Vascular colonization and role of type IV pili retraction in a humanized mouse model of meningococcal sepsis

Neisseria meningitidis (le méningocoque) est une bactérie commensale dont la niche écologique est le rhinopharynx humain. C'est l'agent étiologique des méningites méningococciques et des meningococcémies aiguës, dont la forme la plus sévère est le purpura fulminans. N. meningitidis peut, dans certaines conditions, franchir la barrière épithéliale et atteindre la circulation sanguine. Il est alors capable d'adhérer à l'endothélium et de coloniser les microvaisseaux selon un mécanisme dépendant des pili de type IV. Les pili de type IV sont des facteurs de virulence clefs et sont associés à de nombreuses propriétés dont la motilité bactérienne, l'interaction entre les bactéries et l'adhésion aux cellules hôte. Ce sont des structures dynamiques et rétractables sous l'effet de l'ATPase PilT. Dans ce travail, nous avons tiré parti d'un modèle murin humanisé (souris SCID greffées avec de la peau humaine) pour étudier le rôle de la colonisation vasculaire et de la rétraction des pili de type IV dans la physiopathologie des infections à méningocoque in vivo. Nos résultats montrent que N. meningitidis a une forte affinité pour les microvaisseaux humains et prolifère avec une efficacité remarquable à leur contact. Pour identifier les mécanismes moléculaires impliqués dans la virulence bactérienne pendant la colonisation vasculaire, nous avons réalisé une analyse à haut débit chez des souris greffées et non greffées par séquençage des sites d'insertion d'un transposon d'une librairie de mutants (Tn-seq). Nos résultats montrent que 36 % des gènes importants pour la croissance dans le sang des souris ne sont pas essentiels lorsque les bactéries colonisent les vaisseaux sanguins. Une majorité de ces gènes sont impliqués dans le métabolisme et dans l'import de nutriments, ce qui suggère que les cellules endothéliales humaines constituent une niche nutritionnelle pour N. meningitidis. Nous avons ensuite montré qu'un mutant pilT ne rétractant pas les pili de type IV colonise efficacement le système vasculaire du greffon, mais n'est pas capable de maintenir une bactériémie soutenue et de tuer les souris. Ceci est dû à une altération de la capacité du mutant à se détacher des microcolonies adhérentes à l'endothélium. La colonisation vasculaire dépendante des pili de type IV étant un élément central de la pathogenèse du méningocoque, elle constitue une cible intéressante pour le développement de nouvelles stratégies thérapeutiques. Dans la dernière partie de ce travail, nous avons évalué le potentiel thérapeutique de la trifluopérazine et la thioridazine, médicaments antipsychotiques de première génération appartenant à la famille des phénothiazines, qui affectent la fonctionnalité des pili de type IV. Nos résultats montrent que ces molécules diminuent rapidement la piliation et l'agrégation bactérienne in vitro. In vivo, elles ont un fort effet protecteur en réduisant la colonisation vasculaire méningococcique, en prévenant les dommages vasculaires (thrombose, lésions endothéliales) et en réduisant l'inflammation. De plus, la thioridazine améliore la survie des souris dans notre modèle. Ce travail montre que la colonisation vasculaire est une étape centrale dans la pathogenèse méningococcique in vivo constituant une niche pour la multiplication bactérienne. La rétraction des pili de type IV est nécessaire pour le relargage du méningocoque dans la circulation sanguine et la virulence. Cibler la colonisation vasculaire par des composés phénothiaziniques interférant avec les type IV pili pourrait avoir un effet bénéfique dans le traitement des infections invasives à méningocoque.Neisseria meningitidis (the meningococcus) is a commensal bacterium whose ecological niche is the human nasopharynx. It is the causative agent of meningococcal meningitis and acute meningococcemia, also known as purpura fulminans in its most severe form. N. meningitidis is able under certain conditions to cross the epithelial barrier and invade the bloodstream. From there, N. meningitidis adhere onto and colonize the microvessels in a type IV pili-dependent process. Type IV pili are key virulence factors that are associated with numerous properties including bacterial motility, interaction between bacteria and adhesion onto host cells. They are dynamic structures that are retractable through the effect of PilT ATPase. In this work, we took advantage of a humanized model of SCID mice grafted with human skin to address the role of meningococcal vascular colonization and type IV pilus retraction in the pathogenesis of meningococcal disease in vivo. Our results show that meningococci have a high affinity for human vasculature and proliferate with a remarkable efficacy upon infection within human microvessels. To identify the molecular pathways involved in bacterial virulence during vascular colonization, we performed transposon insertion site sequencing (Tn-seq) in grafted and non-grafted SCID mice. Our results show that 36% of the genes that are important for growth in the blood of mice are dispensable when bacteria colonize blood vessels. Most of those genes are involved in metabolism or nutrient uptake, suggesting that in vivo human endothelial cells constitute a feeding niche for N. meningitidis. We then show that a pilT mutant unable to retract type IV pili colonize efficiently the graft vasculature, but fail to maintain a sustained bacteremia and don't kill the mice. This is due to an impaired ability of pilus-retraction deficient bacteria to release from adhesive microcolonies. As type IV pili-dependent vascular colonization is a central feature in meningococcal pathogenesis, it is an attractive target for the development of novel therapeutic strategies. In the final part of this work, we evaluate trifluoperazine and thioridazine, first generation antipsychotic drugs belonging to the family of phenothiazines, that are affecting type IV pili functionality. We show that these molecules rapidly decrease piliation and bacterial aggregation in vitro. In vivo, they exert a strong protective effect by reducing meningococcal vascular colonization, preventing subsequent vascular damages (thrombosis, endothelial disruption) and reducing inflammation. Furthermore, thioridazine improves the outcome of meningococcal sepsis. Altogether, our work show that vascular colonization is a key feature in meningococcal pathogenesis in vivo providing a niche for meningococcal multiplication and that type IV pili retraction is required for meningococcal spreading through the bloodstream and virulence. Finally, we show that targeting vascular colonization by phenothiazine compounds interfering with type IV pili could beneficially participate in the treatment of invasive meningococcal diseases

