We search for the expressed transcript in mature normal sperm as compared to the infertil one.

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 and senescence, 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.

La PF Transcriptome et Epigenome développe des projets de séquençage à haut débit (collaboration et service) avec des équipes du Campus. Ceux-ci couvrent l'ensemble des thématiques du campus ainsi qu'une large gamme d'organismes (des virus aux mammifères). La plate-forme exerce des activités de biologie humide (construction des librairies et séquençage) et de biologie sèche (analyse bioinformatiques et statistiques). La personne mise a disposition interagira étroitement avec les autres bioinformaticiens du pôle BioMics et du Hub. Ses activités concerneront notamment: - La participation à la conception et à la mise en place des projets avec les équipes demandeuses, la prise en charge des analyses et le reporting aux utilisateurs - La mise en place d'un workflow d'analyse bioinformatique des données de transcriptome /épigénome en étroite collaboration avec le C3BI, la DSI et les autres bioinformaticiens du pole. Ce workflow permettra le contrôle qualité des données, leur prétraitement, le mapping des séquences sur les génomes/transcriptomes de réference, et le comptage des reads pour les différents éléments de l'annotation - L'adaptation du workflow d'analyse aux questions biologiques et aux organismes étudiés dans le cadre des activités de la PF - L'activité de veille technologique et bibliographique (test et validation de nouveaux outils d'analyse, updates d'outils existants...) - La mise en place et le développement d'outils d'analyse adaptés aux futurs projets de la PF: single cell RNAseq, métatranscriptome, ChIPseq, analyse des isoformes de splicing.. Ceci se fera notamment via la réalisation d'analyses dédiées avec certains utilisateurs. Les outils mis en place et validés dans ce cadre seront ensuite utilisés pour l'ensemble des projets. - L'activité de communication et de formation (participation aux réunions du consortium France Génomique,formation permanente à l' Institut Pasteur… - la participation a d autres projets du Pole BioMics (selon disponibilité) Bernd Jagla, qui était le bioinformaticien de la plateforme a rejoint le Hub au 1er janvier 2016. Rachel Legendre est mise a disposition depuis le 2 novembre 2015 et remplace Bernd Jagla. Je souhaite que Rachel Legendre soit mise à disposition de la plateforme pour une durée d'au moins 2 ans.

The Center for Human Immunology (CIH) supports researchers involved in translational research projects by providing access to 16 different cutting edge technologies. Currently, the CIH hosts over 60 scientific projects coming from 8 departments of the Institut Pastuer and 5 external teams. In order to respond to the growing needs of these projects in the area of single cell analysis, the CIH has introduced a significant number of single-cell/single-molecule technologies over the past 2-3 years. These new technologies, such as the Personal Genome Machine (PGM) and Ion Proton sequencers, iSCAN microarray scanner, Nanostring technology for transcriptomics profiling and real-time PCR machine BioMark, give rise to large datasets with high dimensionality. Such trend, in terms of data complexity, is also true for flow cytometry technologies (currently reaching over 20 parameters per cell). The exploration of this data is generally beyond the scope of scientists involved in translational research projects. In order to maximize the research outcomes obtained from the analysis of these rich datasets, and to ensure that the full potential of our technologies can be served to the users of the CIH, we would require a proximity bioinformatics support. A CIH-embedded bioinformatician would: 1) design and implement standard analysis pipelines for each of the data-rich technologies of the CIH; 2) provide regular ‘bioinformatics clinics’ to allow scientists the possibility to customize standard pipelines to their specific needs; 3) run trainings on the ‘R software’ platform and other data analysis tools (such as Qlucore) of interest for the CIH users. The objective would be to empower the users to run exploratory analysis by themselves, and to teach good practices in terms of data management and data analysis.    

Aim : When L. amazonensis (L.am.) amastigotes infect BMDMs, they induce multiple strategies to allow their survival and multiplication. This project aims at deciphering the transcriptional modulations induced after three days of in vitro infection of C57BL/6 BMDMs with L. amazonensis (L.am.) amastigotes, in unstimulated conditions, or conditions that induce NLRP3 inflammasome activation.

We are mapping the interactions between argonaute proteins and their target RNAs at nucleotide resolution to reveals preferential binding sites on RNAs

The Transcriptome & Epigenome Platform is dedicated to the development and use of high throughput approaches for transcriptomics and epigenomics studies. The platform is accessible to any research team from the Pasteur Institute (80% of the projects) as well as from outside. It is involved (most often as collaborator) in several projects funded by the ANR, Microbes and Brain, ERANET and by the Pasteur Institute in the framework of the PTR programs. Next Generation Sequencing (NGS) based on the Illumina technology (HiSeq 2000/2500 sequencers) is used to perform RNA-sequencing experiments for which a large amount of data is generated. After a first step involving bioinformatics, specific statistical methods must be used be analyze rigorously the data. These analyses are most often performed by the statistician(s) of the platform. They are also in charge of bibliographical survey activity.

Tuberculosis (TB) still remains a major public health problem with estimated 9 million incident cases and 1.5 million deaths in 2014 (WHO, Global Tuberculosis Report 2015). More worrisome is the emergence of multi drug resistance (MDR), or even extensively resistant (XDR) M. tuberculosis strains worldwide. The standardized treatment of pan-susceptible tuberculosis is the administration of two antibiotics (rifampicin and isoniazid) for six months, accompanied by two additional antibiotics (pyrazinamid and ethambutol) for the first two months. Although very efficacious, this treatment is very demanding due to the duration and the possible side effects. The treatment of MDR-TB is less standardized, with more toxic and poorly tolerated drugs, resulting in lower cure rates. Therefore, we need not only more molecules with antimycobacterial activity, but also, we urgently need new strategies to increase our therapeutic arsenal for treating MDR-TB. Only three new drugs, bedaquiline, delamanid and PA-824 have been tested in phase2/3 clinical trials.

