A step-by-step in crystallo guide to bond cleavage and 1,6-anhydro-sugar product synthesis by a peptidoglycan-degrading lytic transglycosylase.

Lytic transglycosylases (LTs) are a class of enzymes important for the recycling and metabolism of peptidoglycan (PG). LTs cleave the ß-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and GlcNAc in the PG glycan strand, resulting in the concomitant formation of 1,6-anhydro-N-acetylmuramic acid and GlcNAc. No LTs reported to date have utilized chitins as substrates, despite the fact that chitins are GlcNAc polymers linked via ß-1,4-glycosidic bonds, which are the known site of chemical activity for LTs. Here, we demonstrate enzymatically that LtgA, a non-canonical, substrate-permissive LT from Neisseria meningitidis utilizes chitopentaose ((GlcNAc)5) as a substrate to produce three newly identified sugars: 1,6-anhydro-chitobiose, 1,6-anhydro-chitotriose, and 1,6-anhydro-chitotetraose. Although LTs have been widely studied, their complex reactions have not previously been visualized in the crystalline state because macromolecular PG is insoluble. Here, we visualized the cleavage of the glycosidic bond and the liberation of GlcNAc-derived residues by LtgA, followed by the synthesis of atypical 1,6-anhydro-GlcNAc derivatives. In addition to the newly identified anhydro-chitin products, we identified trapped intermediates, unpredicted substrate rearrangements, sugar distortions, and a conserved crystallographic water molecule bound to the catalytic glutamate of a high-resolution native LT. This study enabled us to propose a revised alternative mechanism for LtgA that could also be applicable to other LTs. Our work contributes to the understanding of the mechanisms of LTs in bacterial cell wall biology.

Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined.

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

Heterogeneity in non-epitope loop sequence and outer membrane protein complexes alters antibody binding to the major porin protein PorB in serogroup B Neisseria meningitidis.

PorB is a well-characterized outer membrane protein that is common among Neisseria species and is required for survival. A vaccine candidate, PorB induces antibody responses that are directed against six variable surface-exposed loops that differ in sequence depending on serotype. Although Neisseria meningitidis is naturally competent and porB genetic mosaicism provides evidence for strong positive selection, the sequences of PorB serotypes commonly associated with invasive disease are often conserved, calling into question the interaction of specific PorB loop sequences in immune engagement. In this report, we provide evidence that antibody binding to a PorB epitope can be altered by sequence mutations in non-epitope loops. Through the construction of hybrid PorB types and PorB molecular dynamics simulations, we demonstrate that loops both adjacent and non-adjacent to the epitope loop can enhance or diminish antibody binding, a phenotype that correlates with serum bactericidal activity. We further examine the interaction of PorB with outer membrane-associated proteins, including PorA and RmpM. Deletion of these proteins alters the composition of PorB-containing native complexes and reduces antibody binding and serum killing relative to the parental strain, suggesting that both intramolecular and intermolecular PorB interactions contribute to host adaptive immune evasion.

Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage.

The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1-3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.

Involvement of Neisseria meningitidis lipoprotein GNA2091 in the assembly of a subset of outer membrane proteins.

GNA2091 of Neisseria meningitidis is a lipoprotein of unknown function that is included in the novel 4CMenB vaccine. Here, we investigated the biological function and the subcellular localization of the protein. We demonstrate that GNA2091 functions in the assembly of outer membrane proteins (OMPs) because its absence resulted in the accumulation of misassembled OMPs. Cell fractionation and protease accessibility experiments showed that the protein is localized at the periplasmic side of the outer membrane. Pulldown experiments revealed that it is not stably associated with the ß-barrel assembly machinery, the previously identified complex for OMP assembly. Thus, GNA2091 constitutes a novel outer membrane-based lipoprotein required for OMP assembly. Furthermore, its location at the inner side of the outer membrane indicates that protective immunity elicited by this antigen cannot be due to bactericidal or opsonic activity of antibodies.

