Premature neonatal gut microbial community patterns supporting an epithelial TLR-mediated pathway for necrotizing enterocolitis.

BACKGROUND: Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data. RESULTS: We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation. CONCLUSIONS: The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.

A Rare Mutation in SPLUNC1 Affects Bacterial Adherence and Invasion in Meningococcal Disease.

BACKGROUND: Neisseria meningitidis (Nm) is a nasopharyngeal commensal carried by healthy individuals. However, invasive infections occurs in a minority of individuals, with devastating consequences. There is evidence that common polymorphisms are associated with invasive meningococcal disease (IMD), but the contributions of rare variants other than those in the complement system have not been determined. METHODS: We identified familial cases of IMD in the UK meningococcal disease study and the European Union Life-Threatening Infectious Disease Study. Candidate genetic variants were identified by whole-exome sequencing of 2 patients with familial IMD. Candidate variants were further validated by in vitro assays. RESULTS: Exomes of 2 siblings with IMD identified a novel heterozygous missense mutation in BPIFA1/SPLUNC1. Sequencing of 186 other nonfamilial cases identified another unrelated IMD patient with the same mutation. SPLUNC1 is an innate immune defense protein expressed in the nasopharyngeal epithelia; however, its role in invasive infections is unknown. In vitro assays demonstrated that recombinant SPLUNC1 protein inhibits biofilm formation by Nm, and impedes Nm adhesion and invasion of human airway cells. The dominant negative mutant recombinant SPLUNC1 (p.G22E) showed reduced antibiofilm activity, increased meningococcal adhesion, and increased invasion of cells, compared with wild-type SPLUNC1. CONCLUSIONS: A mutation in SPLUNC1 affecting mucosal attachment, biofilm formation, and invasion of mucosal epithelial cells is a new genetic cause of meningococcal disease.

Inactivation of NMB0419, Encoding a Sel1-Like Repeat (SLR) Protein, in Neisseria meningitidis Is Associated with Differential Expression of Genes Belonging to the Fur Regulon and Reduced Intraepithelial Replication.

Neisseria meningitidis is a commensal microbe that colonizes the human nasopharynx but occasionally invades the bloodstream to cause life-threatening infection. N. meningitidis MC58 NMB0419 encodes a Sel1-like repeat (SLR)-containing protein, previously implicated in invasion of epithelial cells. A gene-regulatory function was revealed in Escherichia coli expressing plasmid-borne NMB0419 and showing significantly increased epithelial adherence compared to the wild type, due to increased expression of mannose-sensitive type 1 pili. While a meningococcal NMB0419 mutant did not have altered epithelial adherence, in a transcriptome-wide comparison of the wild type and an NMB0419 mutant, a large proportion of genes differentially regulated in the mutant were involved in iron acquisition and metabolism. Fifty-one percent and 38% of genes, respectively, up- and downregulated in the NMB0419 mutant had previously been identified as being induced and repressed by meningococcal Fur. An in vitro growth defect of the NMB0419 mutant under iron restriction was consistent with the downregulation of tbpAB and hmbR, while an intraepithelial replication defect was consistent with the downregulation of tonB, exbB, and exbD, based on a known phenotype of a meningococcal tonB mutant. Disruption of the N-terminal NMB0419 signal peptide, predicted to export the protein beyond the cytoplasmic membrane, resulted in loss of functional traits in N. meningitidis and E. coli Our study indicates that the expression of NMB0419 is associated with transcriptional changes counterbalancing the regulatory function of Fur, offering a new perspective on regulatory mechanisms involved in meningococcal interaction with epithelial cells, and suggests new insights into the roles of SLR-containing genes in other bacteria.

Dysbiosis anticipating necrotizing enterocolitis in very premature infants.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease of premature infants speculatively associated with infection. Suspected NEC can be indistinguishable from sepsis, and in established cases an infant may die within hours of diagnosis. Present treatment is supportive. A means of presymptomatic diagnosis is urgently needed. We aimed to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants. METHODS: Fecal samples and clinical data were collected from a 2-year cohort of 369 premature neonates. Next-generation sequencing of 16S ribosomal RNA gene regions was used to characterize the microbiota of prediagnosis fecal samples from 12 neonates with NEC, 8 with suspected NEC, and 44 controls. Logistic regression was used to determine clinical characteristics and operational taxonomic units (OTUs) discriminating cases from controls. Samples were cultured and isolates identified using matrix-assisted laser desorption/ionization-time of flight. Clostridial isolates were typed and toxin genes detected. RESULTS: A clostridial OTU was overabundant in prediagnosis samples from infants with established NEC (P = .006). Culture confirmed the presence of Clostridium perfringens type A. Fluorescent amplified fragment-length polymorphism typing established that no isolates were identical. Prediagnosis samples from NEC infants not carrying profuse C. perfringens revealed an overabundance of a Klebsiella OTU (P = .049). Prolonged continuous positive airway pressure (CPAP) therapy with supplemental oxygen was also associated with increased NEC risk. CONCLUSIONS: Two fecal microbiota signatures (Clostridium and Klebsiella OTUs) and need for prolonged CPAP oxygen signal increased risk of NEC in presymptomatic infants. These biomarkers will assist development of a screening tool to allow very early diagnosis of NEC. Clinical Trials Registration. NCT01102738.