In this context, the european funded project NAREB has been created. It brings together 14 partners from 8 EU Member and Associated States, and it aims to (i) screen different combinations of antibiotic drugs with nano-carriers (lipid, polymeric, biopolymeric) with and without targeting ligands, (ii) coload antibiotics in order to develop innovative therapeutic combination therapies (iii) test in vitro and in vivo the best therapeutic combinations. In particular, we will analyze more in-depth the effect of bedaquilin, new TB drugs and nano-carriers on the host/bacterial transcriptome using RNAseq.

We are interested in determining the differences in the transcriptome of select developmental stages of the malaria parasite, Plasmodium.

We are interested in the cytoplasmic quality control of gene expression and more especially into the behavior of aberrant peptides which could be generated from non-conform translation events. We are now investigating the role of a Saccharomyces cerevisiae RNA helicase protein that we named Tac4 (for Translation associated Component 4). We showed that this protein is involved in translation. We demonstrated, by sucrose gradient and affinity purification that Tac4 interacts with the ribosome. A first UV cross-linking and cDNA analysis (CRAC) experiment clearly revealed that Tac4 interacts with the 18S rRNA of the 40S ribosomal subunit and we precisely defined the crosslink point. These preliminary results also suggested an enrichment of the 3’-end regions of mRNAs. This implies that Tac4 could not only interact with the small ribosomal subunit but also directly with mRNA. Tac4 is conserved through the evolution and its mammalian homologue is involved in initiation of translation. Therefore, we thought that Tac4 could be associated with the 5’-end rather than with the 3’-end. However, a recent paper from the Rachel Green’s lab showed that translation reinitiation into the 3’-UTR region may occurs when translation termination is affected (Young et al., Cell 2015). The factors and molecular mechanisms implicated in these events are not known. Altogether, our preliminary results suggest that Tac4 is an excellent candidate participating to the unwinding of RNA structure or to the release of some RNA-binding proteins into the 3’-end mRNA. We now would like to 1) confirm that Tac4 preferentially interacts with the 3’-end of mRNA, 2) determine whether Tac4 interacts with a region upstream the Stop codon or in the 3’-UTR of the mRNA, 3) identify the mRNA targets to determine whether Tac4 could have a general role in translation or could only be involved in translation of some specific mRNA.

After the behavioural characterisation of the Shank3 KO cohort, we extracted and dissected different region of the brain: cortex, hippocampus, striatum, cerebellum.

These regions were selected according to the expression level of Shank3 (Peça et al. 2011). Using QUIAGEN miRNAeasy with DNAse, miRNA-enriched RNA was extracted from the brain regions in order to perform RNAseq.

We will ask 3 questions:

• What is the pattern of Shank3 isoforms in different brain regions?

• What are the genes/pathways differentially expressed in wild-type and Shank3 knock-out mice?

• What are the genes/pathways associated with the severity of the self grooming behaviour ?

Beside data driven eperiments, we will test candidate genes/pathways such as glutamatergic receptors and <

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants underlying this response, we characterized the TCR repertoire directed at the most immunodominant CD4 epitope in HIV-1 capsid, Gag293. HIV Controllers from the ANRS CO21 CODEX cohort showed a highly skewed TCR repertoire characterized by a predominance of the TRAV24 and TRBV2 variable gene families. Controllers shared public clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control (Benati D. et al., J Clin Invest 2016).   We propose to test the generality of these findings by characterizing the TCRs specific for a series of immunodominant HIV Gag and Env epitopes, and comparing the frequencies of public clonotypes in groups of HIV Controllers and treated patients. We will then assay the functions of the most prevalent public clonotypes through lentivector-based TCR transfer, and correlate the panel of T cell functions to TCR affinity and frequency.

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 genome of the yellow fever mosquito (Aedes Aegypti) is not fully annoyed, and this project aims at discovering novel transcripts using RNAseq data.

We are analyzing a Listeria protein secreted in the supernatant. From cells transfected with this protein, after crosslinking, we have isolated RNA and found the protein in the RNA fraction. In addition, the protein binds several splicing factors in RNA dependent manner. Furthemore, the protein localizes to the nucleus. In this project, we have isolated the protein from the supernatant and from bacterial extract after growth in BHI. We have extracted the RNA from the supernatant fraction containing the protein and from the bacterial extract containing the protein. We want to alalyze the RNA in these different fractions.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (MTB), is the deadliest disease due to a single infectious agent. Despite considerable efforts to fight the disease, TB remains a major public health problem. Even more worrying for the future, multidrug resistant (MDR) strains of MTB are continually emerging and about 10% of people with MDR-TB have extensively drug-resistant TB (XDR-TB). Drug-sensitive TB can be cured by a 6-month treatment using 4 antibiotics, but MDR-TB and extensively drug-resistant XDR-TB require treatment for up to 2 years with more toxic and costly second- and third-line drugs. Toxicity of these drugs is well described; it includes hepatotoxicity, liver injury, skin reactions, gastrointestinal and neurological disorders. However how MTB drugs influence the host response to MTB infection has been poorly addressed. The main goal of project is to understand how drugs interact with the host in order to improve the treatment.

Our team studies interactions between arboviruses and Aedes aegypti mosquitoes. We recently generated RNAseq data to analyse the response of the mosquito to virus infection. We would now like to analyse that data using Gene Ontology to find pathways or processes that are activated upon infection.