Neisseria meningitidis Opc invasin binds to the sulphated tyrosines of activated vitronectin to attach to and invade human brain endothelial cells.

The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y(56) and Y(59). Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43-68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain (45)RGD(47) was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may enable the bacterium to harness the benefits of the physiological processes in which the host effector molecule participates.

An evaluation of the role of properdin in alternative pathway activation on Neisseria meningitidis and Neisseria gonorrhoeae.

Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against invasive neisserial infections. Recently, commercially available unfractionated properdin was shown to bind to certain biological surfaces, including Neisseria gonorrhoeae, which facilitated C3 deposition. Unfractionated properdin contains aggregates or high-order oligomers, in addition to its physiological "native" (dimeric, trimeric, and tetrameric) forms. We examined the role of properdin in AP activation on diverse strains of Neisseria meningitidis and N. gonorrhoeae specifically using native versus unfractionated properdin. C3 deposition on Neisseria decreased markedly when properdin function was blocked using an anti-properdin mAb or when properdin was depleted from serum. Maximal AP-mediated C3 deposition on Neisseriae even at high (80%) serum concentrations required properdin. Consistent with prior observations, preincubation of bacteria with unfractionated properdin, followed by the addition of properdin-depleted serum resulted in higher C3 deposition than when bacteria were incubated with properdin-depleted serum alone. Unexpectedly, none of 10 Neisserial strains tested bound native properdin. Consistent with its inability to bind to Neisseriae, preincubating bacteria with native properdin followed by the addition of properdin-depleted serum did not cause detectable increases in C3 deposition. However, reconstituting properdin-depleted serum with native properdin a priori enhanced C3 deposition on all strains of Neisseria tested. In conclusion, the physiological forms of properdin do not bind directly to either N. meningitidis or N. gonorrhoeae but play a crucial role in augmenting AP-dependent C3 deposition on the bacteria through the "conventional" mechanism of stabilizing AP C3 convertases.

The macrophage scavenger receptor A is host-protective in experimental meningococcal septicaemia.

Macrophage Scavenger Receptor A (SR-A) is a major non-opsonic receptor for Neisseria meningitidis on mononuclear phagocytes in vitro, and the surface proteins NMB0278, NMB0667, and NMB1220 have been identified as ligands for SR-A. In this study we ascertain the in vivo role of SR-A in the recognition of N. meningitidis MC58 (serogroup B) in a murine model of meningococcal septicaemia. We infected wild-type and SR-A(-/-) animals intraperitoneally with N. meningitidis MC58 and monitored their health over a period of 50 hours. We also determined the levels of bacteraemia in the blood and spleen, and measured levels of the pro-inflammatory cytokine interleukin-6 (IL-6). The health of SR-A(-/-) animals deteriorated more rapidly, and they showed a 33% reduction in survival compared to wild-type animals. SR-A(-/-) animals consistently exhibited higher levels of bacteraemia and increased levels of IL-6, compared to wild-type animals. Subsequently, we constructed a bacterial mutant (MC58-278-1220) lacking two of the SR-A ligands, NMB0278 and NMB1220. Mutation of NMB0667 proved to be lethal. When mice were infected with the mutant bacteria MC58-278-1220, no significant differences could be observed in the health, survival, bacteraemia, and cytokine production between wild-type and SR-A(-/-) animals. Overall, mutant bacteria appeared to cause less severe symptoms of septicaemia, and a competitive index assay showed that higher levels of wild-type bacteria were recovered when animals were infected with a 1ratio1 ratio of wild-type MC58 and mutant MC58-278-1220 bacteria. These data represent the first report of the protective role of SR-A, a macrophage-restricted, non-opsonic receptor, in meningococcal septicaemia in vivo, and the importance of the recognition of bacterial protein ligands, rather than lipopolysaccharide.

Bacterial nitric oxide detoxification prevents host cell S-nitrosothiol formation: a novel mechanism of bacterial pathogenesis.

