The novel biphasic medium for transport, culture and conservation at an ambient temperature of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae.

Bacterial meningitis remains a very important disease worldwide, and the major causative pathogens were Neisseria meningitidis (N. meningitidis), Streptococcus pneumoniae (S. pneumoniae) and Haemophilus influenzae (H. influenzae). In our context, the technical difficulties encountered in the routine practice were associated with the fragility of these bacteria, the high rates of negative culture and the demanding transport conditions. That's why the need to look for a solution to its technical problems and to propose a new proper solution with the local situation. The aim of this study was to develop, perform and evaluate a novel biphasic medium used for the transport, culture and conservation at an ambient temperature of N. meningitidis, S. pneumoniae and H. influenzae. The results showed that this biphasic medium provided more, novels and easy nutriments through the addition of liquid phase and solid phase medium and it was found to be conducive to the growth and conservation of N. meningitidis, S. pneumoniae and H. influenzae at an ambient temperature of a minimum of 40 days. And the ingredients used in the medium are readily available at a low cost as well as the components prepared in large quantities, they could be stored at + 4 ± 1 °C for 2 years without significantly altering their growth and conservation supporting their potential. The survival and recovery for the fastidious bacteria on the biphasic medium and the other media used for comparison in this study were significantly different (P < 0.05). In addition, the Sensitivity, Specificity, Positive and Negative Predictive Value of biphasic medium showed highest among the three bacteria at least 40 days of storage at room temperature in this study. In conclusion, we found the biphasic medium to be low cost and suitable for previously mentioned bacteria from suspected meningitis patients, offering an optimal condition and an increase in the viability of the isolates at ambient temperature. And it was concluded that this biphasic medium could be used as a technical solution in laboratories for the management of meningitis.

Colonization of dermal arterioles by Neisseria meningitidis provides a safe haven from neutrophils.

The human pathogen Neisseria meningitidis can cause meningitis and fatal systemic disease. The bacteria colonize blood vessels and rapidly cause vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, we use a humanized mouse model to show that vascular colonization leads to the recruitment of neutrophils, which partially reduce bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles, adhesion molecules such as E-Selectin are not expressed on the endothelium, and intravascular neutrophil recruitment is minimal. Our results indicate that the colonization of capillaries and arterioles by N. meningitidis creates an intravascular niche that precludes the action of neutrophils, resulting in immune escape and progression of the infection.

Meningococcal carriage among Hajj pilgrims, risk factors for carriage and records of vaccination: a study of pilgrims to Mecca.

OBJECTIVE: The Saudi government requires that all pilgrims receive a quadrivalent meningococcal vaccine at least 10 days before the Hajj. We conducted a study to determine the uptake of meningococcal vaccine and antibiotic use. We also investigated risk factors of meningococcal carriage and carriage of Neisseria meningitidis pathogenic serogroups A, C, W and Y. METHODS: A cross-sectional oropharyngeal carriage survey was conducted in 2973 Hajj pilgrims in September 2017. A real-time polymerase chain reaction (rt-PCR) assay was used to identify N. meningitidis from the oropharyngeal swabs. A questionnaire investigated potential risk factors for carriage of N. meningitidis. RESULTS: Two thousand two hundred forty nine oropharyngeal swabs were obtained. The overall prevalence of carriage of N. meningitidis was 4.6% (95% CI: 3.4%-6%). Carriage of pathogenic serogroups was not associated significantly with any of the meningococcal risk factors evaluated. 77% of pilgrims were vaccinated but 22.58 % said they were carrying unofficial vaccination cards. CONCLUSION: Carriage of serogroups A, C, W and Y was not significantly associated with any of the risk factors investigated. Almost a quarter of pilgrims were unlikely to have been vaccinated, highlighting a need to strengthen compliance with the current policy of vaccination to prevent meningococcal disease outbreaks during and after the Hajj.

Long term storage of fastidious bacteria (Neisseria spp. and Haemophilus spp.) with swab preservation at -80 °C.

Preservation of strains is important to further studies and characterization; we have shown a technique of long-term preservation of Neisseria spp. and Haemophilus spp. by freezing cotton tip swabs impregnated with fastidious organisms at -80 °C without the addition of cryoprotectants.

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

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

Clinical and economic burden of invasive meningococcal disease: Evidence from a large German claims database.