I am interested in gut-brain axis and specifically how a bacterial metabolite, MDP, can be sensed directly by neurons.

The three HP1 proteins (Heterochromatin Protein 1 alpha, -beta, -gamma) are epigenetic markers of heterochromatin, the condensed, repressed form of chromatin. They are typically known to associate to the di-/tri-methylated lysine 9 of histone H3 (H3-K9me2/3), a repressive histone mark, HP1s are therefore linked to chromatin silencing. But on the other hand, HP1s are also linked to activated transcription, for example HP1g has been shown to localize within the body of coding genes in correlation with their transcriptional activity. We focus on the mechanisms linking HP1gamma to regulation of chromatin and transcription in response to cellular stimuli. We are dissecting the functional links between HP1 proteins and chromatin-associated RNA. For this purpose, have identified all RNA populations associated with HP1gamma factor in immortalized cell lines derived from MEF (mouse embryonic fibroblast) with or without stimuli. We are now analysing the functional impact on gene expression levels. This should allow us to understand to what extend HP1gamma is associated to transcriptional processes on the chromatin.

In a previous project (collaboration with PF2 and C3BI) , we determined the transcriptome landscape of the pathogen Leptospira interrogans (Zhukova A, Fernandes LG, Hugon P, Pappas CJ, Sismeiro O, Coppée JY, Becavin C, Malabat C, Eshghi A, Zhang JJ, Yang FX, Picardeau M. Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans.Front Cell Infect Microbiol. 2017 Jan 19;7:10.). In the present project, we performed RNA-seq with the PF2 on the saprophyte Leptospira biflexa and a mutant of a gene encoding a hfq-like protein. Hfq is a chaperone usually involved in the stability of sRNA in other bacteria and we would like to : 1) identify the sRNA ((intergenic regions, antisense RNA, etc) of L. biflexa wt strain 2) compare the level of expression of wt and hfq-like mutant strains.

The RpoS/σS sigma subunit of RNA polymerase is the master regulator of the general stress response in many Gram-negative bacteria. σS also contributes to virulence and biofilm formation of the human pathogen Salmonella enterica serovar Typhimurium. We have used RNA-sequencing to unravel the σS-dependent transcriptome in S. Typhimurium ATCC14028. These studies have revealed a major effect of σS on the remodelling of metabolism and membrane functions, and have highlighted the importance of down-regulation of gene expression by σS. More recently, we have used a mass spectrometry-based proteomics approach to explore the wide σS-dependent proteome of ATCC14028. Our proteomics data, and complementary assays, have unravelled the large impact of σS on the Salmonella proteome in stationary phase. As expected, the majority of genes up-regulated by σS at the protein level were also up-regulated at the transcript level. In sharp contrast, our data have revealed a large number of genes down-regulated at the protein level, but not at the transcript level, suggesting that post-transcriptional regulation plays a larger role in σS gene regulation than previously recognized. In this project, we will determine the impact on genome expression of a single amino-acid substitution in RpoS which confers specific phenotypes.

Entamoeba histolytica is a protozoan parasite and an amitochondriate pathogenic amoeba, which causes amoebiasis (dysentery and liver abscess) in humans. In addition to E. histolytica several species infect the human intestine although these do not cause disease and include in most of cases E. dispar and ocassionnally E. moshkovskii. A phylogenetically close Entamoeba, E. invadens infecting snails, is used as cellular model for Entamoeba cyst formation.

Supported by the National Agency for Research (ANR-10-GENM-0011) we developed a project to firstly study the transcriptional landscape of pathogenic E. histolytica. Among the results we discovered that 60% of ORFs present anti-sense RNAs (NATs) that map to the 3‘ end of genes. Their regulation is modified upon environmental changes. The regulation of NATs is basically governed by genomic sequences within the very short intragenic region of the amoeba genome. Secondly, we have started to conduct comparative genomics and transcriptomics approaches to understand phenotypic differences between Entamoeba species, in particular with respect to virulence.

The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections, including Influenza, HIV and Zika viruses, by inhibiting virus-cell fusion. We showed that IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses. We also reported that amino-terminal mutants of IFITM3 preventing ubiquitination or endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. Our aim is to analyze the RNA levels of the various members of the IFITM family, in various normal or pathological human or animal tissues.

This research project focuses on the characterization of the role of small RNAs and their associated Argonaute proteins in transcriptional and post-transcriptional regulation of germline gene expression. Using the nematode C. elegans, we have recently showed that one of the germline-expressed Argonaute protein, CSR-1, promotes germline transcription. However, CSR-1 also possess an endonucleolytic activity that might participate in post-transcriptional silencing. Therefore, two possible functions of the protein might regulate the germline transcriptome. 1) CSR-1 promotes specific germline transcription programs in the nucleus, and 2) negatively regulates expression of target transcripts in the cytoplasm. To gain mechanistic insights into these two functions, we aim to use RNA-seq, sRNA-seq, ChIP-seq, GRO-seq, Ribo-seq, RIP-seq, iCLIP in wild type worms, knock out and catalytic inactive mutants of CSR-1 protein at different times of germline development.