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 gamma-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.

Comparative genome biology of a serogroup B carriage and disease strain supports a polygenic nature of meningococcal virulence.

Neisseria meningitidis serogroup B strains are responsible for most meningococcal cases in the industrialized countries, and strains belonging to the clonal complex ST-41/44 are among the most prevalent serogroup B strains in carriage and disease. Here, we report the first genome and transcriptome comparison of a serogroup B carriage strain from the clonal complex ST-41/44 to the serogroup B disease strain MC58 from the clonal complex ST-32. Both genomes are highly colinear, with only three major genome rearrangements that are associated with the integration of mobile genetic elements. They further differ in about 10% of their gene content, with the highest variability in gene presence as well as gene sequence found for proteins involved in host cell interactions, including Opc, NadA, TonB-dependent receptors, RTX toxin, and two-partner secretion system proteins. Whereas housekeeping genes coding for metabolic functions were highly conserved, there were considerable differences in their expression pattern upon adhesion to human nasopharyngeal cells between both strains, including differences in energy metabolism and stress response. In line with these genomic and transcriptomic differences, both strains also showed marked differences in their in vitro infectivity and in serum resistance. Taken together, these data support the concept of a polygenic nature of meningococcal virulence comprising differences in the repertoire of adhesins as well as in the regulation of metabolic genes and suggest a prominent role for immune selection and genetic drift in shaping the meningococcal genome.

Meningococcal B vaccine antigen FHbp variants among disease-causing Neisseria meningitidis B isolates, Italy, 2014-2017.

BACKGROUND: Typing of Neisseria meningitidis isolates is crucial for the surveillance of invasive meningococcal disease (IMD). We performed a molecular epidemiology study of N. meningitidis serogroup B (MenB) causing IMD in Italy between 2014 and 2017 to describe circulating strains belonging to this serogroup, with particular regards to the two factor H-binding protein (FHbp) subfamilies present in the bivalent MenB vaccine. MATERIALS AND METHODS: A total of 109 culture positive and 46 culture negative MenB samples were collected within the National Surveillance System (NSS) of IMD in Italy and molecularly analyzed by conventional methods. RESULTS: Overall, 71 MenB samples showed the FHbp subfamily A and 83 the subfamily B. The subfamily variants were differently distributed by age. The most frequent variants, A05 and B231, were associated with cc213 and cc162, respectively. All MenB with the FHbp A05 variant displayed the PorA P1.22,14 and 85.7% of them the FetA F5-5. The majority of MenB with the FHbp B231 variant showed the PorA P1.22,14 (65.4%) and 84.6%, the FetA F3-6. CONCLUSION: MenB circulating in Italy were characterized by a remarkable association between clonal complex and FHbp variants, although a high degree of genetic diversity observed over time. A dynamic trend in clonal complexes distribution within MenB was detected. Our results stress the importance of continued meningococcal molecular surveillance to evaluate the potential vaccine coverage of the available MenB vaccines.

Low efficacy of vaccination against serogroup B meningococci in patients with atypical hemolytic uremic syndrome.

BACKGROUND: The C5 complement inhibitor eculizumab is first-line treatment in atypical hemolytic uremic syndrome (aHUS) going along with a highly increased risk of meningococcal infections. Serogroup B meningococci (MenB) are the most frequently encountered cause for meningococcal infections in Europe. Efficacy of the protein-based MenB-vaccine Bexsero in aHUS has not been determined and testing is only possible in patients off-treatment with eculizumab as a human complement source is required. METHODS: Patients with aHUS were vaccinated with two doses of the protein-based MenB-vaccine Bexsero. Serum bactericidal antibody (SBA) titers against factor H binding protein (fHbp) of MenB were determined in 14 patients with aHUS off-treatment with eculizumab. RESULTS: Only 50% of patients showed protective human serum bactericidal antibody (hSBA) titers (≥1:4) against MenB following two vaccinations. Bactericidal antibody titers were relatively low (≤1:8) in three of seven patients with protective titers. While 71% of patients were on immunosuppressive treatment for either thrombotic microangiopathy or renal transplantation at either first or second vaccination, all four patients not receiving any immunosuppressive treatment showed protective bactericidal antibody response. Time between second vaccination and titer measurement was not significantly different between patients with protective titers compared with those with non-protective titers, while time between first and second vaccination was significantly longer in patients with protective titers going along with a tendency for reduction in immunosuppressive treatment. CONCLUSIONS: Efficacy of vaccination against MenB is insufficient in patients with aHUS. Response to vaccination seems to be hampered by immunosuppression. Therefore, implementation of adequate antibiotic prophylaxis seems pivotal.

