Late Repression of NF-κB Activity by Invasive but Not Non-Invasive Meningococcal Isolates Is Required to Display Apoptosis of Epithelial Cells

Meningococcal invasive isolates of the ST-11 clonal complex are most frequently associated with disease and rarely found in carriers. Unlike carriage isolates, invasive isolates induce apoptosis in epithelial cells through the TNF-α signaling pathway. While invasive and non-invasive isolates are both able to trigger the TLR4/MyD88 pathway in lipooligosaccharide (LOS)-dependant manner, we show that only non-invasive isolates were able to induce sustained NF-κB activity in infected epithelial cells. ST-11 invasive isolates initially triggered a strong NF-κB activity in infected epithelial cells that was abolished after 9h of infection and was associated with sustained activation of JNK, increased levels of membrane TNFR1, and induction of apoptosis. In contrast, infection with carriage isolates lead to prolonged activation of NF-κB that was associated with a transient activation of JNK increased TACE/ADAM17-mediated shedding of TNFR1 and protection against apoptosis. Our data provide insights to understand the meningococcal duality between invasiveness and asymptomatic carriage

Structural Basis for Agonistic Activity and Selectivity toward Melatonin Receptors hMT1 and hMT2

: Glaucoma, a major ocular neuropathy originating from a progressive degeneration of retinal ganglion cells, is often associated with increased intraocular pressure (IOP). Daily IOP fluctuations are physiologically influenced by the antioxidant and signaling activities of melatonin. This endogenous modulator has limited employment in treating altered IOP disorders due to its low stability and bioavailability. The search for low-toxic compounds as potential melatonin agonists with higher stability and bioavailability than melatonin itself could start only from knowing the molecular basis of melatonergic activity. Thus, using a computational approach, we studied the melatonin binding toward its natural macromolecular targets, namely melatonin receptors 1 (MT1) and 2 (MT2), both involved in IOP signaling regulation. Besides, agomelatine, a melatonin-derivative agonist and, at the same time, an atypical antidepressant, was also included in the study due to its powerful IOP-lowering effects. For both ligands, we evaluated both stability and ligand positioning inside the orthosteric site of MTs, mapping the main molecular interactions responsible for receptor activation. Affinity values in terms of free binding energy (ΔGbind) were calculated for the selected poses of the chosen compounds after stabilization through a dynamic molecular docking protocol. The results were compared with experimental in vivo effects, showing a higher potency and more durable effect for agomelatine with respect to melatonin, which could be ascribed both to its higher affinity for hMT2 and to its additional activity as an antagonist for the serotonin receptor 5-HT2c, in agreement with the in silico results

Experimental Meningococcal Sepsis in Congenic Transgenic Mice Expressing Human Transferrin

Severe meningococcal sepsis is still of high morbidity and mortality. Its management may be improved by an experimental model allowing better understanding of its pathophysiology. We developed an animal model of meningococcal sepsis in transgenic BALB/c mice expressing human transferrin. We studied experimental meningococcal sepsis in congenic transgenic BALB/c mice expressing human transferrin by transcriptional profiling using microarray analysis of blood and brain samples. Genes encoding acute phase proteins, chemokines and cytokines constituted the largest strongly regulated groups. Dynamic bioluminescence imaging further showed high blood bacterial loads that were further enhanced after a primary viral infection by influenza A virus. Moreover, IL-1 receptor–associated kinase–3 (IRAK-3) was induced in infected mice. IRAK-3 is a negative regulator of Toll-dependant signaling and its induction may impair innate immunity and hence result in an immunocompromised state allowing bacterial survival and systemic spread during sepsis. This new approach should enable detailed analysis of the pathophysiology of meningococcal sepsis and its relationships with flu infection

Differential Modulation of TNF-α–Induced Apoptosis by Neisseria meningitidis

Infections by Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell–bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods (FITC-Annexin V staining followed by FACS analysis, caspase assays and DNA fragmentation). Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-α is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-α receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-α. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx

Serum Albumin Is Inversely Associated With Portal Vein Thrombosis in Cirrhosis

We analyzed whether serum albumin is independently associated with portal vein thrombosis (PVT) in liver cirrhosis (LC) and if a biologic plausibility exists. This study was divided into three parts. In part 1 (retrospective analysis), 753 consecutive patients with LC with ultrasound-detected PVT were retrospectively analyzed. In part 2, 112 patients with LC and 56 matched controls were entered in the cross-sectional study. In part 3, 5 patients with cirrhosis were entered in the in vivo study and 4 healthy subjects (HSs) were entered in the in vitro study to explore if albumin may affect platelet activation by modulating oxidative stress. In the 753 patients with LC, the prevalence of PVT was 16.7%; logistic analysis showed that only age (odds ratio [OR], 1.024; P = 0.012) and serum albumin (OR, -0.422; P = 0.0001) significantly predicted patients with PVT. Analyzing the 112 patients with LC and controls, soluble clusters of differentiation (CD)40-ligand (P = 0.0238), soluble Nox2-derived peptide (sNox2-dp; P < 0.0001), and urinary excretion of isoprostanes (P = 0.0078) were higher in patients with LC. In LC, albumin was correlated with sCD4OL (Spearman's rank correlation coefficient [r(s)], -0.33; P < 0.001), sNox2-dp (r(s), -0.57; P < 0.0001), and urinary excretion of isoprostanes (r(s), -0.48; P < 0.0001) levels. The in vivo study showed a progressive decrease in platelet aggregation, sNox2-dp, and urinary 8-iso prostaglandin F2 alpha-III formation 2 hours and 3 days after albumin infusion. Finally, platelet aggregation, sNox2-dp, and isoprostane formation significantly decreased in platelets from HSs incubated with scalar concentrations of albumin. Conclusion: Low serum albumin in LC is associated with PVT, suggesting that albumin could be a modulator of the hemostatic system through interference with mechanisms regulating platelet activation

Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity.

International audienceThe ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival

Functional impacts of the diversity of the meningococcal factor H binding protein.

International audienceNeisseria meningitidis is a human pathogenic bacterium responsible for life threatening and rapidly evolving invasive infections. Several bacterial virulence factors may play primordial roles during host-bacteria interactions. The meningococcal factor H binding protein, fHbp, interacts with the complement negative regulator, factor H (fH), to enhance meningococcal survival. fHbp is a major component in recombinant vaccines against meningococci that are under development. In 2010, we detected variations in fhbp gene during an outbreak provoked by serogroup C isolates belonging to the clonal complex, ST-11. We therefore explored 680 meningococcal isolates (88% of all invasive isolates in 2009 and 2010) by DNA sequencing of fhbp gene. The level of fHbp at the bacterial surface was determined by ELISA and flow cytometry using anti-fHbp antibodies. We also analyzed the interaction of fHbp with human fH as well as the deposition of C3b complement component. We observed important sequence diversity of fHbp in particular within regions known to interact with fH. The distribution of fhbp alleles differed among meningococcal serogroups and clonal complexes. This diversity affected directly binding of fH to fHbp and seemed to influence the deposition of the complement C3b component on the bacterial surface. However, bacterial killing by anti-fHbp antibodies was still achieved and required a minimum level of fHbp at the bacterial surface regardless the binding to fH or sequence diversity. These data have impacts on our understanding of the role of fHbp in meningococcal pathogenesis. They also provide data on the diversity of fhbp before the introduction of vaccines targeting fHbp and stress the need to include characterization of fHbp in typing schemes of meningococcal isolates

Analysis in vitro and in vivo of the transcriptional regulator CrgA of Neisseria meningitidis upon contact with target cells

International audienceContact between CrgA, a LysR-like regulatory protein in Neisseria meningitidis, and DNA is involved in the repression of several bacterial genes upon contact with epithelial cells. We used a defined in vitro system containing crgA promoter, purified RNA polymerase (RNAP) and purified CrgA protein to demonstrate that CrgA was directly responsible for this transcriptional repression. Interaction between the C-terminal domain of CrgA and the RNAP led to the production of short abortive transcripts, suggesting that CrgA may act by preventing RNAP from clearing the promoter. We probed the regulation by CrgA of its own production by analysing CrgA-DNA contacts during cell-bacteria interaction by assaying in vivo protection against dimethyl sulphate (DMS) methylation. Comparison of DMS footprints in vitro and in vivo suggested that CrgA repressed transcription through specific base contacts, probably in the major groove of the DNA double helix, resulting in DNA looping. Upon contact with target cells, CrgA was released from the DNA, allowing transcription of the target gene to proceed to elongation and facilitating tight control of the expression of genes regulated by CrgA

Lipocalin 2 in cerebrospinal fluid as a marker of acute bacterial meningitis.

International audienceBACKGROUND: Early differential diagnosis between acute bacterial and viral meningitis is problematic. We aimed to investigate whether the detection of lipocalin 2, a protein of the acute innate immunity response, may be used as a marker for acute bacterial meningitis. METHODS: Transgenic mice expressing the human transferrin were infected by intraperitoneal route and were imaged. Cerebrospinal fluid (CSF) was sampled up to 48hours post- infection to measure lipocalin 2. We also tested a collection of 90 and 44 human CSF with confirmed acute bacterial or acute viral meningitis respectively. RESULTS: Lipocalin 2 was detected after 5 h in CSF during experimental infection in mice. Lipocalin 2 levels were significantly higher (p < 0.0001) in patients with confirmed acute bacterial meningitis (mean 125 pg/mL, range 106-145 pg/mL) than in patients with acute viral meningitis (mean 2 pg/mL, range 0-6 pg/mL) with a sensitivity of 81%, a specificity of 93%, a positive predictive value of 96% and a negative predictive value of 71% in diagnosing acute bacterial meningitis. CONCLUSIONS: Increased levels of lipocalin 2 in cerebrospinal fluid may discriminate between acute bacterial and viral meningitis in patients with clinical syndrome of meningitis