Adult neurogenesis is the process by which adult neural stem cells (NSCs) produce new neuronal and glial cells throughout an animal life. Studies in vertebrates have unveiled the crucial importance of this phenomenon for neural tissue homeostasis and proper brain function. Fundamentally, this process is a balance between maintaining a quiescent NSC pool and recruiting them into the neurogenesis cascade. Using the adult zebrafish anterior brain (telencephalon) as a model, we aim at deciphering the molecular mechanisms governing this balance. We identified a microRNA, miR-9, as a prominent quiescence enforcer. Unexpectedly, miR-9 concentrates into the nucleus of quiescent adult NSCs, together with Argonaute proteins (notably Ago2), effector proteins of microRNAs. This nuclear enrichment of Ago/miR-9 is not observed in embryonic or juvenile fish, being thus a signature of deep adult NSC quiescence. It is also observed in mouse NSCs. We wish to use nuclear miR-9/Ago2 as molecular entry points into the molecular mechanisms controlling this adult-specific deep NSC quiescence state. Within this frame, the present project aims to identify Ago2-bound nuclear targets. Through fractionation experiments, we could detect Ago proteins both in the nuclear soluble and in the chromatin-associated fractions of adult zebrafish NSCs. Thus, we built genetic tools to recover the nuclear Ago2-bound transcripts and genomic loci, ie. using CLIP-seq and DamID approaches, respectively. The enriched transcript sequences will be screened for potential miRNA-binding motifs. Putative genomic targets will be screened, in addition, for specific motifs and for their coincidence with defined functional regions (eg. coding or regulatory). The data will be cross-matched with the transcriptome and proteome of miR-9-positive versus –negative adult NSCs. Together, these data should help elaborate hypotheses on the molecular mode(s) of action of nuclear miR-9/AgAgo2 when controlling adult NSC quiescence.

There is a wide variation in both animal and human risk and outcome of infection, generally encompassing asymptomatic, to more severe and sometimes lethal cases. Dengue virus, a re-emerging arbovirus is a great examples of this, as it lead to a very wide spectrum of disease severity. Genetic variation associated with intrahost populations has been postulated to influence viral fitness and disease pathogenesis. This project aims to characterize the complexity of viral populations in samples from patients affected with different degree of dengue disease severity.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by a biallelic mutation of the Ataxia Telangiectasia Mutated (ATM) gene. The ATM protein is involved in a very large number of cellular functions (over 700 substrates), mainly DNA repair, cell cycle regulation, mitochondrial and oxidative metabolism, as well as regulation of gene expression. The majority of hematological malignancies occurring in AT patients are non-Hodgkin's B lymphomas and Hodgkin's lymphomas. The association with the Epstein-Barr virus (EBV) is found in 50 to 100% of these cases according to the histological subtype. EBV is a widespread virus in human populations with a prevalence greater than 90% and is associated with the development of lymphomas in immunocompromised patients. We will study the transcriptome of EBV-infected cells in ATM patients, in order to determine the impact of ATM on EBV infection control.

Despite the worldwide importance of malaria due to Plasmodium vivax, there is currently almost no data on the molecular responses of the Anopheles mosquito vectors to this parasite species. Understanding these responses will contribute to identify relevant strategies to interrupt the transmission of P. vivax by targeting the mosquito vector. Such approaches are urgently needed, as P. vivax is difficult to target on the long term in humans as a consequence of the hypnozoite stage that is responsible for relapses. This project will investigate the molecular response of Anopheles arabiensis, member of the Anopheles gambiae complex, to P. vivax from experimental infections performed in Madagascar where both mosquito and parasite are present. This is a unique situation that will capitalize on the strong knowledge and tools available for An. gambiae sensu lato. The response will be compared to the one triggered by P. falciparum, also present in Madagascar. From mosquitoes infected in a field setting, a transcriptomic (RNAseq) approach will be used to identify common and unique pathways to both parasite species. These analyses will further contribute to identify targets for interrupting transmission of each or both parasites simultaneously. However, a pilot study performed with the PF transcriptomics & epigenomics revealed that the current release of the An. arabiensis genome is poorly annotated. Therefore, to be able to make sens of the RNAseq analyses per se, a strong support from the C3BI Hub for bioinformatics and biostatistics is critical.

The development of the mammary gland occurs in five distinct phases: embryogenesis, puberty, pregnancy, lactation, and involution. Due to its extraordinary regenerative capacity, the mammary epithelium is a fantastic system to study the physiological regulation of cellular plasticity in vivo. It comprises two major cellular lineages, the outer myoepithelial (also called basal) and inner luminal cell layers. Although the existence of post-natal bipotent mammary stem cells (MaSCs) remains debatable, it is well accepted that there are subtypes of epithelial cells (progenitors, unipotent and/or bipotent stem cells) that are responsible for the remodeling and renewal of the mammary gland. Moreover, these cells have also been suggested to be the cell of breast cancer origin. Therefore, it is crucial to further understand how mammary gland maintains its proper cellular plasticity, especially during the cycles of pregnancy and involution, which might shed new lights on the initiation, progression, and metastasis of breast cancer.

Pathogen leptospires are responsible for the zoonotic disease leptospirosis. This neglected but emerging infectious disease has a worldwide distribution and affects people from developing countries, mostly under tropical areas. The clinical manifestations of this infection range from a febrile state to a severe life-threatening form characterized by multiple organ hemorrhages. More than one million cases of leptospirosis are currently reported annually in the word, with 10% of mortality. Leptospira penetrate hosts and rapidly disseminate to target organs (including kidney, liver, lungs) throughout the bloodstream. They are not obligatory intracellular pathogen but they can transiently persist inside macrophages. Due to the difficulty of gene inactivation in pathogen Leptospira, their study is hampered and limited. Thus, their virulence mechanisms and how they survive inside hosts remain largely unknown. During infection, Leptospira are confronted with dramatic adverse environmental changes such as deadly reactive oxygen species (ROS). Defenses against ROS, e.g. peroxidase activity, are crucial for Leptospira virulence. In previous studies, we have identified by RNASeq the cellular factors solicited by Leptospira interrogans to adapt to an oxidative stress and determined the regulons of the two peroxide stress regulators PerR1 and PerR2. We aim now at studying how small non coding RNAs participate in the adaptive response to oxidative stress in pathogen Leptospira. Regulation of any predicted small non coding RNAs will be examined in the RNASeq data we have already obtained.

