of antigens and adjuvant for immunological preparation against N. meningitidis B, based on cross reactivity with N. lactamica outer membrane vesicles (OMV) antigens and the (DDA-BF) adjuvant. Complexes of 25 µg of OMV in 0.1 mM of DDA-BF were colloidally stable, exhibiting a mean diameter and charge optimal for antigen presentation. Immunogenicity tests for these complexes were performed in mice. A single dose of OMV/DDA-BF was sufficient to induce a (DTH) response, while the same result was achieved only after two doses of OMV/alum. In addition, to achieve total IgG levels that are similar to a single immunization with OMV/DDA-BF, it was necessary to give the mice a second dose of OMV/alum. Moreover, the antibodies induced from a single immunization with OMV/DDA-BF had an intermediate avidity, but antibodies with a similar avidity were only induced by OMV/alum after two immunizations. The use of this novel cationic adjuvant for the first time with a N. lactamica OMV preparation revealed good potential for future vaccine design.

The two-partner secretion (TPS) systems of Gram-negative bacteria consist of a largesecreted exoprotein (TpsA) and a transporter protein (TpsB) located in the outer membrane. TpsA targets TpsB for transport across the membrane via its ~30-kDa TPS domain locatedat its N terminus, and this domain is also the minimal secretory unit. Neisseria meningitidis genomes encode up to five TpsAs and two TpsBs. Sequence alignments of TPS domains suggested that these are organized into three systems, while there are two TpsBs, which raised questions on their system specificity. We show here that the TpsB2 transporter of Neisseria meningitidis is able to secrete all types of TPS domains encoded in N. meningitidisand the related species Neisseria lactamica but not domains of Haemophilus influenzae andPseudomonas aeruginosa. In contrast, the TpsB1 transporter seemed to be specific for its cognate N. meningitidis system and did not secrete the TPS domains of other meningococcalsystems. However, TpsB1 did secrete the TPS2b domain of N. lactamica, which is related tothe meningococcal TPS2 domains. Apparently, the secretion depends on specific sequences within the TPS domain rather than the overall TPS domain structure. © 2013, American Society for Microbiology.

The 2,272,351–base pair genome ofNeisseria meningitidisstrain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior ofN. meningitidiscan be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally,N. meningitidiscontains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Natural immunity againstNeisseria meningitidisis thought to develop following nasopharyngeal colonization with this bacterium or other microbes expressing cross-reactive antigens.Neisseria lactamicais a commensal of the upper respiratory tract which is often carried by infants and young children; epidemiological evidence indicates that colonization with this bacterium can elicit serum bactericidal activity (SBA) againstNeisseria meningitidis, the most validated correlate of protective immunity. Here we demonstrate experimentally that immunization of mice with liveN. lactamicaprotects animals against lethal meningococcal challenge and that some, but not all, strains ofN. lactamicaelicit detectable SBA in immunized animals regardless of the serogroup ofN. meningitidis. While it is unlikely that immunization with liveN. lactamicawill be implemented as a vaccine against meningococcal disease, understanding the basis for the induction of cross-protective immunity and SBA should be valuable in the design of subunit vaccines for the prevention of this important human infection.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited - © 2006 Snyder and Saunders; licensee BioMed Central Ltd. Background: Neisseria meningitidis causes the life-threatening diseases meningococcal meningitis and meningococcal septicemia. Neisseria gonorrhoeae is closely related to the meningococcus, but is the cause of the very different infection, gonorrhea. A number of genes have been implicated in the virulence of these related yet distinct pathogens, but the genes that define and differentiate the species and their behaviours have not been established. Further, a related species, Neisseria lactamica is not associated with either type of infection in normally healthy people, and lives as a harmless commensal. We have determined which of the genes so far identified in the genome sequences of the pathogens are also present in this non-pathogenic related species. Results: Thirteen unrelated strains of N. lactamica were investigated using comparative genome hybridization to the pan-Neisseria microarray-v2, which contains 2845 unique gene probes. The presence of 127 'virulence genes' was specifically addressed; of these 85 are present in N. lactamica. Of the remaining 42 'virulence genes' only 11 are present in all four of the sequenced pathogenic Neisseria. Conclusion: Assessment of the complete dataset revealed that the vast majority of genes present in the pathogens are also present in N. lactamica. Of the 1,473 probes to genes shared by all four pathogenic genome sequences, 1,373 hybridize to N. lactamica. These shared genes cannot include genes that are necessary and sufficient for the virulence of the pathogens, since N. lactamica does not share this behaviour. This provides an essential context for the interpretation of gene complement studies of the pathogens. This study is supported by the Wellcome Trust.

Background Neisseria meningitidis colonizes humans and transmits mainly by asymptomatic carriage. We sought to determine the prevalence and epidemiology of meningococcal carriage in Ethiopia prior to the introduction of MenAfriVac, a serogroup A meningococcal conjugate vaccine. Methods A cross-sectional meningococcal carriage study was conducted in Arba Minch, southern Ethiopia. A total of 7479 oropharyngeal samples were collected from 1 to 29 year old volunteers, between March and October, 2014. The swabs were cultured for N. meningitidis and Neisseria lactamica in Ethiopia. N. meningitidis isolates were confirmed and characterized by their serogroup, sequence type (ST) and PorA:FetA profile in Norway. Results Overall carriage prevalence was 6.6 %. There was no significant difference in overall carriage between male (6.7 %) and female (6.4 %) participants. Highest carriage prevalence (10.9 %) for females was found in the 15–19 years of age, while prevalence among males was highest (11.3 %) in the 20–24 age group. Non-groupable isolates dominated (76.4 %), followed by serogroups X (14.0 %) and W (5.9 %) isolates. No serogroup A was found. Most non-groupable isolates were ST-192. Serogroup W isolates were assigned to the ST-11 clonal complex, and serogroup X isolates to the ST-181 and ST-41/44 clonal complexes. Overall carriage prevalence of N. lactamica was 28.1 %. Carriage of N. meningitidis and N. lactamica varied depending on age and geographic area, but there was no association between carriage of the two species. Conclusions Epidemic strains of serogroups W and X were circulating in this area of Ethiopia. As no serogroup A was found among the carriage isolates the immediate impact of mass-vaccination with MenAfriVac on transmission of N. meningitidis in this population is expected to be marginal.