Modeling Neisseria meningitidis B metabolism at different specific growth rates.

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. At the Netherlands Vaccine Institute (NVI) a vaccine against serogroup B organisms is currently being developed. This study describes the influence of the growth rate of N. meningitidis on its macro-molecular composition and its metabolic activity and was determined in chemostat cultures. In the applied range of growth rates, no significant changes in RNA content and protein content with growth rate were observed in N. meningitidis. The DNA content in N. meningitidis was somewhat higher at the highest applied growth rate. The phospholipid and lipopolysaccharide content in N. meningitidis changed with growth rate but no specific trends were observed. The cellular fatty acid composition and the amino acid composition did not change significantly with growth rate. Additionally, it was found that the PorA content in outer membrane vesicles was significantly lower at the highest growth rate. The metabolic fluxes at various growth rates were calculated using flux balance analysis. Errors in fluxes were calculated using Monte Carlo Simulation and the reliability of the calculated flux distribution could be indicated, which has not been reported for this type of analysis. The yield of biomass on substrate (Y(x/s)) and the maintenance coefficient (m(s)) were determined as 0.44 (+/-0.04) g g(-1) and 0.04 (+/-0.02) g g(-1) h(-1), respectively. The growth associated energy requirement (Y(x/ATP)) and the non-growth associated ATP requirement for maintenance (m(ATP)) were estimated as 0.13 (+/-0.04) mol mol(-1) and 0.43 (+/-0.14) mol mol(-1) h(-1), respectively. It was found that the split ratio between the Entner-Doudoroff and the pentose phosphate pathway, the sole glucose utilizing pathways in N. meningitidis, had a minor effect on ATP formation rate but a major effect on the fluxes going through for instance the citric-acid cycle. For this reason, we presented flux ranges for underdetermined parts of metabolic network rather than presenting single flux values, which is more commonly done in literature.

PorA types in Neisseria meningitidis serogroup B isolated in Argentina from 2001 to 2003: implications for the design of an outer membrane protein-based vaccine.

Identification of Neisseria meningitidis PorA types remains important, as the PorA protein is a major immunogenic component of several meningococcal vaccines under development. In this study, 191 N. meningitidis serogroup B isolates collected in Argentina through active laboratory-based surveillance from 2001 to 2003 were serosubtyped. Nucleotide sequences of the porA variable region 1 (VR1) and VR2 regions were determined in 52 non-serosubtypeable isolates. A substantial number of distinct VR types were identified, and a new VR2 variant from the P1.16 family was described. This is the first report describing PorA types in N. meningitidis serogroup B isolates in Argentina. Furthermore, the wide diversity of subtypes detected by serosubtyping and genosubtyping reveals the difficulty in designing a useful outer-membrane vaccine applicable in this country. A possible mechanism responsible for altered PorA expression was analysed in two PorA types.

The glycointeractome of serogroup B Neisseria meningitidis strain MC58.