The three HP1 proteins (Heterochromatin Protein 1 alpha, -beta, -gamma) are epigenetic markers of heterochromatin, the condensed, repressed form of chromatin. They are typically known to associate to the di-/tri-methylated lysine 9 of histone H3 (H3-K9me2/3), a repressive histone mark, HP1s are therefore linked to chromatin silencing. But on the other hand, HP1g has been shown to localize within the body of coding genes in correlation with their transcriptional activity. We have identified functional links between HP1g and chromatin-associated RNA on gene models, but we are now dissecting these links on a genome-wide basis.

A number of mammalian cell types are susceptible to Salmonella Typhimurium infection, including epithelial cells, fibroblasts and macrophages. It has recently been demonstrated that Salmonella display specific lifestyles in a host cell type-specific manner, where these lifestyles are remarkable for the subcellular localization and replication rate of Salmonella. During epithelial cells infection, Salmonella are first uptaken by the host cell and being encapsulated in a unique endocytic compartment termed Salmonella-containing vacuole (SCV). Within the SCV, Salmonella could remodel the SCV into a viable replicative niche or rupture the SCV and hyper-replicate in the host cytosol. Salmonella replicate at distinct rates in the two localizations, the Salmonella replication in SCV commences from 5 hours post-infection (pi) and infected host cell harbors less then 10 bacteria at 8h pi. While the cytosolic Salmonella begins replication within the first hour of infection at a rate of 30 minutes per division and accumulates up to over 50 bacteria in the host at 8h pi. To understand how Salmonella achieves the two distinct intracellular lifestyles as well as the pathophysiological impacts of the two lifestyles, the primitive goal of my project is to develop a bacterial-borne fluorescent reporter that clearly depicts the subcellular localization and replication rate of Salmonella. With this multiplex fluorescent reporter, we will set to explore the fundamental biological questions: to determine the immune response of epithelial cells against the two Salmonella lifestyles.

Cocaine is the most widely used illicit stimulant in Europe1, with a recent increase in use in the French general population. Cocaine addiction (CocAdd) is recognised as a public health priority worldwide, affecting 3% of the US general population, with high burden for individuals and societies1 (4 to 8-fold increase in standardised mortality rates). Clinically, (cocaine) addiction (or substance use disorder) is defined as the compulsive use of a substance causing clinically and functionally significant impairment (health problems, disability, and failure to meet major responsibilities at work, school, or home). These features constitute the basis of diagnostic criteria. There is no approved medication to treat CocAdd, despite significant advances regarding the mechanisms underpinning the neurobiology of chronic cocaine self-administration in rodents. CocAdd is defined as a maladaptive and compulsive reward-seeking behaviour related to cocaine. The loss-of-control over drug use and associated urges (craving) is such that 75-90% people with CocAdd report that they have relapsed within one year after inpatient detoxification. It is thus crucial to develop innovative and precise biomarkers to predict the liability to relapse if we are to develop efficient treatment strategies against this devastating disorder. Relapse is a key player in the chronicity of addiction in general and, as such, is the core therapeutic target of any therapy against addiction . Attached to an ongoing study of the neuroimaging signature of CocAdd relapse, we implemented an auxiliary study to draw blood samples from patients with CocAdd undergoing hospital detoxification and followed for up to three months after, so as to collect genetic material. The 'OBSCOC' protocol thus provides biological samples at entry and 15, 30 and 90 days after. These samples are aimed for characterising the whole RNome, miRNome and ChipSeq profile of these patients, who all provided written informed consent for the genetic study and for extensive clinical and sociodemographic assessment as well. We hypothesised that specific changes of gene expression between time points could either predict increased risk for/precocity of or announce imminent relapse. Thus they could serve, on the longer-term, as biomarkers and/or therapeutic targets.

Microbes are prone to rapid changes and they can either exploit or countervail their variation in a context-dependent manner. To this purpose, both genetic diversity and non-genetic phenotypic variation exist. However, while the overall mutational evolution occurs over lengthy timescales, epigenetic changes take place on a large scale and more rapidly. Collectively this implies that the diversity we observe is profoundly driven by non-genetic variation. This is particularly relevant for the WHO Priority Pathogen Mycobacterium tuberculosis, whose lack of lateral gene transfer and low mutation rate make phenotypic variation an important means of adaptation to stressful conditions. A few studies, including ours, have begun to explore this phenomenon at the single-cell level in M. tuberculosis in axenic and host conditions, which are technically very challenging. This project is based on the assumption that M. tuberculosis can successfully endure harsh environmental conditions thanks to its phenotypic variation. In our view a better understanding of the drivers of phenotypic variation will improve the design and development of original strategies for tuberculosis control. Here we investigate the physiology of M. tuberculosis at the single-cell and subpopulation scale, striving to demystify the bases of phenotypic diversity and the implications for adaptation and persistence. Previously we examined by real-time imaging a fluorescent reporter of ribosomal expression (rRNA-GFP) as a gauge for cellular activity, and found that M. tuberculosis displays phenotypic heterogeneity under optimal growth conditions, which is enhanced in the host, in long-term stationary phase and upon drug exposure. Remarkably we could also detect subpopulations of quiescent bacilli, whose molecular characteristics have yet to be determined, which is the aim of this project. Here we constructed a dual fluorescent reporter of metabolic activity/quiescence in M. tuberculosis, by using our rRNA-GFP reporter as a background strain, further modified with a red fluorescent marker of cellular quiescence. We carried out snapshot microscopy and single-cell analysis during optimal growth conditions as compared with stressful conditions. We found that the cell-activity marker decreases, whereas the cell-quiescence marker is induced under different host-mimetic conditions. We also observed significant intracellular variation during infection assays. Now we envision carrying out a comprehensive analysis of M. tuberculosis phenotypic variation by RNA sequencing. We aim to reveal the molecular differences between subpopulations of bacilli that exhibit discrete metabolic potential, based on their fluorescence output. We have recreated the most interesting conditions on a bulk scale, and sorted active versus quiescent subpopulations, aiming to compare their transcriptional profiles, and to ultimately identify subpopulation-specific biomarkers of persistence towards more accurate diagnostics.

