Screening for NSm function in infected mammalian cells by proteomic analysis.

Lassa virus (LASV) is an arenavirus causing hemorrhagic fever in human. 300 000 to 500 000 cases of LASV infection are reported every year in western Africa, including 5 000 to 6 000 deaths. LASV is highly pathogenic, and no vaccine or treatment is available in endemic areas. LASV pathogenesis mechanisms are not well documented, and further investigations are needed to understand viral and immunological factors involved during infection. As previously shown by studies conducted on patients and non-human primates infected by LASV, type I interferon (IFN-I) is important for an effective response to LASV infection. As plasmacytoid DC are specialized in IFN-I response, they could be important for an efficient response to LASV infection.

The post-translational modification by SUMO is an essential regulatory mechanism of protein function that is involved in most challenges faced by eukaryotic cells. Gene expression is particularly regulated by sumoylation as many SUMO substrates are transcription factors and chromatin-associated proteins, including histones. The emerging paradigm for the proposed work is that sumoylation controls multiple aspects of chromatin structure and function in response to external cues. According to this view, sumoylation is expected to impact both global and specific transcriptional programs thereby affecting constitutive and inducible expression of both coding and non coding genes. Recently, we found SUMO as an integral and instructive component of chromatin in cell growth, senescence and cancer, thus establishing sumoylation as a new and paradigmatic chromatin modification. This work now paves the way for detailed understanding of the contribution of SUMO as a multifaceted modifier of chromatin.

Gene ontology analysis of RNAseq data from uninfected and Leishmania-infected mouse macrophages.  Scientific context During the course of cutaneous or visceral disease in humans or experimental animal models, the resolution of leishmanial infections or the control of parasite growth is dependent on appropriate innate and adaptive immune responses developed by the parasitized host. Leishmania largely evades and subverts these responses by its intracellular life style inside the mammalian host, where the parasites develop into amastigotes mainly within macrophages (BMDMs). We have focused our interest in the BMDM inflammasome and the way Leishmania amastigotes interfere or subvert BMDM inflammatory responses. Our recent data are in favor of an absence of stimulation, even a down-regulation of the inflammasome in BMDMs harboring replicating amastigotes at the gene and protein expression levels. To go further on this, we have performed RNAseq experiments on uninfected and infected BMDMs. This project was done at the “Transcriptome and Epigenome” platform and in close collaboration with the C3BI for normalization and statistical analysis procedures. Objective In the present proposal we will perform a deep analysis of the repartition of modulated genes between the different conditions using these RNAseq data. Using C3Bi expertise we will classify known functions of modulated genes into GO aspects i.e. molecular function, cellular component and biological process, visualize gene annotations and perform statistical analyses for the distribution of the annotated genes over the GO hierarchy for the different gene lists analyzed. Hopefully, these analyses will bring us a better understanding of the mechanisms underlying the subversion of BMDM functions in the innate and adaptive immune response to Leishmania infection which is a prerequisite to design novel anti-parasitic intervention strategies targeting the infected host cell rather than the parasite.

Because of their increasing incidence, dramatic severity, lack of treatment or vaccine, complicated diagnosis, misreading of the pathogenesis, and need for a maximum containment, Viral Hemorrhagic Fevers (VHF) constitute a major public health problem. There is therefore an urgent need to further study VHF to understand the pathogenesis of the severe disease and the host responses involved in their control or in the dramatic damages. Among VHF, Lassa fever (LF) is probably the most worrying one because of its endemicity and the large number of cases. LF is caused by the Old-World arenavirus Lassa virus (LASV). It is endemic to West Africa and is responsible for 300,000 cases and 5,000 to 6,000 deaths each year. We propose here to study the pathogenesis of VHF by using LF in cynomolgus monkeys as a paradigm, with a particular emphasis on the very early events. The viral tropism, pathophysiological mechanisms, and immune responses will be studied during the course of infection, including the incubation period. Powerful approaches will be used to (1) identify early biological markers of infection, to be able to confirm infection and isolate patients; (2) determine the viral tropism and dynamics during the course of infection to understand the natural history of virus into its host. (3) characterize the early pathogenic events that lead to the severe hemorrhagic syndrome to fully understand the pathophysiogenesis of VHF and identify new therapeutic targets. (4) identify the immune responses involved in the control of infection or in the fatal outcome, to reveal the involvement of immunopathological mechanisms and help to design a vaccine approach. This ambitious and unprecedented project will allow to develop therapeutic and prophylactic approaches but also to identify early biological markers of infection and improve the early diagnosis to optimize the management of outbreaks in the field and increase the survival rate in patients.