Neisseria meningitidis express numerous virulence factors that enable it to interact with diverse microenvironments within the host, during both asymptomatic nasopharyngeal colonization and invasive disease. Many of these interactions involve bacterial or host glycans. In order to characterise the meningococcal glycointeractome, glycan arrays representative of structures found on human cells, were used as a screening tool to investigate host glycans bound by N. meningitidis. Arrays probed with fluorescently labelled wild-type MC58 revealed binding to 223 glycans, including blood group antigens, mucins, gangliosides and glycosaminoglycans. Mutant strains lacking surface components, including capsule, lipooligosaccharide (LOS), Opc and pili, were investigated to identify the factors responsible for glycan binding. Surface plasmon resonance and isothermal calorimetry were used to confirm binding and determine affinities between surface components and host glycans. We observed that the L3 LOS immunotype (whole cells and purified LOS) bound 26 structures, while L8 only bound 5 structures. We further demonstrated a direct glycan-glycan interaction between purified L3 LOS and Thomsen-Friedenreich (TF) antigen, with a KD of 13 nM. This is the highest affinity glycan-glycan interaction reported to date. These findings highlight the diverse glycointeractions that may occur during different stages of meningococcal disease, which could be exploited for development of novel preventative and therapeutic strategies.

Identification of vaccine antigens using integrated proteomic analyses of surface immunogens from serogroup B Neisseria meningitidis.

Meningococcal surface proteins capable of evoking a protective immune response are candidates for inclusion in protein-based vaccines against serogroup B Neisseria meningitidis (NmB). In this study, a 2-dimensional (2-D) gel-based platform integrating surface and immune-proteomics was developed to characterize NmB surface protein antigens. The surface proteome was analyzed by differential 2-D gel electrophoresis following treatment of live bacteria with proteinase K. Alongside, proteins recognized by immune sera from mice challenged with live meningococci were detected using 2-D immunoblots. In combination, seventeen proteins were identified including the well documented antigens PorA, OpcA and factor H-binding protein, previously reported potential antigens and novel potential immunogens. Results were validated for the macrophage infectivity potentiator (MIP), a recently proposed NmB vaccine candidate. MIP-specific antisera bound to meningococci in whole-cell ELISA and facilitated opsonophagocytosis and deposition of complement factors on the surface of meningococcal isolates of different serosubtypes. Cleavage by proteinase K was confirmed in western blots and shown to occur in a fraction of the MIP expressed by meningococci suggesting transient or limited surface exposure. These observations add knowledge for the development of a protein NmB vaccine. The proteomic workflow presented here may be used for the discovery of vaccine candidates against other pathogens. BIOLOGICAL SIGNIFICANCE: This study presents an integrated proteomic strategy to identify proteins from N. meningitidis with desirable properties (i.e. surface exposure and immunogenicity) for inclusion in subunit vaccines against bacterial meningitis. The effectiveness of the method was demonstrated by the identification of some of the major meningococcal vaccine antigens. Information was also obtained about novel potential immunogens as well as the recently described potential antigen macrophage infectivity potentiator which can be useful for its consideration as a vaccine candidate. Additionally, the proteomic strategy presented in this study provides a generic 2-D gel-based platform for the discovery of vaccine candidates against other bacterial infections.

Cysteine depletion causes oxidative stress and triggers outer membrane vesicle release by Neisseria meningitidis; implications for vaccine development.

Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis.

Outer membrane vesicles (OMV) production of Neisseria meningitidis serogroup B in batch process.

Serogroup B outer membrane vesicles (OMV) with iron regulated proteins (IRP) from Neisseria meningitidis constitute the antigen for the vaccine against the disease caused by this bacterium. Aiming to enhance final OMV concentration, seven batch experiments were carried out under four different conditions: (i) with original Catlin medium; (ii) with original Catlin medium and lactate and amino acids pulse at the 6th cultivation hour; (iii) with Catlin medium with double initial concentrations of lactate and amino acids and (iv) Catlin medium without glycerol and with double initial concentrations of lactate and amino acids. The cultivation experiments were carried out in a 7-L bioreactor under the following conditions: 36°C, 0.5atm, overlay air 1L/min, agitation: 250-850 rpm, and O(2) control at 10%, 20 h. After lactate and amino acids exhaustion, cell growth reached stationary phase and a significant release increase of OMV was observed. According to the Luedeking & Piret model, OMV liberation is non-growth associated. Glycerol was not consumed during cultivation. The maximum OMV concentration value attained was 162 mg/L with correspondent productivity of 8.1mg/(Lh) employing Catlin medium with double initial concentrations of lactate and amino acids. The obtained OMV satisfied constitution and protein pattern criteria and were suitable for vaccine production.

