The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus.

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.

Receptor recognition by meningococcal type IV pili relies on a specific complex N-glycan.

Bacterial infections are frequently based on the binding of lectin-like adhesins to specific glycan determinants exposed on host cell receptors. These interactions confer species-specific recognition and tropism for particular host tissues and represent attractive antibacterial targets. However, the wide structural diversity of carbohydrates hampers the characterization of specific glycan determinants. Here, we characterized the receptor recognition of type IV pili (Tfp), a key adhesive factor present in numerous bacterial pathogens, using Neisseria meningitidis as a model organism. We found that meningococcal Tfp specifically recognize a triantennary sialylated poly-N-acetyllactosamine-containing N-glycan exposed on the human receptor CD147/Basigin, while fucosylated derivatives of this N-glycan impaired bacterial adhesion. Corroborating the inhibitory role of fucosylation on receptor recognition, adhesion of the meningococcus on nonhuman cells expressing human CD147 required prior defucosylation. These findings reveal the molecular basis of the selective receptor recognition by meningococcal Tfp and thereby, identify a potential antibacterial target.

Angiopoietin-like 4 protects against endothelial dysfunction during bacterial sepsis.

Loss of endothelial integrity and vascular leakage are central features of sepsis pathogenesis; however, no effective therapeutic mechanisms for preserving endothelial integrity are available. Here we show that, compared to dermal microvessels, brain microvessels resist infection by Neisseria meningitidis, a bacterial pathogen that causes sepsis and meningitis. By comparing the transcriptional responses to infection in dermal and brain endothelial cells, we identified angiopoietin-like 4 as a key factor produced by the brain endothelium that preserves blood-brain barrier integrity during bacterial sepsis. Conversely, angiopoietin-like 4 is produced at lower levels in the peripheral endothelium. Treatment with recombinant angiopoietin-like 4 reduced vascular leakage, organ failure and death in mouse models of lethal sepsis and N. meningitidis infection. Protection was conferred by a previously uncharacterized domain of angiopoietin-like 4, through binding to the heparan proteoglycan, syndecan-4. These findings reveal a potential strategy to prevent endothelial dysfunction and improve outcomes in patients with sepsis.

Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues.

Methylation of cytosine residues within the CpG dinucleotide in mammalian cells is an important mediator of gene expression, genome stability, X-chromosome inactivation, genomic imprinting, chromatin structure, and embryonic development. The majority of CpG sites in mammalian cells is methylated in a nonrandom fashion, raising the question of how DNA methylation is distributed along the genome. Here, we focused on the functions of DNA methyltransferase-3b (Dnmt3b), of which deregulated activity is linked to several human pathologies. We generated Dnmt3b hypomorphic mutant mice with reduced catalytic activity, which first revealed a deregulation of Hox genes expression, consistent with the observed homeotic transformations of the posterior axis. In addition, analysis of deregulated expression programs in Dnmt3b mutant embryos, using DNA microarrays, highlighted illegitimate activation of several germ-line genes in somatic tissues that appeared to be linked directly to their hypomethylation in mutant embryos. We provide evidence that these genes are direct targets of Dnmt3b. Moreover, the recruitment of Dnmt3b to their proximal promoter is dependant on the binding of the E2F6 transcriptional repressor, which emerges as a common hallmark in the promoters of genes found to be up-regulated as a consequence of impaired Dnmt3b activity. Therefore, our results unraveled a coordinated regulation of genes involved in meiosis, through E2F6-dependant methylation and transcriptional silencing in somatic tissues.

Ebselen oxide and derivatives are new allosteric HER2 inhibitors for HER2-positive cancers.