BACKGROUND: Limited data is available to describe clinical characteristics, long-term outcomes, healthcare resource use and the attributable costs of invasive meningococcal disease (IMD) in Germany. We aimed to examine demographic and clinical characteristics as well as healthcare resource use and related costs. METHODS: We conducted a retrospective cohort study based on the InGef database in patients with IMD between 2009 and 2015. Cases were identified based on hospital main discharge diagnoses of IMD. Demographics, clinical characteristics, 30-day and 1-year mortality as well as IMD-related complications and sequelae in IMD cases were examined. In addition, short and long-term costs and healthcare resource use in IMD cases were analyzed and compared to an age- and sex-matched control group without IMD. RESULTS: The study population comprised 164 IMD cases between 2009 and 2015. The mean length of the IMD-related hospitalization was 13 days and 38% of all cases presented with meningitis only, 35% with sepsis only, 16% with both and 11% with other IMD. The 30-day and one-year mortality were 4.3% and 5.5%, respectively. Approximately 13% of IMD cases had documented IMD-related complications at hospital discharge and 24% suffered from sequelae during follow-up. The IMD-related hospitalization was associated with mean costs of € 9,620 (standard deviation: € 22,197). The difference of mean costs between IMD cases and matched non-IMD controls were € 267 in the first month and € 1,161 from one month to one year after discharged from IMD-related hospitalization. During the later follow-up period, the mean overall costs and costs associated with individual healthcare sectors were also higher for IMD cases without reaching statistical significance. CONCLUSIONS: IMD resulted in severe complications and sequelae and was associated with extensive costs and increased healthcare resource use in Germany, especially in the first year after IMD diagnosis and due the IMD-related hospitalization.

Neisseria meningitidis Induces Pathology-Associated Cellular and Molecular Changes in Trigeminal Schwann Cells.

Neisseria meningitidis, a common cause of sepsis and bacterial meningitis, infects the meninges and central nervous system (CNS), primarily via paracellular traversal across the blood-brain barrier (BBB) or blood-cerebrospinal fluid barrier. N. meningitidis is often present asymptomatically in the nasopharynx, and the nerves extending between the nasal cavity and the brain constitute an alternative route by which the meningococci may reach the CNS. To date, the cellular mechanisms involved in nerve infection are not fully understood. Peripheral nerve glial cells are phagocytic and are capable of eliminating microorganisms, but some pathogens may be able to overcome this protection mechanism and instead infect the glia, causing cell death or pathology. Here, we show that N. meningitidis readily infects trigeminal Schwann cells (the glial cells of the trigeminal nerve) in vitro in both two-dimensional and three-dimensional cell cultures. Infection of trigeminal Schwann cells may be one mechanism by which N. meningitidis is able to invade the CNS. Infection of the cells led to multinucleation and the appearance of atypical nuclei, with the presence of horseshoe nuclei and the budding of nuclei increasing over time. Using sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics followed by bioinformatics pathway analysis, we showed that N. meningitidis induced protein alterations in the glia that were associated with altered intercellular signaling, cell-cell interactions, and cellular movement. The analysis also suggested that the alterations in protein levels were consistent with changes occurring in cancer. Thus, infection of the trigeminal nerve by N. meningitidis may have ongoing adverse effects on the biology of Schwann cells, which may lead to pathology.

Continuous production of Neisseria meningitidis outer membrane vesicles.

Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application.

Contribution of σ70 and σN Factors to Expression of Class II pilE in Neisseria meningitidis.

Neisseria meningitidis expresses multicomponent organelles called type four pili (Tfp), which are key virulence factors required for attachment to human cells during carriage and disease. Pilin (PilE) is the main component of Tfp, and N. meningitidis isolates either have a class I pilE locus and express pilins that undergo antigenic variation or have a class II pilE locus and express invariant pilins. The transcriptional regulation of class I pilE has been studied in both N. meningitidis and Neisseria gonorrhoeae, while the control of expression of class II pilE has been elucidated in the nonpathogenic species Neisseria elongata However, the factors that govern the regulation of the class II pilE gene in N. meningitidis are not known. In this work, we have bioinformatically and experimentally identified the class II pilE promoter. We confirmed the presence of conserved σ70 and σN-dependent promoters upstream of pilE in a collection of meningococcal genomes and demonstrated that class II pilE expression initiates from the σ70 family-dependent promoter. By deletion or overexpression of sigma factors, we showed that σN, σH, and σE do not affect class II pilin expression. These findings are consistent with a role of the housekeeping σD in expression of this important component of Tfp. Taken together, our data indicate that the σ-dependent network responsible for the expression of class II pilE has been selected to maintain pilE expression, consistent with the essential roles of Tfp in colonization and pathogenesis.IMPORTANCE The type four pilus (Tfp) of Neisseria meningitidis contributes to fundamental processes such as adhesion, transformation, and disease pathology. Meningococci express one of two distinct classes of Tfp (class I or class II), which can be distinguished antigenically or by the major subunit (pilE) locus and its genetic context. The factors that govern transcription of the class II pilE gene are not known, even though it is present in isolates that cause epidemic disease. Here we show that the transcription of class II pilE is maintained throughout growth and under different stress conditions and is driven by a σ70-dependent promoter. This is distinct from Tfp regulation in nonpathogenic Neisseria spp. and may confer an advantage during host-cell interaction and infection.