Differential subcellular distribution and transcriptional activity of sigmaE3, sigmaE4, and sigmaE3-4 isoforms of the rat estrogen receptor-alpha.

E3, E4, and E3-4 are naturally occurring estrogen receptor (ER) isoforms, generated through differential splicing of the ERalpha primary transcript and abundantly expressed in embryonic rat pituitary. Studies in COS cells transfected with full-length ERalpha or its three splice variants fused to green fluorescent protein (GFP), revealed a different subcellular localization for each isoform. In the absence of estradiol, full-length ERalpha-GFP was predominantly nuclear, and E3-GFP and E4-GFP were present both in cytoplasm and nucleus, whereas E3-4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistribution of full-length ERalpha-GFP and E3-GFP, from a diffuse to punctate pattern, occurred within the nucleus. In contrast, the distribution of E4-GFP and E3-4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-alpha and E3 displayed the same hormone-induced ability to tether to nuclear matrix, whereas nuclear E4 appeared to remain loosely associated to functional nuclear constituents. When cotransfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER-negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, E3-4 exhibited a very weak estrogen-dependent transactivation activity, whereas E3 had an inhibitory effect on full-length ER action. Conversely, E4 displayed estrogen-independent transcriptional activity in ER-negative cells, and in ER-positive cells, enhanced the estrogen-induced gene expression as efficiently as full-length ERalpha. In a gel mobility shift assay, phosphorylated E4 was able to form a specific complex with a consensus ERE, while E3 and E3-4 never did bind by themselves. The observed inhibitory action of E3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodimers with full-length ERalpha, as suggested by immunoprecipitation followed by Western blotting. These data suggest that E3 and E4 may play a physiologically relevant role as negative or constitutively positive modulators of transcription, in the developing rat pituitary

Raf-1 and B-Raf proteins have similar regional distributions but differential subcellular localization in adult rat brain.