Transcriptional profiling of Neisseria meningitidis interacting with human epithelial cells in a long-term in vitro colonization model.

Neisseria meningitidis is a commensal of humans that can colonize the nasopharyngeal epithelium for weeks to months and occasionally invades to cause life-threatening septicemia and meningitis. Comparatively little is known about meningococcal gene expression during colonization beyond those first few hours. In this study, the transcriptome of adherent serogroup B N. meningitidis strain MC58 was determined at intervals during prolonged cocultivation with confluent monolayers of the human respiratory epithelial cell line 16HBE14. At different time points up to 21 days, 7 to 14% of the meningococcal genome was found to be differentially regulated. The transcriptome of adherent meningococci obtained after 4 h of coculture was markedly different from that obtained after prolonged cocultivation (24 h, 96 h, and 21 days). Genes persistently upregulated during prolonged cocultivation included three genes (hfq, misR/phoP, and lrp) encoding global regulatory proteins. Many genes encoding known adhesins involved in epithelial adherence were upregulated, including those of a novel locus (spanning NMB0342 to NMB0348 [NMB0342-NMB0348]) encoding epithelial cell-adhesive function. Sixteen genes (including porA, porB, rmpM, and fbpA) encoding proteins previously identified by their immunoreactivity to sera from individuals colonized long term with serogroup B meningococci were also upregulated during prolonged cocultivation, indicating that our system models growth conditions in vivo during the commensal state. Surface-expressed proteins downregulated in the nasopharynx (and thus less subject to selection pressure) but upregulated in the bloodstream (and thus vulnerable to antibody-mediated bactericidal activity) should be interesting candidate vaccine antigens, and in this study, three new proteins fulfilling these criteria have been identified: NMB0497, NMB0866, and NMB1882.

Regulation of pga operon expression and biofilm formation in Actinobacillus pleuropneumoniae by sigmaE and H-NS.

Clinical isolates of the porcine pathogen Actinobacillus pleuropneumoniae often form adherent colonies on agar plates due to expression of an operon, pgaABCD, encoding a poly-beta-1,6-N-acetyl-D-glucosamine (PGA) extracellular matrix. The adherent colony phenotype, which correlates with the ability to form biofilms on the surfaces of polystyrene plates, is lost following serial passage in broth culture, and repeated passage of the nonadherent variants on solid media does not result in reversion to the adherent colony phenotype. In order to investigate the regulation of PGA expression and biofilm formation in A. pleuropneumoniae, we screened a bank of transposon mutants of the nonadherent serovar 1 strain S4074(T) and identified mutations in two genes, rseA and hns, which resulted in the formation of the adherent colony phenotype. In other bacteria, including the Enterobacteriaceae, H-NS acts as a global gene regulator, and RseA is a negative regulator of the extracytoplasmic stress response sigma factor sigma(E). Transcription profiling of A. pleuropneumoniae rseA and hns mutants revealed that both sigma(E) and H-NS independently regulate expression of the pga operon. Transcription of the pga operon is initiated from a sigma(E) promoter site in the absence of H-NS, and upregulation of sigma(E) is sufficient to displace H-NS, allowing transcription to proceed. In A. pleuropneumoniae, H-NS does not act as a global gene regulator but rather specifically regulates biofilm formation via repression of the pga operon. Positive regulation of the pga operon by sigma(E) indicates that biofilm formation is part of the extracytoplasmic stress response in A. pleuropneumoniae.

A proteomics-based method for identifying antigens within immune complexes.

A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.

Evidence for capsule switching between carried and disease-causing Neisseria meningitidis strains.