Dengue virus (DENV) induces strong T and B cell responses upon infection. However, there is currently neither vaccine nor specific treatment against DENV, which is spreading worldwide causing 400 million new infections every year, of which 100 million cases are symptomatic, ranging from a self-limiting febrile illness named dengue fever (DF) to more severe life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). One of the major obstacles of dengue vaccine development is the cross-reactivity among antibodies against the different DENV serotypes (designed as DENV1-4) that are 67-75% identical at the amino acid level. Indeed, while a primary infection by one DENV serotype can induce a lifelong immunity against re-infection by the same serotype, subsequent infections by heterologous serotypes increase the risk of developing severe dengue, a phenomenon due to non-neutralizing or sub-neutralizing antibodies and called antibody-dependent enhancement (ADE). To avoid the induction of such enhancing antibodies, and given the identification of CD4+ and CD8+ T cell epitopes from previously dengue virus (DENV)-infected donors, we have designed a minimal DENV antigen (called DENV1-NS, patent EP14305984.8 filled on June 23 2014), which is enriched in conserved and highly antigenic epitopes. Using this minimal DENV1-NS antigen, we have first established in vivo its immunogenicity in transgenic mice expressing HLA class II and class I alleles (with the activation of DENV-specific CD4 and CD8 T cells). We have also shown that a prime-boost DNA immunization of these HLA transgenic mice induces a strong T cell immunity, with a significant protection against DENV1 infection, in the absence of neutralizing or sub-neutralizing anti-DENV antibodies (Roth et al., accepted for publication). Our proposal aims, therefore, to identify early transcriptional signatures correlated with the development of memory CD4 and CD8 T cells in vaccinated animals, which promote enhanced anti-dengue immunity.

This projects aims at characterizing at the single cell level the stromal microenvironment of the skeletal muscle, to get a better insight into the heterogeneity and function of different cell populations involved in skeletal muscle homeostasis and regeneration.

Gene-modified T cells expressing a chimeric antigen receptor (CAR) targeting the CD19 molecule have demonstrated promising clinical efficacy in the treatment of B cell malignancies. However, the frequent relapses and toxic adverse events such as cytokine release syndrome represent hurdles to the success of CAR T cell therapies. In most clinical settings, CAR T cells are generated from a mixture of autologous CD4+ and CD8+ T cells before being infused into patients. This inter-patient heterogeneity within the composition of CAR T cell products renders the large variety of response efficacy and toxicity difficult to interpret. Using a model of B cell aggressive lymphoma developing in the bone marrow, we investigate the differential contributions of CD4+ and CD8+ anti-CD19 CAR T cells to tumor outcome and changes in the tumor microenvironment. Our first in vivo imaging and flow cytometry results suggest that CD4+ CAR T cells have poor cytotoxic potential compared to CD8+ CAR T cell. On the other hand, CD4+ CAR T cells were largely responsible for the cytokine release syndrome and have a unique role in boosting the accumulation of NK cells at the tumor site. Using single cell RNAseq, we aim to identify the changes in the bone marrow tumor microenvironment induced by CD4+ and CD8+ CAR T cells, focusing on the recruitment of host specific immune cell populations and their activation status. Identifying specific contributions of the CD4+ and CD8+ CAR T cells to immune cell recruitment and tumor outcome would help designing optimal CAR T cell products, with important clinical implications.

The enteropathogen, Shigella, is highly virulent and remarkably adjusted to the intestinal environment of its almost exclusive human host. Key for Shigella pathogenicity is the injection of virulence effectors into the host cell, via its type three secretion system (T3SS), initiating disease onset and progression. T3SS-mediated host cell targeting is associated with Shigella invasion and dissemination in epithelial cells. Yet, our group reported the direct targeting of human lymphocytes by an injection-only mechanisms, i.e. the injection of Shigella T3SS effectors without subsequent cell invasion. To further investigate the scope and selectivity of this targeting mechanism, we utilised human lamina propria mononuclear cells (LPMCs) isolated from colonic explants and peripheral blood mononuclear cells (PBMCs) and assessed the mode of Shigella targeting by the use of a T3SS reporter stain and multiparametric CyTOF technology. In human LPMCs, a population of CD38hi plasma cells/ plasmablasts was identified as primarily targeted and presented up to 80% of all targeted LPMCs. Interestingly, plasma cells constitute the primary IgA-producing cell type and IgA has been previously identified as important mediator of Shigella pathogenicity. The functional implications on plasma cell targeting by Shigella is subject of current investigations.