The Raf kinases play an important and specific role in the activation of extracellular signal-regulated kinases (ERK) cascade. Beside its role in the control of proliferation and differentiation, the ERK cascade has also been implicated in neuron-specific functions. In order to gain clues on the function of Raf kinases in the adult central nervous system (CNS), we performed a comparative analysis of the distribution and subcellular localization of the different Raf kinases in rat brain with antibodies specific for the different Raf kinases. We show that B-Raf and Raf-1 proteins are present in most brain areas, whereas A-Raf is not detected. Interestingly, the two Raf proteins have an approximately similar pattern of distribution with a rostro-caudal decreasing gradient of expression. These two kinases are colocalized in neurons but they are differentially located in subcellular compartments. Raf-1 is localized mainly in the cytosolic fraction around the nucleus, whereas B-Raf is widely distributed in the cell bodies and in the neuritic processes. In addition, we demonstrated that numerous B-Raf isoforms are present in the brain. These isoforms have a differential pattern of distribution, some of them being ubiquitously expressed whereas others are localized to specific brain areas. These isoforms also have a clear differential subcellular localization, specially in Triton-insoluble fractions, but also in synaptosomal, membrane and cytosolic compartments. Altogether these results suggest that each Raf protein could have a distinct signalling regulatory function in the brain with regard to its subcellular localization

N-terminal deletion in the src gene of Rous sarcoma virus results in synthesis of a 45,000-Mr protein with mitogenic activity.

International audienceExpression of the v-src gene of Rous sarcoma virus in avian embryo neuroretina cells results in transformation and sustained proliferation of these normally resting cells. Transformed neuroretina cells are also tumorigenic upon inoculation into immunodeficient hosts. We have previously described conditional mutants of Rous sarcoma virus encoding p60v-src proteins which induce proliferation of neuroretina cells in the absence of transformation and tumorigenicity. These results suggest that p60v-src is composed of functionally distinct domains which may interact with multiple cellular targets. In this study, we describe a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion of 278 base pairs in the 5' portion of the v-src gene but which has retained the ability to induce proliferation of quail neuroretina cells. The deleted v-src gene encodes a 45,000-molecular-weight phosphoprotein which contains both phosphoserine and phosphotyrosine, is myristylated, and possesses tyrosine kinase activity indistinguishable from that of wild-type p60v-src. Molecular cloning and sequence analysis of the mutant v-src gene have shown that this deletion extends from amino acid 33 to 126 of the wild-type p60v-src. Therefore, this portion of the v-src protein is dispensable for the mitogenic activity of Rous sarcoma virus in neuroretina cells

Characterization of Pseudomonas aeruginosa L,D-transpeptidases and evaluation of their role in peptidoglycan adaptation to biofilm growth

International audiencePeptidoglycan is an essential component of the bacterial cell envelope that sustains the turgor pressure of the cytoplasm, determines cell shape, and acts as a scaffold for the anchoring of envelope polymers such as lipoproteins. The final cross-linking step of peptidoglycan polymerization is performed by classical d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family and by l,d-transpeptidases (LDTs), which are dispensable for growth in most bacterial species and whose physiological functions remain elusive. In this study, we investigated the contribution of LDTs to cell envelope synthesis in Pseudomonas aeruginosa grown in planktonic and biofilm conditions. We first assigned a function to each of the three P. aeruginosa LDTs by gene inactivation in P. aeruginosa, heterospecific gene expression in Escherichia coli, and, for one of them, direct determination of its enzymatic activity. We found that the three P. aeruginosa LDTs catalyze peptidoglycan cross-linking (LdtPae1), the anchoring of lipoprotein OprI to the peptidoglycan (LdtPae2), and the hydrolysis of the resulting peptidoglycan-OprI amide bond (LdtPae3). Construction of a phylogram revealed that LDTs performing each of these three functions in various species cannot be assigned to distinct evolutionary lineages, in contrast to what has been observed with PBPs. We showed that biofilm, but not planktonic bacteria, displayed an increase proportion of peptidoglycan cross-links formed by LdtPae1 and a greater extent of OprI anchoring to peptidoglycan, which is controlled by LdtPae2 and LdtPae3. Consistently, deletion of each of the ldt genes impaired biofilm formation and potentiated the bactericidal activity of EDTA. These results indicate that LDTs contribute to the stabilization of the bacterial cell envelope and to the adaptation of peptidoglycan metabolism to growth in biofilm. IMPORTANCE Active-site cysteine LDTs form a functionally heterologous family of enzymes that contribute to the biogenesis of the bacterial cell envelope through formation of peptidoglycan cross-links and through the dynamic anchoring of lipoproteins to peptidoglycan. Here, we report the role of three P. aeruginosa LDTs that had not been previously characterized. We show that these enzymes contribute to resistance to the bactericidal activity of EDTA and to the adaptation of cell envelope polymers to conditions that prevail in biofilms. These results indicate that LDTs should be considered putative targets in the development of drug-EDTA associations for the control of biofilm-related infections