Human epidermal growth factor receptor 2 (ErbB2/HER2) is a tyrosine kinase receptor that is overexpressed in 25% of primary human breast cancers, as well as in multiple other cancers. HER2-targeted therapies improved progression-free and overall survival in patients with HER2+ breast cancers. However, associated resistance mechanisms and toxicity highlight the need for new therapeutic approaches for these cancers. We recently established that, in normal cells, HER2 is stabilized in a catalytically repressed state by direct interaction with members of the ezrin/radixin/moesin (ERM) family. In HER2-overexpressing tumors, the low expression of moesin contributes to the aberrant activation of HER2. Through a screen designed to find moesin-mimicking compounds, we identified ebselen oxide. We show that ebselen oxide, and some derivatives, conferred an efficient allosteric inhibition of overexpressed HER2, as well as mutated and truncated oncogenic forms of HER2, which are resistant to current therapies. Ebselen oxide selectively inhibited anchorage-dependent and -independent proliferation of HER2+ cancer cells and showed a significant benefit in combination with current anti-HER2 therapeutic agents. Finally, ebselen oxide significantly blocked HER2+ breast tumor progression in vivo. Collectively, these data provide evidence that ebselen oxide is a newly identified allosteric inhibitor of HER2 to be considered for therapeutic intervention on HER2+ cancers.

Exposure of human cerebral microvascular endothelial cells hCMEC/D3 to laminar shear stress induces vascular protective responses.

Endothelial cells (ECs) are constantly submitted in vivo to hemodynamical forces derived from the blood circulation, including shear stress (SS). ECs are able to detect SS and consequently adapt their phenotype, thus affecting many endothelial functions. If a plethora of shear stress-regulated molecular networks have been described in peripheral ECs, less is known about the molecular responses of microvascular brain ECs which constitute the blood-brain barrier (BBB). In this work, we investigated the response of human cerebral microvascular ECs to laminar physiological shear stress using the well characterized hCMEC/D3 cell line. Interestingly, we showed that hCMEC/D3 cells responded to shear stress by aligning perpendicularly to the flow direction, contrary to peripheral endothelial cells which aligned in the flow direction. Whole proteomic profiles were compared between hCMEC/D3 cells cultured either in static condition or under 5 or 10 dyn.cm-2 SS for 3 days. 3592 proteins were identified and expression levels were significantly affected for 3% of them upon both SS conditions. Pathway analyses were performed which revealed that most proteins overexpressed by SS refer to the antioxidant defense, probably mediated by activation of the NRF2 transcriptional factor. Regarding down-regulated proteins, most of them participate to the pro-inflammatory response, cell motility and proliferation. These findings confirm the induction of EC quiescence by laminar physiological SS and reveal a strong protective effect of SS on hCMEC/D3 cells, suggesting a similar effect on the BBB. Our results also showed that SS did not significantly increase expression levels nor did it affect the localization of junctional proteins and did not afect either the functional activity of several ABC transporters (P-glycoprotein and MRPs). This work provides new insights on the response of microvascular brain ECs to SS and on the importance of SS for optimizing in vitro BBB models.

Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase.

Non-coding RNAs are emerging as key players in many fundamental biological processes, including specification of higher-order chromatin structure. We examined the implication of RNA transcribed from mouse centromeric minor satellite repeats in the formation and function of centromere-associated complexes. Here we show that the levels of minor satellite RNA vary during cell-cycle progression, peaking in G2/M phase, concomitant with accumulation of proteins of the chromosomal passenger complex near the centromere. Consistent with this, we describe that murine minor satellite RNA are components of CENP-A-associated centromeric fractions and associate with proteins of the chromosomal passenger complex Aurora B and Survivin at the onset of mitosis. Interactions of endogenous Aurora B with CENP-A and Survivin are sensitive to RNaseA. Likewise, the kinase activity of Aurora B requires an RNA component. More importantly, Aurora B kinase activity can be potentiated by minor satellite RNA. In addition, decreased Aurora B activity after RNA depletion can be specifically rescued by restitution of these transcripts. Together, our data provide new functional evidence for minor satellite transcripts as key partners and regulators of the mitotic kinase Aurora B.

Meningococcus, this famous unknown.

Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for two devastating forms of invasive diseases: purpura fulminans and meningitis. Since the first description of the epidemic nature of the illness at the dawn of the nineteenth century, the scientific knowledge of meningococcal infection has increased greatly. Major advances have been made in the management of the disease with the advent of antimicrobial therapy and the implementation of meningococcal vaccines. More recently, an extensive knowledge has been accumulated on meningococcal interaction with its human host, revealing key processes involved in disease progression and new promising therapeutic approaches.

Neisseria meningitidis (méningocoque) est une bactérie à Gram négatif responsable de deux formes gravissimes de maladies invasives : le purpura fulminans et la méningite. Depuis la première description du caractère épidémique de la maladie à l'aube du 19e siècle, les connaissances scientifiques sur les infections méningococciques ont considérablement augmenté. Des progrès majeurs ont été réalisés dans la gestion de la maladie avec l'avènement des agents antimicrobiens et le développement de vaccins contre le méningocoque. De nombreuses connaissances ont récemment été accumulées sur son interaction avec l'être humain, son unique hôte, révélant les processus clés impliqués dans la progression de la maladie et de nouvelles approches thérapeutiques prometteuses.

Allosteric Inhibition of HER2 by Moesin-Mimicking Compounds Targets HER2-Positive Cancers and Brain Metastases.

Therapies targeting the tyrosine kinase receptor HER2 have significantly improved survival of patients with HER2+ cancer. However, both de novo and acquired resistance remain a challenge, particularly in the brain metastatic setting. Here we report that, unlike other HER tyrosine kinase receptors, HER2 possesses a binding motif in its cytosolic juxtamembrane region that allows interaction with members of the Ezrin/Radixin/Moesin (ERM) family. Under physiologic conditions, this interaction controls the localization of HER2 in ERM-enriched domains and stabilizes HER2 in a catalytically repressed state. In HER2+ breast cancers, low expression of Moesin correlated with increased HER2 expression. Restoring expression of ERM proteins in HER2+ breast cancer cells was sufficient to revert HER2 activation and inhibit HER2-dependent proliferation. A high-throughput assay recapitulating the HER2-ERM interaction allowed for screening of about 1,500 approved drugs. From this screen, we found Zuclopenthixol, an antipsychotic drug that behaved as a Moesin-mimicking compound, because it directly binds the juxtamembrane region of HER2 and specifically inhibits HER2 activation in HER2+ cancers, as well as activation of oncogenic mutated and truncated forms of HER2. Zuclopenthixol efficiently inhibited HER2+ breast tumor progression in vitro and in vivo and, more importantly, showed significant activity on HER2+ brain tumor progression. Collectively, these data reveal a novel class of allosteric HER2 inhibitors, increasing the number of approaches to consider for intervention on HER2+ breast cancers and brain metastases. SIGNIFICANCE: This study demonstrates the functional role of Moesin in maintaining HER2 in a catalytically repressed state and provides novel therapeutic approaches targeting HER2+ breast cancers and brain metastasis using Moesin-mimicking compounds.

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.

Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/ß2-adrenoceptor clusters assembled by alpha-actinin-4.

Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist haemodynamic forces. Two endothelial receptors, CD147 and the ß2-adrenergic receptor (ß2AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and ß2AR form constitutive hetero-oligomeric complexes. The scaffolding protein α-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147-ß2AR complexes in highly ordered clusters at bacterial adhesion sites. This multimolecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host-pathogen interaction.

Comprehensive Identification of Meningococcal Genes and Small Noncoding RNAs Required for Host Cell Colonization.