Pathogen leptospires are responsible for the zoonotic disease leptospirosis. During infection, Leptospira are confronted with dramatic adverse environmental changes such as deadly reactive oxygen species (ROS). Defenses against ROS, e.g. peroxidase activity, are crucial for Leptospira virulence and the adaptive response to ROS is controlled by PerR regulators. We aim at studying how small non-coding RNA participate in the adaptive response to oxidative stress in pathogen Leptospira.

Viruses have evolved powerful countermeasures to evade host innate immunity, which produces immediate, but non-specific, immune response during infection. Among viruses possessing RNA genomes, the order of negative-single-strand viruses (Mononegavirales) encompasses many human and animal pathogens that cause severe disease, including measles virus, mumps virus and rabies virus. Rabies virus is known for its neurotropic retrograde progression from the site of transmission to brain parenchyma, and towards salivary glands, different organs linked through parasympathetic nervous system. In the cases of human infection, the exhibited symptoms such as hallucination, diplopia, hydrophobia, unsteadiness or paralysis all indicate that there is causality between rabies virus infection and dysfunction of neural activity. However, lack of pathological brain lesions observed at the point of autopsy or noncytolytic propagation devoid of apoptosis suggest that rabies virus possesses mechanisms to evade or delay immune responses and cell death at least for the duration of replication and transmission. Even though the details in molecular perspective of these discoveries are well laid out now, how these proteins work in coordination or if there are hidden components which connect them all together leading toward deterioration of neural cells on the benefit of virus is largely unclear. Moreover, considering the complexity of brain cell composition and how important the neighboring cells are to shape one neuron’s specialization and dependency onto others in homeostasis, which result in the astounding heterogeneity of gene expression, an integrated and holistic approach is mandatory to get a fully comprehensive view of the mechanisms involved. Consequently, we performed an RNASeq analysis in human interneuron cells derived from induced pluripotent stem cells and infected by two recombinant rabies viruses (Tha virus, isolated from a dog in Thailand and Th4M, a less pathogenic virus which is mutated on 4 different residues of the M gene; this virus can no longer escape the NF-KB pathway) in order to obtain transcriptome data by comparison with uninfected cells, and to have an overview of the temporal dynamics of the genes expression.

The aim of this RNA-seq project is to characterize three transcriptional regulators of Streptococcus agalactiae, the main etiological agent of bacterial meningitis in neonate, and one transcriptional factor of Enterococcus faecalis, a second Gram-positive opportunistic pathogen.

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.

Chlamydia trachomatis is an intracellular bacterium. We have observed that some of the epigenetic changes in the host that occur in infection can be reversed by modifying the culture conditions. We will measure the impact this has on the global transcriptional response of the host to infection.

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

This RNA-seq project aims to characterize the function of genes regulated by the master regulator of virulence in Group B Streptococcus.

Anti-TNF therapy has proven effective to reduce inflammation and clinical symptoms in SpA, however, the high rate of non-responsiveness (30-40%) to TNFi exposes a substantial fraction of patients to side effects without clinical benefit, and it is still not possible to determine which patients will respond to TNF inhibitors (TNFi) before treatment initiation. The recent introduction of antibodies blocking IL-17A has expanded the therapeutic options for axial SpA (axSpA), as well as psoriasis and psoriatic arthritis. It is therefore important to develop tools to guide treatment decisions for patients affected by SpA and other chronic inflammatory diseases, to optimize clinical care and contain health care costs. This project builds on our recent work performed in collaboration with the Bioinfo Hub, in which we have defined the mechanisms of action of TNF-blockers and identified immune signatures correlating with therapeutic responses to anti-TNF therapy in SpA patients (Menegatti et al., 2020). We have measured whole blood immune responses to microbial and pathway-specific stimuli using TruCulture assays in 20 axSpA patients before or after treatment with IL-17A-inhibitors and 20 patients before and after anti-TNF therapy. Proteins in supernatants have been measured by Olink technology for protein profiling. Gene expression in cell pellets has been analyzed by RNA sequencing at the Biomics platform. A preliminary analysis of the RNA-Seq data has been performed by Etienne Kornobis and Thomas Cokelaer.

Dans les années récentes, l’utilisation de données RNA-Seq nous a permis de ré-annoter le génome des trois souches de références de, respectivement, C. neoformans, C. deneoformans et C. deuterogattii (JANBON et al. 2014; GONZALEZ-HILARION et al. 2016; FERRAREZE et al. 2020). En utilisant des données de TSS-Seq et 3UTR-Seq, nous avons identifié les extrémités de chaque gène codant, ce qui nous a permis de décrire en détail la structure des Transcript Leader (TL) (WALLACE et al. 2020). Les séquences TL chez Cryptococcus contiennent un grand nombre de codons uATG (plus de 10 000) qui ne sont pas utilisées pour coder le protéome de ces levures. Nous avons pu montrer que les ATG « codants » étaient caractérisés par un motif proche de celui identifié par Marylin Kozak en étudiant des cellules de singes dans les années 80 (KOZAK 1986). Chez Cryptococcus, le nombre, la position et le motif associé aux uATG régulent l’expression des gènes. Les motifs associés aux ATG peuvent aussi réguler la diversité protéique et notamment leur adressage dans différentes organelles lorsque deux ATG sont en phase et incluant ou excluant, respectivement une séquence d’adressage. (WALLACE et al. 2020). Lors de cette étude, nous avons identifié un grand nombre de clusters de TSS alternative associés au gènes codant chez Cryptococcus. Leur nombre dépend des conditions de cultures mais par exemple, plus de 16 000 clusters de TSS sont associés aux 6800 gènes codants de C. neoformans quand les cellules sont cultivées à 30°C en phase exponentielle. Ces TSS alternatifs peuvent altérer la taille de la séquence TL mais aussi la séquence N-terminale et donc l’adressage de la protéine codée. Certains d’entre de ces TSS alternatifs sont positionnés au milieu de la partie codante des gènes et contrôlent la transcription de mRNAs dont la fonction est inconnue. Les conditions de culture régulent l’utilisation de ces TSS, certains étant régulés par la température d’autres par la phase de croissance. L’étude de la régulation de l’utilisation des TSS alternatifs et de leurs conséquences sur la biologie de Cryptococcus est un sujet majeur de l’unité. Une partie de cette étude demande l’identification et la description de TSS alternatifs et de leur régulation. Le but est de tester différents modèles et/ou pipelines afin de premierement caratétiser ce qu’est un cluster de TSS type chez C. neoformans puis dans un deuxième temps d’analyser la régulation de leurs usages en fonction des conditions de culture.

