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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.

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 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.

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.

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.

OBJECTIVES: Escherichia coli is the leading cause of bacteraemia. In an era of emerging multi-drug-resistant strains, development of effective preventative strategies will be informed by knowledge of strain diversity associated with specific infective syndromes/patient groups. We hypothesised that the number of virulence factor (VF) genes amongst bacteraemia isolates from neutropaenic patients would be lower than isolates from immunocompetent patients.METHODS: Immunocompetent and neutropaenic adults with E. coli bacteraemia were recruited prospectively and the source of bacteraemia determined. VF gene profiles were established in silico following whole genome sequencing.RESULTS: Isolates from individual patients were monoclonal. Strains from immunocompetent patients with urinary tract infective foci (UTIF) harboured more VF genes (median number of VF genes 16, range 8-24) than isolates from both immunocompetent patients with non-UTIF (10, 2-22, p = 0.0058) and neutropaenic patients with unknown focus of infection (NPUFI) (8, 3-13, p < 0.0001). Number of VF genes (OR 1.21, 95% CIs 1.01-1.46, p = 0.039) and urinary catheter/recurrent urinary tract infection (OR 12.82, 95% CIs 1.24-132.65, p = 0.032) were independent predictors of bacteraemia secondary to UTIF vs. non-UTIF in immunocompetent patients. papA, papC, papE/F, papG, agn43, tia, iut, fyuA, kpsM and sat were significantly more prevalent amongst UTIF- vs non-UTIF-originating isolates amongst immunocompetent patients, while papC, papE/F, papG, agn43, tia, fyuA, hlyA, usp and clb were significantly more prevalent amongst UTIF- vs NPUFI-associated isolates.CONCLUSIONS: Bacteraemia-associated E. coli strains originating from UTIF have distinct VF gene profiles from strains associated with non-UTIF- and NPUFI. This diversity must be addressed in the design of future vaccines to ensure adequate coverage of strains responsible for site-specific disease.

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.

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Neisseria lactamica (Nlac) is a commensal of the human oropharynx. Colonisation with Nlac significantly reduces colonisation by N. meningitidis (Nmen). We hypothesised that cross-reactive adaptive cellular immune responses were responsible for this effect. 31 volunteers were inoculated intra-nasally with 105 colony-forming units of Nlac or vehicle control. Nlac/Nmen colonisation status was assessed at baseline and 7-, 14- and 28-days post-inoculation. Nlac/Nmen-specific plasma cells (PCs)/memory B-cells were quantified in PBMCs at all time points using ELISpot assays. Amongst Nlac-colonised volunteers (n=17), Nlac-specific PC frequencies/2×105 PBMCs increased significantly. Median baseline vs. post-colonisation peak Nlac-specific PC frequencies were 0 (range 0–0.5) vs. 2 (0–31) for IgM, 0 (0–1) vs. 5 (0–20.5) for IgA, and 0 (0–1) vs. 3 (0–27) for IgG (all p These data suggest that the previously observed protective effect of Nlac on Nmen colonisation may have an immunological basis. We plan to assess the frequency/effector phenotype of Nlac-specific CD4+ memory T-cells following Nlac colonisation and hypothesise that cross-reactive CD4+ memory T-cells directed towards Nmen will be Th17.

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 nmb1612 (NEIS1533) gene encoding the ~27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)3 and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre4) in a human serum bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same protein was killed (median titres of 16-64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015,91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines.

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%.

open access article Non-typeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that plays a major role in a number of respiratory tract infections, including otitis media, cystic fibrosis and chronic obstructive pulmonary disease. Biofilm formation has been implicated in both NTHi colonization and disease, and is responsible for the increased tolerance of this pathogen towards antibiotic treatment. Targeting metabolic pathways that are important in NTHi biofilm formation represents a potential strategy to combat this antibiotic recalcitrance. A previous investigation demonstrated increased expression of a putative D-methionine uptake protein following exposure of NTHi biofilms to the ubiquitous signalling molecule, nitric oxide. We therefore hypothesized that treatment with exogenous D-methionine would impact on NTHi biofilm formation and increase antibiotic sensitivity. Treatment of NTHi during the process of biofilm formation resulted in a reduction in biofilm viability, increased biomass, changes in the overall biofilm architecture and the adoption of an amorphous cellular morphology. Quantitative proteomic analyses identified 124 proteins that were differentially expressed following D-methionine treatment, of which 51 (41 %) were involved in metabolic and transport processes. Nine proteins involved in peptidoglycan synthesis and cell division showed significantly increased expression. Furthermore, D-methionine treatment augmented the efficacy of azithromycin treatment and highlighted the potential of D-methionine as an adjunctive therapeutic approach for NTHi biofilm-associated infections.