Neisseria meningitidis serogroup B lipooligosaccharide genotyping reveals high prevalence of L2 strains in Spain and unexpected relationship with factor H-binding protein expression.

Neisseria meningitidis may be classified according to the lipooligosaccharide immunotype. We show that this classification can be achieved by PCR genotyping of the genes involved in the lipooligosaccharide inner-core biosynthesis, lpt3, lpt6, lgtG and lot3. Genotyping data correlated well (90-100%) with mass spectrometry data and was, therefore, applied to screen a random subset of recent N. meningitidis serogroup B isolates from Europe. Analysis of the proportion of the different lipooligosaccharide types highlighted the predominance of L3 strains. Surprisingly, high rates of L2 type strains were found in Spain (17%, versus 2.5% in Germany and 1.9% in the United Kingdom). Therefore, we also investigated further these Spanish L2 strains in an attempt to explain such prevalence despite the known sensitivity of L2 immunotype to complement. We explored the hypothesis that these strains express high amounts of factor H-binding protein (fHbp), but we found, on the contrary, that L2 strains express low or undetectable amounts of fHbp. Our findings suggest that, in addition to a genetic analysis, a multivalent approach may be necessary to estimate the effectiveness of a N. meningitidis serogroup B vaccine.

LPS from Neisseria meningitidis is crucial for inducing monocyte- and microparticle-associated tissue factor activity but not for tissue factor expression.

Neisseria meningitidis causes sepsis with coagulopathy. The present study evaluated the tissue factor (TF)-inducing capacity of bacterial LPS in different presentation forms, i.e. membrane-bound LPS versus purified LPS, and of non-LPS components of N. meningitidis. By using a wild-type N. meningitidis, a mutant N. meningitidis lacking LPS (LPS-deficient N. meningitidis), purified LPS from N. meningitidis and Escherichia coli, we measured TF-expression and TF-activity on human monocytes and microparticles (MPs). The effect of TF-modulators, such as phosphatidylserine (PS), tissue factor pathway inhibitor (TFPI) and recombinant IL-10 (rhIL-10) was investigated. In plasmas from meningococcal patients, fibrinopeptide A (FPA), LPS and IL-10 were quantified. Monocytes and MPs exposed to purified LPS or wild-type N. meningitidis had much higher TF-activity than monocytes and MPs exposed to LPS-deficient N. meningitidis (clot formation assay). Incubation with wild-type N. meningitidis, but also LPS-deficient N. meningitidis, resulted in TF-expression on monocytes (flow cytometry, qRT-PCR). Increased cellular TF-activity is associated with coincident surface-exposure of PS and the number of monocytes positive for both PS and TF was significantly higher for monocytes exposed to wild-type N. meningitidis (7.6%) compared with monocytes exposed to LPS-deficient N. meningitidis (1.8%). Treatment with rhIL-10 reduced monocyte- and MP-associated TF-activity, the number of monocytes positive for both TF and PS, and microvesiculation. Patients with meningococcal septicemia had significantly higher levels of LPS, FPA and IL-10 than patients with distinct meningitis. Our results indicate that LPS from N. meningitidis is crucial for inducing TF-activity, but not for monocyte- and MP-associated TF-expression. TF-activity seems to require coincident expression of TF and PS on monocytes, and LPS induces such double-positive monocytes.