The liver is the biggest internal organ that is responsible for metabolism, detoxification and the defense between the host and the external environment. Thus the liver is also an immune organ, which constantly confronts conflicting demands of immunity against pathogens and tolerance to antigens metabolized locally and bacteria products derived from the gut. In homeostasis, many mechanisms ensure suppression of immune response in the liver, which include low expression levels of MHC molecules in liver antigen presenting cells (APC), increased expression of inhibitory molecules and sustained expression of suppressive cytokines such as IL-10 and TGF-b. Thus, several pathogens including hepatitis B virus (HBV) shrewdly exploit the tolerant immune environment in the liver to establish chronic infection. However, inflammation can elicit efficient immune response. To investigate immune response to HBV in the context of inflammation, we used Mdr2^-/- mice, which develop cholestatic inflammation in the liver. We show that after injection of HBV to the mice, WT animals develop chronic infection whereas Mdr2^-/- mice eliminate the virus. We detected robust viral-specific antibody production in Mdr2^-/- mice but not in WT mice. Kupffer cells (KC) are liver-resident macrophages and important APCs, expressing MHC I, MHC II and costimulatory molecules required for T cell activation. The efficient immune response against HBV observed in Mdr2^-/- mice prompts us to profile gene expression in Kupffer cells derived from these mice and compare to those from WT mice at the naïve state.

Blood cells were collected in the field from patients admited in the Ebola treatment center of Macenta (Guinea). After red cells lysis, blood cells were frozen and RNA was extracted after the shipment of samples in France. Despite poor RNA quality, Affimetrix chips have been performed and transcriptomic data are available. The aim of this study is to compare the transcriptomic data from patients who survived and those who died and also to perform a kinetic analysis of the infection in the two groups of patients.  Samples from febrile patients who were not infected with Ebola virus have been used as negative controls.

Innate lymphoid cells (ILCs) are the most recently identified components of the innate immune system. ILCs colonize different tissue sites and react promptly to microenvironmental perturbations. Due to their high plasticity, ILCs can shape their functional output in response to local cues. As such, ILCs play roles under homeostatic conditions and in the context of infection, chronic inflammation, metabolic diseases and cancer. Diverse ILC subsets (NK cells, ILC2) have been shown to regulate the metabolic homeostasis. Metabolic states affect cellular functions and have been shown to play an important role in the regulation of adaptive immunity. In contrast, almost nothing is known about innate lymphocytes metabolism and the importance of energy regulation for ILC function. This project will study metabolic profiles in human ILC subsets under diverse environmental conditions. Enhancing or interfering with ILC activity could ultimately represent a novel useful therapy for chronic inflammatory diseases.

Ce projet a pour but de réaliser une analyse par voies fonctionnelles d’environ 600 gènes impliqués dans la réponse immunitaire chez l’homme avec une attention particulière vis-à-vis des gènes impliqués dans le contrôle de l’inflammation et de l’auto-immunité.

Streptococcus gallolyticus sous espèce gallolyticus, autrefois dénommée Streptococcus bovis biotype I, est une bactérie de la flore intestinale qui constitue une cause émergente de septicémies et d’endocardites chez les personnes âgées. Depuis plus de 50 ans, les études épidémiologiques indiquent l’existence d’une forte association entre les infections invasives à S. gallolyticus et le CCR (cancer colorectal) (Pasquereau-Kotula et al., 2018). Néanmoins, le rôle exact de S.gallolyticus dans le développement du cancer colorectal n'est toujours pas déterminé. Dans notre projet, nous voulons déterminer si S.gallolyticus, une bactérie commensale du colon, est à l’origine du cancer colorectal comme Helicobacter pylori est l’agent étiologique du cancer de l’estomac. Précédemment, notre l’équipe a montré que cette bactérie est aussi capable de profiter de l’environnement tumoral pour se multiplier au détriment d’autres bactéries présentes dans le microbiote de souris comme Enterococcus faecalis (Aymeric et al., 2018). L'hypothèse alternative étant que cette bactérie profite de l’environnement tumoral pour se multiplier au détriment des autres populations bactériennes et provoquer des endocardites suite au relâchement de la barrière intestinale provoquée par la tumeur. L’objectif principal de ce projet de recherche est de mieux comprendre le rôle de S. gallolyticus dans les CCR. L’étude transcriptomique va nous permettre de déterminer impact de S. gallolyticus sur les cellules intestinalles non transformées vs cellules tumorales. Cette l’étude aidera à identifier les gènes altérés par S. gallolyticus qui pourraient à long terme contribuer à la progression de CCR et/ou à oncogénique des cellules normales. La réponse transcriptionnelle induite par S.gallolyticus sur les cellules humaines (normale et canceresuse) permettra de mieux comprendre les mécanismes moléculaires qui sous-tendent le rôle de S.gallolyticus dans le développement du cancer colorectal.