Targeting Type IV pili as an antivirulence strategy against invasive meningococcal disease.

Bacterial virulence factors are attractive targets for the development of therapeutics. Type IV pili, which are associated with a remarkable array of properties including motility, the interaction between bacteria and attachment to biotic and abiotic surfaces, represent particularly appealing virulence factor targets. Type IV pili are present in numerous bacterial species and are critical for their pathogenesis. In this study, we report that trifluoperazine and related phenothiazines block functions associated with Type IV pili in different bacterial pathogens, by affecting piliation within minutes. Using Neisseria meningitidis as a paradigm of Gram-negative bacterial pathogens that require Type IV pili for pathogenesis, we show that piliation is sensitive to altered activity of the Na+ pumping NADH-ubiquinone oxidoreductase (Na+-NQR) complex and that these compounds probably altered the establishment of the sodium gradient. In vivo, these compounds exert a strong protective effect. They reduce meningococcal colonization of the human vessels and prevent subsequent vascular dysfunctions, intravascular coagulation and overwhelming inflammation, the hallmarks of invasive meningococcal infections. Finally, they reduce lethality. This work provides a proof of concept that compounds with activity against bacterial Type IV pili could beneficially participate in the treatment of infections caused by Type IV pilus-expressing bacteria.

The CRISPR/Cas system in Neisseria meningitidis affects bacterial adhesion to human nasopharyngeal epithelial cells.

Neisseria meningitidis, a commensal ß-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.

The Meningococcal Cysteine Transport System Plays a Crucial Role in Neisseria meningitidis Survival in Human Brain Microvascular Endothelial Cells.

While Neisseria meningitidis typically exists in an asymptomatic nasopharyngeal carriage state, it may cause potentially lethal diseases in humans, such as septicemia or meningitis, by invading deeper sites in the body. Since the nutrient compositions of human cells are not always conducive to meningococci, N. meningitidis needs to exploit nutrients from host environments. In the present study, the utilization of cysteine by the meningococcal cysteine transport system (CTS) was analyzed for the pathogenesis of meningococcal infections. A N. meningitidis strain deficient in one of the three cts genes annotated as encoding cysteine-binding protein (cbp) exhibited approximately 100-fold less internalization into human brain microvascular endothelial cells (HBMEC) than the wild-type strain. This deficiency was restored by complementation with the three cts genes together, and the infectious phenotype of HBMEC internalization correlated with cysteine uptake activity. However, efficient accumulation of ezrin was observed beneath the cbp mutant. The intracellular survival of the cbp mutant in HBMEC was markedly reduced, whereas equivalent reductions of glutathione concentrations and of resistance to reactive oxygens species in the cbp mutant were not found. The cbp mutant grew well in complete medium but not in synthetic medium supplemented with less than 300 µM cysteine. Taking cysteine concentrations in human cells and other body fluids, including blood and cerebrospinal fluid, into consideration, the present results collectively suggest that the meningococcal CTS is crucial for the acquisition of cysteine from human cells and participates in meningococcal nutrient virulence.IMPORTANCENeisseria meningitidis colonizes at a nasopharynx of human as a unique host and has many strains that are auxotrophs for amino acids for their growth. To cause invasive meningococcal diseases (IMD) such as sepsis and meningitis, N. meningitidis passes through epithelial and endothelial barriers and infiltrates into blood and cerebrospinal fluid as well as epithelial and endothelial cells. However, meningococcal nutrients, including cysteine, become less abundant when it more deeply infiltrates the human body even during inflammation, such that N. meningitidis has to acquire nutrients in order to survive/persist, disseminate, and proliferate in humans. This was the first study to examine the relationship between meningococcal cysteine acquisition and the pathogenesis of meningococcal infections. The results of the present study provide insights into the mechanisms by which pathogens with auxotrophs acquire nutrients in hosts and may also contribute to the development of treatments and prevention strategies for IMD.