The formation and degradation of S-nitrosothiols (SNOs) are important mechanisms of post-translational protein modification and appear to be ubiquitous in biology. These processes play well-characterized roles in eukaryotic cells, including a variety of pathologies and in relation to chronic conditions. We know little of the roles of these processes in pathogenic and other bacteria.It is clear, mostly from growth and transcriptional studies, that bacteria sense and respond to exogenous SNOs. These responses are phenotypically and mechanistically distinct from the responses of bacteria to nitric oxide (NO) and NO-releasing agents, as well as peroxynitrite. Small SNOs, such as S-nitrosoglutathione (GSNO), are accumulated by bacteria with the result that intracellular S-nitrosoproteins (the 'S-nitrosoproteome') are detectable. Recently, conditions for endogenous SNO formation in enterobacteria have been described.The propensity of intracellular proteins to form SNOs is presumably constrained by the same rules of selectivity that have been discovered in eukaryotic systems, but is also influenced by uniquely bacterial NO detoxification systems, exemplified by the flavohemoglobin Hmp in enterobacteria and NO reductase of meningococci. Furthermore, the bacterial expression of such proteins impacts upon the formation of SNOs in mammalian hosts.The impairment during bacterial infections of specific SNO events in the mammalian host is of considerable interest in the context of proteins involved in innate immunity and intracellular signalling. In bacteria, numerous mechanisms of S-nitrosothiol degradation have been reported (e.g., GSNO reductase); others are thought to operate, based on consideration of their mammalian counterparts. The nitrosothiols of bacteria and particularly of pathogens warrant more intensive investigation.

S-nitrosylation is an important mediator of multiple nitric oxide-dependent biological processes, including eukaryotic cellular events such as macrophage apoptosis and proinflammatory signaling. Many pathogenic bacteria possess NO detoxification mechanisms, such as the nitric oxide reductase (NorB) of Neisseria meningitidis and the flavohemoglobins (Hmp) of Salmonella enterica and Escherichia coli, which serve to protect the microorganism from nitrosative stress within the intracellular environment. In this study, we demonstrate that expression of meningococcal NorB increases the rate at which low-molecular-weight S-nitrosothiol (SNO) decomposes in vitro. To determine whether this effect occurs in cells during infection by bacteria, we induced SNO formation in murine macrophages by activation with lipopolysaccharide and γ-interferon and observed a reduced abundance of SNO during coincubation with N. meningitidis, S. enterica, or E. coli. In each case, this effect was shown to be dependent on bacterial NO detoxification genes, which act to prevent SNO formation through the removal of NO. This may represent a novel mechanism of host cell injury by bacteria.—Laver, J. R., Stevanin, T. M., Messenger, S. L., Dehn Lunn, A., Lee, M. E., Moir, J. W. B., Poole, R. K., Read, R. C. Bacterial nitric oxide detoxification prevents host cell S-nitrosothiol formation: a novel mechanism of bacterial pathogenesis.

The exclusive reservoir of the genus Neisseria is the human. Of the broad range of species that comprise the Neisseria, only two are frequently pathogenic, and only one of those is a resident of the nasopharynx. Although Neisseria meningitidis can cause severe disease if it invades the bloodstream, the vast majority of interactions between humans and Neisseria are benign, with the bacteria inhabiting its mucosal niche as a non-invasive commensal. Understandably, with the exception of Neisseria gonorrhoeae, which preferentially colonises the urogenital tract, the neisseriae are extremely well adapted to survival in the human nasopharynx, their sole biological niche. The purpose of this review is to provide an overview of the molecular mechanisms evolved by Neisseria to facilitate colonisation and survival within the nasopharynx, focussing on N. meningitidis. The organism has adapted to survive in aerosolised transmission and to attach to mucosal surfaces. It then has to replicate in a nutrition-poor environment and resist immune and competitive pressure within a polymicrobial complex. Temperature and relative gas concentrations (nitric oxide and oxygen) are likely to be potent initial signals of arrival within the nasopharyngeal environment, and this review will focus on how N. meningitidis responds to these to increase the likelihood of its survival.