Streptococcus gallolyticus sous espèce gallolyticus, autrefois dénommée Streptococcus bovis biotype I, est une bactérie de la flore intestinale qui constitue une cause émergente de septicémies et d’endocardites chez les personnes âgées. Depuis plus de 50 ans, les études épidémiologiques indiquent l’existence d’une forte association entre les infections invasives à S. gallolyticus et le CCR (cancer colorectal) (Pasquereau-Kotula et al., 2018). Néanmoins, le rôle exact de S.gallolyticus dans le développement du cancer colorectal n'est toujours pas déterminé. L’objectif principal de ce projet de recherche est de mieux comprendre le rôle de S. gallolyticus dans les CCR. Pour vérifier le rôle du S. gallolyticus dans le dévalement du CCR on a utilisé un modèle de cancer colorectal chez la souris (A/J + AOM). Grace à ce model o a peux démonter que S. gallolyticus accélère la tumorigenèse. Pour essayer de trouver un mechanism responsable de ce effect nous avons réalisé un protéome et un phosphoprotéome complets sur des tissus de souris colonisés par bactérie contrôle ou S. gallolyticus. L'analyse des changements dans le total et le phospho protéome aidera à mettre en évidence le mécanisme pertinent de la cancérogenèse induite par le S. gallolyticus.

Listeriolysin S (LLS) is a bacteriocin from Listeria monocytogenes that targets other Gram-positive bacteria, including Listeria monocytogenes itself. In order to understand the mechanism of action of LLS on target bacteria, we exposed the LLS-susceptible L. monocytogenes strain 10430S to the bacteriocin. We identified the proteome of the target bacteria with the goal of understanding which metabolic pathways are upregulated upon exposure to LLS.

Left-right asymmetry of the heart is essential for establishing the double blood circulation. Impairment of left-right embryo patterning is associated with severe congenital heart defects. During embryonic development, the heart initially forms as a straight tube. While it elongates, it acquires the shape of a rightward helix, a process referred to as heart looping. Previous work suggests that dynamic signaling in the heart precursor cells are essential for heart looping completion, but so far only a few genes have been identified as asymmetrically expressed. In order to investigate the dynamic spatiotemporal patterning of heart precursors, and screen for novel players in asymmetric heart morphogenesis, we aim to perform several transcriptomic analyses.

The aim of this study is based on the analysis of the transcriptome during the infection with European bat lyssavirus type 1 (EBLV-1) in torpid bat. This study will be conducted using an in vitro model using another bat cell line from an European bat.

This project aims at characterizing the transcriptional signature of skeletal muscle during regeneration.

Bacterial toxins can induce a wide range of life threatening diseases and are at risk of bioweapon development due to their extreme toxicity and ease of production. Some AB-like toxins exploit the host cell endocytic machinery to enter into the cell cytosol where they exert their poisoning effects. Host-targeted therapy involving the development of small molecules interfering with key vesicular trafficking components hijacked by bacterial toxins offers to counteract toxin action. We have identified the small compound C910, in a cell-based High-throughput screen (HTS) followed by orthogonal screens described in (Mahtal N et al., 2018), for its cell-protective activity against CNF1 toxin from pathogenic Escherichia coli, as well as diphtheria and Shiga toxins. Therefore, to characterize C910 mechanism of action and find its cellular target we have realized an siRNA wide-genome screening on primary human cells. This screening aims to find genes that can interfere with the trafficking of the CNF1 toxin.

Nod2 is expressed on brain neurons, however the downstream effects of its activation by its ligand MDP are not know. We used RNAseq strategy to answer this question