UNLABELLED: Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N. meningitidis life cycle that is poorly understood. Here, highly saturated random transposon insertion libraries of N. meningitidis were engineered, and the fitness of mutations during routine growth and that of colonization of endothelial and epithelial cells in a flow device were assessed in a transposon insertion site sequencing (Tn-seq) analysis. This allowed the identification of genes essential for bacterial growth and genes specifically required for host cell colonization. In addition, after having identified the small noncoding RNAs (sRNAs) located in intergenic regions, the phenotypes associated with mutations in those sRNAs were defined. A total of 383 genes and 8 intergenic regions containing sRNA candidates were identified to be essential for growth, while 288 genes and 33 intergenic regions containing sRNA candidates were found to be specifically required for host cell colonization. IMPORTANCE: Meningococcal meningitis is a common cause of meningitis in infants and adults. Neisseria meningitidis (meningococcus) is also a commensal bacterium of the nasopharynx and is carried by 3 to 30% of healthy humans. Under some unknown circumstances, N. meningitidis is able to invade the bloodstream and cause either meningitis or a fatal septicemia known as purpura fulminans. The onset of symptoms is sudden, and death can follow within hours. Although many meningococcal virulence factors have been identified, the mechanisms that allow the bacterium to switch from the commensal to pathogen state remain unknown. Therefore, we used a Tn-seq strategy coupled to high-throughput DNA sequencing technologies to find genes for proteins used by N. meningitidis to specifically colonize epithelial cells and primary brain endothelial cells. We identified 383 genes and 8 intergenic regions containing sRNAs essential for growth and 288 genes and 33 intergenic regions containing sRNAs required specifically for host cell colonization.

Jak2 and proteasome activities control the availability of cell surface growth hormone receptors during ligand exposure.

Several mechanisms participate in the down-regulation of growth hormone receptor (GHR) signalling under ligand exposure. In CHO cells expressing GHR, we show that ligand stimulation induces degradation of the total cell GHR content. Experiments with 125I-hGH indicate that ligand-bound internalized receptors are not immediately replaced. Using cell surface biotinylation, we demonstrate for the first time that, concomitantly with the degradation of cell surface receptors, GHRs from the intracellular compartments are also degraded. We thus suggest that under prolonged ligand exposure, some GHRs are targeted to the cell surface, while others are routed to degradation compartments. Inhibitors of Jak2 and of the proteasome partially inhibited degradation of cell surface receptors, while these compounds completely inhibit the degradation of intracellular GHRs, resulting in their accumulation. We therefore propose that Jak2 and proteasome activities control the amount of intracellular GHRs, and thus the availability of receptors at the cell surface, during ligand exposure.

Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization.

Neisseria meningitidis is a cause of meningitis epidemics worldwide and of rapidly progressing fatal septic shock. A crucial step in the pathogenesis of invasive meningococcal infections is the adhesion of bloodborne meningococci to both peripheral and brain endothelia, leading to major vascular dysfunction. Initial adhesion of pathogenic strains to endothelial cells relies on meningococcal type IV pili, but the endothelial receptor for bacterial adhesion remains unknown. Here, we report that the immunoglobulin superfamily member CD147 (also called extracellular matrix metalloproteinase inducer (EMMPRIN) or Basigin) is a critical host receptor for the meningococcal pilus components PilE and PilV. Interfering with this interaction potently inhibited the primary attachment of meningococci to human endothelial cells in vitro and prevented colonization of vessels in human brain tissue explants ex vivo and in humanized mice in vivo. These findings establish the molecular events by which meningococci target human endothelia, and they open new perspectives for treatment and prevention of meningococcus-induced vascular dysfunctions.

Accumulation of small murine minor satellite transcripts leads to impaired centromeric architecture and function.

RNAs have been implicated in the assembly and stabilization of large-scale chromatin structures including centromeric architecture; unidentified RNAs are integral components of human pericentric heterochromatin and are required for localization of the heterochromatin protein HP1 to centromeric regions. Because satellite repeats in centromeric regions are known to be transcribed, we assessed a role for noncoding centromeric RNAs in the structure and function of the centromere. We identified minor satellite transcripts of 120 nt in murine cells that localize to centromeres and accumulate upon stress or differentiation. Forced accumulation of 120-nt transcripts leads to defects in chromosome segregation and sister-chromatid cohesion, changes in hallmark centromeric epigenetic markers, and mislocalization of centromere-associated proteins essential for centromere function. These findings suggest that small centromeric RNAs may represent one of many pathways that regulate heterochromatin assembly in mammals, possibly through tethering of kinetochore- and heterochromatin-associated proteins.