Changing antigenic structure such as with capsule polysaccharide is a common strategy for bacterial pathogens to evade a host immune system. The recent emergence of an invasive W:2a:P1.7-2,4 sequence type 11 (ST-11) strain of Neisseria meningitidis in New Zealand, an uncommon serogroup/serotype in New Zealand disease cases, was investigated for its genetic origins. Molecular typing of 107 meningococcal isolates with similar serotyping characteristics was undertaken to determine genetic relationships. Results indicated that the W:2a:P1.7-2,4 strain had emerged via capsule switching from a group C strain (C:2a:P1.7-2,4). Neither the upstream nor downstream sites of recombination could be elucidated, but sequence analysis demonstrated that at least 45 kb of DNA was involved in the recombination, including the entire capsule gene cluster. The oatWY gene carried by the W:2a:P1.7-2,4 strain contained the insertion sequence element IS1301, one of five variants of oatWY found in group W135 strains belonging to the carriage-associated ST-22 clonal complex. This suggested that the origin of the capsule genes carried by the invasive W:2a:P1.7-2,4 strain is carriage associated. These results provide novel evidence for the long-standing dogma that disease-associated strains acquire antigenic structure from carriage-associated strains. Moreover, the capsule switch described here has arisen from the exchange of the entire capsule locus.

Antibiotic resistance potential of the healthy preterm infant gut microbiome.

BACKGROUND: Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected. RESULTS: Dominant clinically important species identified within the microbiomes included C. perfringens, K. pneumoniae and members of the Staphylococci and Enterobacter genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of mecA and high levels of Staphylococci within some infants. We were able to demonstrate that in a third of the infants the S. aureus identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples. CONCLUSIONS: We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.

[Automated mapping of urban forests' disturbance and recovery in Nanjing, China].

Using Landsat TM/ETM dense time series observations spanning from 1987 to 2011, taking Laoshan forest farm and Purple Mountain as the research objects, the landsat ecosystem disturbance adaptive processing system (Ledaps) algorithm was used to generate surface reflectance datasets, which were fed to the vegetation change tracker model (VCT) model to derive urban forest disturbance and recovery products over Nanjing, followed by an intensive validation of the products. The results showed that there was a relatively high spatial agreement for forest disturbance products mapped by VCT, ranging from 65.4% to 95.0%. There was an apparent fluctuating forest disturbance and recovery rate over time, and the change trend of forest disturbance occurring at the two sites was roughly similar, but forest recovery was obviously different. Forest coverage in Purple Mountain was less than that in Laoshan forest farm, but the forest disturbance and recovery rates in Laoshan forest farm were larger than those in Purple Mountain.

Latitude in sample handling and storage for infant faecal microbiota studies: the elephant in the room?

BACKGROUND: In this manuscript, we investigate the "stones best left unturned" of sample storage and preparation and their implications for the next-generation sequencing of infant faecal microbial communities by the 16S ribosomal ribonucleic acid (rRNA) gene. We present a number of experiments that investigate the potential effects of often overlooked methodology factors, establishing a "normal" degree of variation expected between replica sequenced samples. Sources of excess variation are then identified, as measured by observation of alpha diversity, taxonomic group counts and beta diversity magnitudes between microbial communities. RESULTS: Extraction of DNA from samples on different dates, by different people and even using varied sample weights results in little significant difference in downstream sequencing data. A key assumption in many studies is the stability of samples stored long term at -80 °C prior to extraction. After 2 years, we see relatively few changes: increased abundances of lactobacilli and bacilli and a reduction in the overall OTU count. Where samples cannot be frozen, we find that storing samples at room temperature does lead to significant changes in the microbial community after 2 days. Mailing of samples during this time period (a common form of sample collection from outpatients for example) does not lead to any additional variation. CONCLUSIONS: Important methodological standards can be drawn from these results; painstakingly created archives of infant faecal samples stored at -80 °C are still largely representative of the original community and varying factors in DNA extraction methodology have comparatively little effect on overall results. Samples taken should ideally be either frozen at -80 °C or extracted within 2 days if stored at room temperature, with mail samples being mailed on the day of collection.

Late-Onset Bloodstream Infection and Perturbed Maturation of the Gastrointestinal Microbiota in Premature Infants.