Anticoagulants impact on innate immune responses and bacterial survival in whole blood models of Neisseria meningitidis infection.

Neisseria meningitidis (meningococcus) causes invasive diseases such as meningitis or septicaemia. Ex vivo infection of human whole blood is a valuable tool to study meningococcal virulence factors and the host innate immune responses. In order to consider effects of cellular mediators, the coagulation cascade must be inhibited to avoid clotting. There is considerable variation in the anticoagulants used among studies of N. meningitidis whole blood infections, featuring citrate, heparin or derivatives of hirudin, a polypeptide from leech saliva. Here, we compare the influence of these three different anticoagulants, and additionally Mg/EGTA, on host innate immune responses as well as on viability of N. meningitidis strains isolated from healthy carriers and disease cases, reflecting different sequence types and capsule phenotypes. We found that the anticoagulants significantly impact on cellular responses and, strain-dependently, also on bacterial survival. Hirudin does not inhibit complement and is therefore superior over the other anticoagulants; indeed hirudin-plasma most closely reflects the characteristics of serum during N. meningitidis infection. We further demonstrate the impact of heparin on complement activation on N. meningitidis and its consequences on meningococcal survival in immune sera, which appears to be independent of the heparin binding antigens Opc and NHBA.

The helicase DinG responds to stress due to DNA double strand breaks.

Neisseria meningitidis (Nm) is a Gram-negative nasopharyngeal commensal that can cause septicaemia and meningitis. The neisserial DNA damage-inducible protein DinG is a helicase related to the mammalian helicases XPD and FANCJ. These helicases belong to superfamily 2, are ATP dependent and exert 5' → 3' directionality. To better understand the role of DinG in neisserial genome maintenance, the Nm DinG (DinGNm) enzymatic activities were assessed in vitro and phenotypical characterization of a dinG null mutant (NmΔdinG) was performed. Like its homologues, DinGNm possesses 5' → 3' directionality and prefers DNA substrates containing a 5'-overhang. ATPase activity of DinGNm is strictly DNA-dependent and DNA unwinding activity requires nucleoside triphosphate and divalent metal cations. DinGNm directly binds SSBNm with a Kd of 313 nM. Genotoxic stress analysis demonstrated that NmΔdinG was more sensitive to double-strand DNA breaks (DSB) induced by mitomycin C (MMC) than the Nm wildtype, defining the role of neisserial DinG in DSB repair. Notably, when NmΔdinG cells grown under MMC stress assessed by quantitative mass spectrometry, 134 proteins were shown to be differentially abundant (DA) compared to unstressed NmΔdinG cells. Among the DNA replication, repair and recombination proteins affected, polymerase III subunits and recombinational repair proteins RuvA, RuvB, RecB and RecD were significantly down regulated while TopA and SSB were upregulated under stress condition. Most of the other DA proteins detected are involved in metabolic functions. The present study shows that the helicase DinG is probably involved in regulating metabolic pathways as well as in genome maintenance.

A virulence-associated filamentous bacteriophage of Neisseria meningitidis increases host-cell colonisation.

Neisseria meningitidis is a commensal of human nasopharynx. In some circumstances, this bacteria can invade the bloodstream and, after crossing the blood brain barrier, the meninges. A filamentous phage, designated MDAΦ for Meningococcal Disease Associated, has been associated with invasive disease. In this work we show that the prophage is not associated with a higher virulence during the bloodstream phase of the disease. However, looking at the interaction of N. meningitidis with epithelial cells, a step essential for colonization of the nasopharynx, we demonstrate that the presence of the prophage, via the production of viruses, increases colonization of encapsulated meningococci onto monolayers of epithelial cells. The analysis of the biomass covering the epithelial cells revealed that meningococci are bound to the apical surface of host cells by few layers of heavily piliated bacteria, whereas, in the upper layers, bacteria are non-piliated but surrounded by phage particles which (i) form bundles of filaments, and/or (ii) are in some places associated with bacteria. The latter are likely to correspond to growing bacteriophages during their extrusion through the outer membrane. These data suggest that, as the biomass increases, the loss of piliation in the upper layers of the biomass does not allow type IV pilus bacterial aggregation, but is compensated by a large production of phage particles that promote bacterial aggregation via the formation of bundles of phage filaments linked to the bacterial cell walls. We propose that MDAΦ by increasing bacterial colonization in the mucosa at the site-of-entry, increase the occurrence of diseases.