Transformation-defective mutants with 5′ deletions of the src gene are frequently generated during replication of rous sarcoma virus in established quail fibroblasts

International audienceReplication of Rous sarcoma virus (RSV) in avian fibroblasts leads to the generation of replication-competent variants that are defective for cell transformation (td virus). These td variants contain deletions affecting various portions of the v-src gene. We compared the rate of td virus production in Q3B cells, a quail cell line established by mutagen treatment, and in normal quail fibroblasts. Twenty-five days after infection with an RSV stock containing only transforming virions, Q3B cells harbor similar amounts of v-src-containing and v-src-deleted proviruses. However, these cells synthesize very low levels of p60v-src and generate large excess of td variants, as determined by biological assays. Unlike Q3B cells, normal quail fibroblasts infected with the same virus stock produce td variants only after multiple passages of undiluted virus on fresh cells. Restriction analysis showed that the td virus produced by Q3B cells is composed of two types of genomes: one lacking the entire v-src gene and the other carrying partial deletions of this gene predominantly located in the amino-terminal portion of the coding region of v-src. To study the mechanisms of these partial deletions, we molecularly cloned and sequenced the v-src genes of several td proviruses. We show that these mutants carry single or multiple v-src deletions of limited size, presumably generated by multiple mechanisms. Two deletions of 170 and 112 bp located in the 5' portion of v-src are frequently generated during RSV replication in Q3B cells and may represent preferential sites for v-src deletion in these cells

An ADAM-10 dependent EPCR shedding links meningococcal interaction with endothelial cells to purpura fulminans.

Purpura fulminans is a deadly complication of Neisseria meningitidis infections due to extensive thrombosis of microvessels. Although a Disseminated Intra-vascular Coagulation syndrome (DIC) is frequently observed during Gram negative sepsis, it is rarely associated with extensive thrombosis like those observed during meningococcemia, suggesting that the meningococcus induces a specific dysregulation of coagulation. Another specific feature of N. meningitidis pathogenesis is its ability to colonize microvessels endothelial cells via type IV pili. Importantly, endothelial cells are key in controlling the coagulation cascade through the activation of the potent anticoagulant Protein C (PC) thanks to two endothelial cell receptors among which the Endothelial Protein C Receptor (EPCR). Considering that congenital or acquired deficiencies of PC are associated with purpura fulminans, we hypothesized that a defect in the activation of PC following meningococcal adhesion to microvessels is responsible for the thrombotic events observed during meningococcemia. Here we showed that the adhesion of N. meningitidis on endothelial cells results in a rapid and intense decrease of EPCR expression by inducing its cleavage in a process know as shedding. Using siRNA experiments and CRISPR/Cas9 genome edition we identified ADAM10 (A Disintegrin And Metalloproteinase-10) as the protease responsible for this shedding. Surprisingly, ADAM17, the only EPCR sheddase described so far, was not involved in this process. Finally, we showed that this ADAM10-mediated shedding of EPCR induced by the meningococcal interaction with endothelial cells was responsible for an impaired activation of Protein C. This work unveils for the first time a direct link between meningococcal adhesion to endothelial cells and a severe dysregulation of coagulation, and potentially identifies new therapeutic targets for meningococcal purpura fulminans