Establishment of a left-right asymmetry is essential for the function of the heart, which is to ensure a double blood circulation. This asymmetry is initiated during an embryonic process, in which the heart tube acquires a loop shape. The molecular cascade at the origin of left-right asymmetry is well established and involved in the severe heart defects of the heterotaxy syndrome. However, another 20% of heart malformations display cardiac chamber misalignment and their origin is poorly understood. Using a mutant mouse model and expertise in heart morphogenesis, we want to provide novel insight into malformations displaying an abnormal alignment of ventricles.

To better understand the vector specificity of Aedes aegypti mosquito in the transmission of CHIK virus, we try to identify genes involved in the primary responses of the viral infection when the virus is still in the midgut of mosquitoes, prior to the crossing of the gut epithelium. This investigation will be performed via mRNAseq from sequences of pools of 8 mosquitoes that fed on blood (controls) or on blood +CHIKV (infected mosquitoes).

The chronic inflammatory diseases (CID), as psoriasis, psoriatic arthritis (PsA) and axial spondyloarthritis (SpA), are clinically heterogeneous conditions that share common inflammatory pathways and derive from aberrant immune responses. GWAS strongly suggests that the IL-23/IL-17 pathway plays a key role in several CID. The successful treatment of psoriasis, PsA and axial SpA with IL-17A inhibitors has been validated. Interestingly, in literature, a strong GWAS association of IL23R with SpA was shown and IL-17A was shown to be downstream of IL-23 in murine CD4+ T cells. Surprisingly, a recent phase 2 study testing the IL-23 inhibitor risankizumab did not show any clinically improvement compared to placebo in patients with active axial SpA, whereas it has proven effective for the treatment of psoriasis and PsA. The reasons underlying the failure of IL-23 blocking in axial SpA are not known. Our understanding of the pathogenic mechanisms of these diseases is therefore limited, and the role of IL-23 needs to be studied. The specific question we want to address with this project is what are the gene induced by IL-23 in immune cell populations.

To better understand the vector specificity of Aedes aegypti mosquito in the transmission of the alphaviruses CHIKV and ONNV, we are using an in vitro system to specifically depict critical viral infection steps at the molecular level: transcription and replication of the viral genome. This in vitro system requires mosquito cell transfections followed by luciferase quantification. For better describe the viral replication in mosquito cells, we need to characterize our mosquito cell lines since it is well established that some insect specific viruses (ISVs) modulate arbovirus infection in vectors. In order to determine whether our mosquito cell lines are infected by ISVs , we have already sequenced respectively total and small RNAs of our cell lines: Aedes aegypti Aag2 cells and Anopheles cells ( 4A3a, 4A3a and SUA4). The analysis of these data will furnish global and comprehensive cell features regarding the presence of ISVs which may interfere with viral infections in mosquitoes.

Bacteria use two-component systems (TCS) to sense and adapt to their environment. The aim of this project is to characterize the regulatory network of each of the 20 TCS in the neonatal pathogen Streptococcus agalactiae.

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

The recent propagation of resistance mechanisms to insecticides constitutes a major challenge in the fight against the transmission of vector-borne diseases. Even though alternatives are being explored in mosquitoes, there are few studies focusing on the role that these molecules could have or play in the transmission of arboviruses. Using an in cellulo model, this project aims to determine the impact that an insecticide selection can have on an infection caused by the chikungunya virus.

African trypanosomes are flagellated protist parasites transmitted to mammals by the infectious bite of the tsetse fly. They are responsible for sleeping sickness in humans and nagana in cattle. Trypanosomes first proliferate freely in the blood, and then, about six hours after being inoculated, leave the bloodstream to invade various organs, including the skin, which is an anatomic reservoir for the parasites. Few details are known on the metabolic aspects of the different parasite stages in the blood and in the skin, as well as the immune response of the mammalian host against the different stages of development of dermal parasites. Two strains of trypanosomes (“AnTat1.1E” and “Lister 427”) with different genetic profiles were studied here: one having only one developmental stage named "SL" or slender in reference to their tapered shape in mice, the second having two developmental stages "SL", and "ST" for stumpy in reference to their stocky shape in mice. This will allow us to distinguish the transcriptomic signature of parasites at the ST stage. Mice were sampled 5 days or 4 weeks after experimental infection in order to evaluate how the parasite transcriptome evolves over the course of an infection. In all these conditions, blood and skin samples were taken in order to compare the transcriptomes of blood and dermal parasites on the one hand, and transcriptomes of murine blood and dermal tissues on the other. We will especially scrutinize the metabolic pathways of the two parasite strains in the blood and the skin, as well as the immune response of the host in each compartment, two crucial elements determining the development of the infection.