BACKGROUND: Late-onset bloodstream infection (LO-BSI) is a common complication of prematurity, and lack of timely diagnosis and treatment can have life-threatening consequences. We sought to identify clinical characteristics and microbial signatures in the gastrointestinal microbiota preceding diagnosis of LO-BSI in premature infants. METHOD: Daily faecal samples and clinical data were collected over two years from 369 premature neonates (<32 weeks gestation). We analysed samples from 22 neonates who developed LO-BSI and 44 matched control infants. Next-generation sequencing of 16S rRNA gene regions amplified by PCR from total faecal DNA was used to characterise the microbiota of faecal samples preceding diagnosis from infants with LO-BSI and controls. Culture of selected samples was undertaken, and bacterial isolates identified using MALDI-TOF. Antibiograms from bloodstream and faecal isolates were compared to explore strain similarity. RESULTS: From the week prior to diagnosis, infants with LO-BSI had higher proportions of faecal aerobes/facultative anaerobes compared to controls. Risk factors for LO-BSI were identified by multivariate analysis. Enterobacteriaceal sepsis was associated with antecedent multiple lines, low birth weight and a faecal microbiota with prominent Enterobacteriaceae. Staphylococcal sepsis was associated with Staphylococcus OTU faecal over-abundance, and the number of days prior to diagnosis of mechanical ventilation and of the presence of centrally-placed lines. In 12 cases, the antibiogram of the bloodstream isolate matched that of a component of the faecal microbiota in the sample collected closest to diagnosis. CONCLUSIONS: The gastrointestinal tract is an important reservoir for LO-BSI organisms, pathogens translocating across the epithelial barrier. LO-BSI is associated with an aberrant microbiota, with abundant staphylococci and Enterobacteriaceae and a failure to mature towards predominance of obligate anaerobes.

Increased transcription of a potential sigma factor regulatory gene Rv1364c in Mycobacterium bovis BCG while residing in macrophages indicates use of alternative promoters.

Alternative sigma factors are key global regulators that coordinate bacterial responses to environmental changes necessary for adaptation and survival. In turn these sigma factors are controlled by regulators such as anti-sigma and anti-anti-sigma factors. In this report, using a cDNA-total RNA subtractive hybridisation strategy that we have developed previously, we identified increased transcription of a potential sigma factor regulatory gene, Rv1364c, in Mycobacterium bovis BCG upon phagocytosis by macrophages and this was confirmed by Northern blot analysis. Primer extension analysis revealed the use of alternative promotors, P1 and P2, and that the increased expression inside macrophages coincided with promoter switching from P2 to P1. Rv1364c (653 amino acids), originally annotated as RsbU, contains structural domains homologous to the PAS redox sensor, the protein phosphatases anti-anti-sigma factor RsbU/SpoIIE, the protein kinase anti-sigma factor RsbW/SpoIIAB and the anti-anti-sigma factor RsbV/SpoIIAA found in other bacteria. These findings have important implications for understanding coordination of the expression of sigma factors under intra-macrophage conditions. Other potentially differentially expressed genes, including genes for fatty acid metabolism, membrane transportors, heat shock proteins, potential sigma factors and energy metabolic pathways are also listed and their biological significance discussed.

[Assessing dynamic patterns of forest fragmentation based on a landscape mosaic indicator: a case study of Oregon State, USA].

Effectively assessing landscape pattern characteristics and predicting their dynamics have been a basic prerequisite for more reasonably regulating and managing forest landscape, and maintaining landscape security patterns. In this study, based on three U. S. National Land Cover Databases (1992, 2001 and 2006), the landscape mosaic indicator in combination with the Markov model was adopted to analyze forest fragmentation patterns and changes in the characteristics of spatial interactions between forests and other land use types in Oregon State, USA. The results showed that conversion from the development-dominated type D to the single development type DD in landscape mosaic model had the highest transition probability 0.319, indicating that urbanization has been the major force responsible for the change of regional landscape patterns. In the forest security model, the highest rates of forest loss occurred in agriculture and the developed landscape mosaic type (ad), showing that in the development and agriculture dominated landscapes, encroaching upon forests was at the highest likelihood. The areal percentage of forest over the total study area was less than 50% when reaching a steady-state distribution, with an accelerating rate of forest fragmentation, and the landscape spatial distribution tended to be a mixed landscape pattern. The Kappa coefficient between the simulated values and the observed values from the 2006 landscape mosaic model was estimated at 0.82, indicating this model had a high precision. However, the accuracy of the forest security model was poor, with a Kappa coefficient of 0.21.

Transcriptional profiling of serogroup B Neisseria meningitidis growing in human blood: an approach to vaccine antigen discovery.