The exclusive reservoir of the genus Neisseria is the human. Of the broad range of species that comprise the Neisseria, only two are frequently pathogenic, and only one of those is a resident of the nasopharynx. Although Neisseria meningitidis can cause severe disease if it invades the bloodstream, the vast majority of interactions between humans and Neisseria are benign, with the bacteria inhabiting its mucosal niche as a non-invasive commensal. Understandably, with the exception of Neisseria gonorrhoeae, which preferentially colonises the urogenital tract, the neisseriae are extremely well adapted to survival in the human nasopharynx, their sole biological niche. The purpose of this review is to provide an overview of the molecular mechanisms evolved by Neisseria to facilitate colonisation and survival within the nasopharynx, focussing on N. meningitidis. The organism has adapted to survive in aerosolised transmission and to attach to mucosal surfaces. It then has to replicate in a nutrition-poor environment and resist immune and competitive pressure within a polymicrobial complex. Temperature and relative gas concentrations (nitric oxide and oxygen) are likely to be potent initial signals of arrival within the nasopharyngeal environment, and this review will focus on how N. meningitidis responds to these to increase the likelihood of its survival.

Nitric oxide (NO) is a ubiquitous gas with potent biological effects, including vasodilation, neuronal signaling, and antimicrobial activity. NO is a free radical and can readily react with other molecules, in particular, iron centers and oxygen. At physiological concentrations in aqueous solutions, even in the presence of oxygen, NO is reasonably stable. Under these conditions, NO is oxidized almost exclusively to nitrite (NO2-). In cell lysates and tissue extracts with iron-containing proteins, however, NO is postulated to have a very short half-life, with the major oxidation product being nitrate (NO3-). In mammalian cells, NO is generated via the action of the NO synthases (NOS), of which there are three known isotypes. NO can also be generated from the chemical decomposition of S-nitrosothiols, and there is some indication that naturally occurring S-nitrosothiols, such as S-nitrosoalbumin, may be natural reservoirs of NO in vivo. Here we describe a methodology to measure variations in NO in liquid samples using chemiluminescence. The protocols described allow us to distinguish between various products of NO chemistry, thus providing a sensitive method of measurement of NO concentration within a sample. They also allow us to distinguish between the various products that may be generated when NO reacts with molecules in complex biological samples such as cell lysates and supernatants.

Nitric oxide (NO) is a ubiquitous gas with potent biological effects, including vasodilation, neuronal signaling, and antimicrobial activity. NO is a free radical and can readily react with other molecules, in particular, iron centers and oxygen. At physiological concentrations in aqueous solutions, even in the presence of oxygen, NO is reasonably stable. Under these conditions, NO is oxidized almost exclusively to nitrite (NO2-). In cell lysates and tissue extracts with iron-containing proteins, however, NO is postulated to have a very short half-life, with the major oxidation product being nitrate (NO3-). In mammalian cells, NO is generated via the action of the NO synthases (NOS), of which there are three known isotypes. NO can also be generated from the chemical decomposition of S-nitrosothiols, and there is some indication that naturally occurring S-nitrosothiols, such as S-nitrosoalbumin, may be natural reservoirs of NO in vivo. Here we describe a methodology to measure variations in NO in liquid samples using chemiluminescence. The protocols described allow us to distinguish between various products of NO chemistry, thus providing a sensitive method of measurement of NO concentration within a sample. They also allow us to distinguish between the various products that may be generated when NO reacts with molecules in complex biological samples such as cell lysates and supernatants.

Macrophages produce nitric oxide (NO) via the inducible nitric oxide synthase as part of a successful response to infection. The gene norB of Neisseria meningitidis encodes a NO reductase which enables utilization and consumption of NO during microaerobic respiration and confers resistance to nitrosative stress-related killing by human monocyte-derived macrophages (MDM). In this study we confirmed that NO regulates cytokine and chemokine release by resting MDM: accumulation of TNF-alpha, IL-12, IL-10, CCL5 (RANTES) and CXCL8 (IL-8) in MDM supernatants was significantly modified by the NO-donor S-nitroso-N-penicillamine (SNAP). Using a protein array, infection of MDM with N. meningitidis was shown to be associated with secretion of a wide range of cytokines and chemokines. To test whether NO metabolism by N. meningitidis modifies release of NO-regulated cytokines, we infected MDM with wild-type organisms and an isogenic norB strain. Resulting expression of the cytokines TNF-alpha and IL-12, and the chemokine CXCL8 was increased and production of the cytokine IL-10 and the chemokine CCL5 was decreased in norB-infected MDM, in comparison to wild-type. Addition of SNAP to cultures infected with wild-type mimicked the effect observed in cultures infected with the norB mutant. In conclusion, NorB-catalysed removal of NO modifies cellular release of NO-regulated cytokines and chemokines.