Biological Functions of the Secretome of Neisseria meningitidis.

Neisseria meningitidis is a Gram-negative bacterial pathogen that normally resides as a commensal in the human nasopharynx but occasionally causes disease with high mortality and morbidity. To interact with its environment, it transports many proteins across the outer membrane to the bacterial cell surface and into the extracellular medium for which it deploys the common and well-characterized autotransporter, two-partner and type I secretion mechanisms, as well as a recently discovered pathway for the surface exposure of lipoproteins. The surface-exposed and secreted proteins serve roles in host-pathogen interactions, including adhesion to host cells and extracellular matrix proteins, evasion of nutritional immunity imposed by iron-binding proteins of the host, prevention of complement activation, neutralization of antimicrobial peptides, degradation of immunoglobulins, and permeabilization of epithelial layers. Furthermore, they have roles in interbacterial interactions, including the formation and dispersal of biofilms and the suppression of the growth of bacteria competing for the same niche. Here, we will review the protein secretion systems of N. meningitidis and focus on the functions of the secreted proteins.

Comparison of culture and PCR methods in the diagnosis of bacterial meningitis

Abstract Our aim in this study is to compare the standard culture method with the multiplex PCR and the Speed-Oligo® Bacterial Meningitis Test (SO-BMT) – a hybridization-based molecular test method – during the CSF examination of the patients with the pre-diagnosis of acute bacterial meningitis. For the purposes of this study, patients with acute bacterial meningitis treated at the Dicle University Medical Faculty Hospital, Infectious Diseases and Clinical Microbiology Clinic between December 2009 and April 2012 were retrospectively evaluated. The diagnosis of bacterial meningitis was made based on the clinical findings, laboratory test anomalies, CSF analysis results, and the radiological images. Growth was observed in the CSF cultures of 10 out of the 57 patients included in the study (17.5%) and Streptococcus pneumoniae was isolated in all of them. The CSF samples of 34 patients (59.6%) were positive according to the SO-BMT and S. pneumoniae was detected in 33 of the samples (97.05%), while Neisseria meningitidis was found in 1 sample (2.95%). In a total of 10 patients, S. pneumoniae was both isolated in the CSF culture and detected in the SO-BMT. The culture and the SO-BMT were negative in 23 of the CSF samples. There was no sample in which the CSF culture was positive although the SO-BMT was negative. While SO-BMT seems to be a more efficient method than bacterial culturing to determine the pathogens that most commonly cause bacterial meningitis in adults, further studies conducted on larger populations are needed in order to assess its efficiency and uses.

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.

A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood.

Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood.

The role of apolipoprotein N-acyl transferase, Lnt, in the lipidation of factor H binding protein of Neisseria meningitidis strain MC58 and its potential as a drug target.

BACKGROUND AND PURPOSE: The level of cell surface expression of the meningococcal vaccine antigen, Factor H binding protein (FHbp) varies between and within strains and this limits the breadth of strains that can be targeted by FHbp-based vaccines. The molecular pathway controlling expression of FHbp at the cell surface, including its lipidation, sorting to the outer membrane and export, and the potential regulation of this pathway have not been investigated until now. This knowledge will aid our evaluation of FHbp vaccines. EXPERIMENTAL APPROACH: A meningococcal transposon library was screened by whole cell immuno-dot blotting using an anti-FHbp antibody to identify a mutant with reduced binding and the disrupted gene was determined. KEY RESULTS: In a mutant with markedly reduced binding, the transposon was located in the lnt gene which encodes apolipoprotein N-acyl transferase, Lnt, responsible for the addition of the third fatty acid to apolipoproteins prior to their sorting to the outer membrane. We provide data indicating that in the Lnt mutant, FHbp is diacylated and its expression within the cell is reduced 10 fold, partly due to inhibition of transcription. Furthermore the Lnt mutant showed 64 fold and 16 fold increase in susceptibility to rifampicin and ciprofloxacin respectively. CONCLUSION AND IMPLICATIONS: We speculate that the inefficient sorting of diacylated FHbp in the meningococcus results in its accumulation in the periplasm inducing an envelope stress response to down-regulate its expression. We propose Lnt as a potential novel drug target for combination therapy with antibiotics. LINKED ARTICLES: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.