Neisseria meningitidis is a nasopharyngeal commensal of humans which occasionally invades the blood to cause septicaemia. The transcriptome of N. meningitidis strain MC58 grown in human blood for up to 4 hours was determined and around 10% of the genome was found to be differentially regulated. The nuo, pet and atp operons, involved in energy metabolism, were up-regulated, while many house-keeping genes were down-regulated. Genes encoding protein chaperones and proteases, involved in the stress response; complement resistant genes encoding enzymes for LOS sialylation and biosynthesis; and fHbp (NMB1870) and nspA (NMB0663), encoding vaccine candidates, were all up-regulated. Genes for glutamate uptake and metabolism, and biosynthesis of purine and pyrimidine were also up-regulated. Blood grown meningococci are under stress and undergo a metabolic adaptation and energy conservation strategy. The localisation of four putative outer membrane proteins encoded by genes found to be up-regulated in blood was assessed by FACS using polyclonal mouse antisera, and one (NMB0390) showed evidence of surface expression, supporting its vaccine candidacy.

Improved detection of bifidobacteria with optimised 16S rRNA-gene based pyrosequencing.

The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, 'bifidobacteria-optimised' universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.

Genome wide expression profiling reveals suppression of host defence responses during colonisation by Neisseria meningitides but not N. lactamica.

Both Neisseria meningitidis and the closely related bacterium Neisseria lactamica colonise human nasopharyngeal mucosal surface, but only N. meningitidis invades the bloodstream to cause potentially life-threatening meningitis and septicaemia. We have hypothesised that the two neisserial species differentially modulate host respiratory epithelial cell gene expression reflecting their disease potential. Confluent monolayers of 16HBE14 human bronchial epithelial cells were exposed to live and/or dead N. meningitidis (including capsule and pili mutants) and N. lactamica, and their transcriptomes were compared using whole genome microarrays. Changes in expression of selected genes were subsequently validated using Q-RT-PCR and ELISAs. Live N. meningitidis and N. lactamica induced genes involved in host energy production processes suggesting that both bacterial species utilise host resources. N. meningitidis infection was associated with down-regulation of host defence genes. N. lactamica, relative to N. meningitidis, initiates up-regulation of proinflammatory genes. Bacterial secreted proteins alone induced some of the changes observed. The results suggest N. meningitidis and N. lactamica differentially regulate host respiratory epithelial cell gene expression through colonisation and/or protein secretion, and that this may contribute to subsequent clinical outcomes associated with these bacteria.

Meningococcal biofilm growth on an abiotic surface - a model for epithelial colonization?

Neisseria meningitidis colonizes the human nasopharynx asymptomatically, often for prolonged periods, but occasionally invades from this site to cause life-threatening infection. In the nasopharynx aggregated organisms are closely attached to the epithelial surface, in a state in which the expression of components of the bacterial envelope differs significantly from that found in organisms multiplying exponentially in liquid phase culture or in the blood. We and others have hypothesized that here they are in the biofilm state, and to explore this we have investigated biofilm formation by the serogroup B strain MC58 on an abiotic surface, in a sorbarod system. Transcriptional changes were analysed, focusing on alteration in gene expression relevant to polysaccharide capsulation, lipooligosaccharide and outer-membrane protein synthesis - all phenotypes of importance in epithelial colonization. We report downregulation of genes controlling capsulation and the production of core oligosaccharide, and upregulation of genes encoding a range of outer-membrane components, reflecting phenotypic changes that have been established to occur in the colonizing state. A limited comparison with organisms recovered from an extended period of co-cultivation with epithelial cells suggests that this model system may better mirror natural colonization than do short-term meningococcal/epithelial cell co-cultivation systems. Modelling prolonged meningococcal colonization with a sorbarod system offers insight into gene expression during this important, but experimentally relatively inaccessible, phase of human infection.

A Neisseria meningitidis NMB1966 mutant is impaired for invasion of respiratory epithelial cells, survival in human blood and for virulence in vivo.

We sought to determine whether NMB1966, encoding a putative ABC transporter, has a role in pathogenesis. Compared to its isogenic wild-type parent strain Neisseria meningitidis MC58, the NMB1966 knockout mutant was less adhesive and invasive for human bronchial epithelial cells, had reduced survival in human blood and was attenuated in a systemic mouse model of infection. The transcriptome of the wild-type and the NMB1966 mutant was compared. The data are consistent with a previous functional assignment of NMB1966 being the ABC transporter component of a glutamate transporter operon. Forty-seven percent of all the differentially regulated genes encoded known outer membrane proteins or pathways generating complex surface structures such as adhesins, peptidoglycan and capsule. The data show that NMB1966 has a role in virulence and that remodelling of the outer membrane and surface/structures is associated with attenuation of the NMB1966 mutant.