Rationale and hypothesis Increased bacterial load in meningococcal sepsis is positively correlated with severe disease and increased concentrations of circulating nitric oxide (NO) metabolites. As a vasorelaxant, NO binds to the soluble guanylyl cyclase (sGC), found in abundance in vascular smooth muscle and activates cGMP, a secondary messenger important for vasodilation, platelet inhibition etc. We previously showed that NO detoxification mechanisms of N. meningitidis reduce abundance of NO species in mammalian cells, so we tested the hypothesis that this activity alters cellular cGMP activity. Objectives To examine the effect of bacterial NO metabolism on cGMP produced by Human macrophages (MDM) and endothelial (HMEC-1) cells. Methodology Human MDMs and HMEC-1 cells were exposed to NO donor spermine NONOate, together with the phosphodiesterase inhibitor IBMX and a soluble guanyl cyclase sensitiser BAY412275. Cells were infected with wild type Neisseria meningitidis (serogroup B strain MC58), a mutant strain for putative Arginine Decarboxylase (ΔNMB0468) and an isogenic mutant unable to detoxify NO (ΔnorB). After two hours, cells were lysed and cGMP was measured by ELISA. Findings In the presence of spermine NONOate, IBMX and BAY 412275, the level of cGMP was detected at 5.69 pmol/mg protein in human MDMs; without these agonists no cGMP was detected (p

Nitric oxide (NO) has a broad spectrum of signalling and regulatory functions and multiple molecular targets. Recently, the intrabacterial toxicity of NO and mechanisms for NO resistance have been intensively investigated. Here we report for the first time that NO elicits release of zinc from a bacterial protein. Using the zinc-responsive expression of zntA (encoding a Zn-exporting P-type ATPase) fused to lacZ, i.e. Phi(zntA-lacZ), to monitor intracellular zinc, and SmtA (the Synechococcus metallothionein) as zinc store, we have shown that the NO donors NOC-5 and NOC-7 elicit zinc ejection. No increase in Phi(zntA-lacZ) activity was observed in a zntR mutant, indicating the specificity of the zntA promoter response to zinc ions.

Rationale and hypothesis Meningococcal septicaemia is characterised by high bacterial titres in blood and an increased production of circulating nitric oxide metabolites, including nitrates NO 3 - , nitrites NO 2 - and S-nitrosothiol [SNO]. The formation of SNO, termed S-nitrosylation, is an important form of post-translational protein modification akin to phosphorylation. Previously, bacterial NO detoxification was demonstrated to reduce the concentration of host-cell SNO. We hypothesise that bacterial NO detoxification during sepsis interferes with host NO homeostasis within the liver, a vital organ in physiological response to infection, and contributes toward pathogenesis. Objectives 1. To establish an in vivo murine model of acute fulminant meningococcal sepsis. 2. To study the link between bacterial NO detoxification and circulating NO metabolites in murine liver. Methodology Female C57 Bl/6 mice were infected by intraperitoneal injection of wild type Neisseria meningitidis (serogroup B strain MC58) or an isogenic mutant unable to detoxify NO (ΔnorB). Eight hours after infection, livers were extracted and lysates were subjected to ozone-based chemiluminescence-based measurement of multiple NO metabolites. We obtained reproducibly high bacterial titres both in blood and liver lysates. Findings 1. Increased bacterial burden in liver lysates is positively and significantly correlated with hepatic nitrite concentrations NO 2 - (Spearman’s rank correlation, r = 0.5682, p = 0.0020, n = 9), negatively and significantly correlated with the hepatic concentration of all NO [NOx] deriviatives (r = −0.4390, p = 0.0220, n = 9) 2. In an acute fulminant meningococcal sepsis model, the production of NO metabolites was found to be unperturbed by the presence of bacterial NO detoxification machinery (p = 0.2704, n = 9).