Candida albicans is responsible for the majority of life-threatening fungal infections occurring in hospitalized patients and is also the most frequently isolated fungal commensal of humans. Microevolution of C. albicans isolates has been observed in a number of instances, being in particular characterized by loss-of-heterozygosity events. Yet, most studies that have investigated such microevolutions have not used whole-genome sequencing. In this project, we aim to characterize C. albicans microevolution at the genome-wide level. To this aim, we will take advantage of multiple isolates collected at the same time in healthy individuals and that share the same molecular type, thus providing information on the extent of genetic diversity of commensal isolates. We will also take advantage of series of isolates collected in patients with different forms of candidiasis and/or that have received antifungal therapy, thus providing information of the impact of pathogenic interaction and antifungal treatment on genome dynamics.

Legionella pneumophila is from a genomic point of view a very diverse species, however, only few clones are responsible for over 50% of all human disease cases. Thus we aim to understand the evolution and emergence of these 5 major disease related clones. We have sequenced a large number L. pneumophila strains belonging to these clones and are undertaking comparative and phylogenetic genome analyses.

A major program of evolutionary and comparative genomics of yeasts has been in progress in my laboratory for many years (see publications). In the next few months (before summer 2015) I need to finish a few comparisons about a new clade to publish as soon as possible.

Comparative genomics approaches in microbiology now use thousands of genomes to analyze a given species in different environmental or medical contexts. By collecting and comparing these genomic sequences, many  studies are focusing on  the overall gene content of a species (i.e. the pan-genome) to understand their evolution in terms of core and accessory parts. Variable regions are of primary importance to understand the adaptive potential of bacteria and contain genomic regions that are exchanged between strains by horizontal gene transfer (i.e. the mobilome). As recently suggested (Chan et al., Genome Biology 2015), a consensus representation of multiple genomes would provide a better analysis framework than using individual reference genomes. Here, we propose to extend this concept by giving it a formal mathematical representation using a graph model and by applying statistical methods to cluster gene families.

The genome of C. tetani contains a chromosome of approximately 2,8Mb and a large size plasmid (74 kbp) harboring the tetanus-toxin gene. The genome of the strain E88 was sequenced and annotated (PNAS 2003, 100:1316-1321). We have sequenced and performed a first comparison of the genomes of three additional strains (Res Microbiol 2015, 166:326-331). Fourteen additional C. tetani strains were sequenced including historical strains (1952-1968) and recent French clinical isolates. We have the raw data obtained by Illumina sequencing. Sequence comparaison of chromosome and plasmid will be done. For this, in a first time the assembly of sequence read of each strain will be done, in a second time a comparaison of chromosome and the plasmid of these 14 strains by BLAST approach will be made. Finally, a phylogenetic tree will be generated allowing us to see the evolution of this bacteria.

Whooping cough is a vaccine-preventable disease due to Bordetella pertussis. Even if vaccination has allowed the control of the disease, isolates are still circulating and cyclic increases of incidence are observed every 3 to 5 years even in vaccinated countries. Most developed countries now use acellular vaccines containing 3 to 5 vaccine antigens (pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN) fimbrial proteins (FIM2/FIM3)) that have replaced whole cell vaccines. In regions vaccinating with acellular vaccines with a high coverage, isolates no more producing some vaccine antigens (mainly PRN) have been reported in the last years.   Bordetella pertussis reference genome has been fully annotated in 2003 by the Sanger Institute. Analysis and comparison of different B.pertussis genomic sequences showed that circulating B.pertussis isolates differ from vaccine and reference strains. Genome evolution is characterized by gene deletions, antigenic divergences, SNP accumulations…Recent genomic analysis gathering isolates from different countries showed that the worldwide B. pertussis population has evolved in the last 60 years,. Gene categories under selection were identified underlying that Bvg-activated genes and genes coding for surface-exposed proteins were important for adaptation. However these analyses concerned only overall vaccine antigen producing isolates.   The PTMMH Unit includes the National Center of reference for Bordetellosis. In the last years some particular B.pertussis French isolates no more producing PRN but also FHA or PT have been collected, analyzed and sequenced. We would like to further analyze these genomic data with a focus on the vaccine antigen deficient isolates through a SNP-based comparison of these isolates vs co-circulating isolates producing all vaccine antigens and vs a reference strain.

One project of our laboratory is to characterize the function of small proteins specifically expressed in nongrowing Salmonella cells under the tight control of SigmaS.  Our current works focus on small proteins secreted or targeted to the membrane since their characterization might reveal novel aspects of membrane functions and secretion in persistent bacteria, including pathogens. The proposed project is an extension of a former project on the phylogenetic distribution of the small orfs, and aims at analyzing the genomic context of orthologs.

Recherche de SNP chez une souche de leptospira biflexa devenue résistante à un bactériophage.

We submitted an article about the diversity of Clostridium botulinum strains based on MLST analysis. One reviewer of the article asked further study of our NGS data.

We are interested to look at evolution of bacteria in presence of bacteriophages within the gastrointestinal tract of animals.    

Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and more serious infections. Moreover, emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon and Côte d’Ivoire. These strains are particular, because although they belong to the B. cereus species genetically speaking, they harbor two plasmids, pBCXO1 and pBCOX2, that are very similar to the pOX1 and pOX2 plasmids of Bacillus anthracis that encode the toxins and the polyglutamate capsule, respectively. Around one hundred strains of Bacillus cereus from different origins (environment, clinical and food) deposited at the Collection de l’Institut Pasteur (CIP)  are studied and whole genome sequencing has been performed in order to characterize the pathogenicity of the strains by looking for virulence genes and by comparing the strains (genes of interest), depending on their origin.

Identification de SNP entre 2 souches bactériennes la souche sauvage et le révertant (Pseudomonas aeruginosa)

The microbiota of the gastrointestinal tract is a diverse mixture different species and strains of bacteria. We aim to identify regions of dissimilarity between two different bacterial strains to be able to quantitate their relative abundance and thus obtain information about their ability to cohabitate a common environment. In addition, we are interested in assessing the surface localised protein repertoir of specific species through a combination bioinformatic analysis and proteomics.

Escherichia coli is one of the major bacterial pathogens that are responsible for numerous nosocomial infections. While most of the E. coli infections are rather related to colonisation of the urinary tract there are rare, but complex to resolve, cases of E. coli infection of central veinous catheter. To determine whether the E. coli strains that colonize central veinous catheters have specific properties we have started a preliminary project where few E. coli strains responsible for such infections have been collected (4 strains) and a comparative phenotypic analysis has been initiated. To deepen this analysis the genome of these 4 strains has been sequenced and we expected to analyse these genomes to identify potential interesting features that can be link to the clinical context of these strains. Notably, it is expected that these strains of E. coli might carry some specific adhesion factors, virulence genes and potential antibiotic resistance. This project could lead to the identification of some interesting factors that can explain their tropism for central veinous catheters. It is envisionned that this project can be expanded to the analysis of more E. coli strains responsible for central veinous catheter-related infections, and thus could open the way for the development of either biological markers of such infection or fighting strategies against catheter-related infections.

Trichosporon asahii is a yeast responsible of human invasive infection worldwide. Actually, no genotyping method is available to determine relationship between clinical isolates. At the NRCMA we have more than 40 clinical isolates and 2 collection strains associated with clinical data. Thanks to P2M facility, whole genome for 33 isolates was sequenced. The aim of this project is to study the genetic diversity of Trichosporon asahii and the potential relationship with clinical and/or phenotypic data and finally propose a new genotyping method that could be usefull for clinician in case of local or national outbreak.

Alterations of the cellular proteome over time due to spontaneous or enzymatic deamidation of glutamine (Gln) and asparagine (Asn) residues is a probable source of aging-related diseases. In particular, deamidation of a conserved glutamine in all GTPases of the Ras superfamily that are essential for cellular GTP turnover, confers to these molecular switches gain-of-function properties that can stimulate oncogenic signaling pathways. The CNF1 toxin produced by pathogenic Escherichia coli, a prevalent resident in human gut microbiota, is an example of a bacterial factor with the potential to cause somatic “oncogenic mutation” of Rho GTPases through deamidation. Development of holistic approaches for personalized medicine to monitor healthy microbiota could led to improved public health and increased lifespans.

- The Institut Pasteur genomic taxonomy database of microbial strains (“Pasteur MLST”) is a free, publicly-accessible resource that hosts nucleotide sequence-based definitions of microbial strains, along with information on bacterial isolates (provenance data) and their genomic sequences. The Pasteur MLST database provides universal nomenclatures that are largely adopted for important pathogens (Klebsiella, Listeria, …), and represent a unifying language on strains for microbial population biology. - Unified strain taxonomies facilitate the coordinated international surveillance of bacterial pathogens. Several hundred research laboratories and public health agencies worldwide have deposited novel strain types, sequences and provenance data on their bacterial isolates. - Pasteur MLST is powered by the Open source GPL3 BIGSdb web application developed at Oxford University (Keith Jolley & Martin Maiden). (http://bigsdb.pasteur.fr ). Its evolution in terms of functionality is tightly linked to the developments of the software at Oxford U. Its evolution in terms of contents is managed by dedicated international teams of curators for each bacterial pathogenic species, coordinated by the PasteurMLST team. - The genomic taxonomies hosted at Pasteur MLST represent unique, authoritative resources that are highly valued by the community, as testified by the routine use of Pasteur MLST strain tags (e.g., K. pneumoniae ST258) in the scientific literature. Several labs (National Reference Centers or Units) of Institut Pasteur are coordinating the curation of genomic taxonomies (Klebsiella, Listeria, Corynebacteria, Bordetella, Leptospira, Yersinia, ...). The aim of the project is to obtain support from the C3BI HUB for the maintenance of the BIGSdb instance at Pasteur: deployment, upgrades, installation of API functionality developed by our partner, coping with future IT evolutions, ...

Helicobacter pylori is a Gram-negative pathogen whose infection results in various gastric diseases including gastric cancer in Humans. Current drug therapy against the bacteria involves a combination of two antibiotics, proton-pump inhibitor and a Bismuth salt. Introduction of bismuth has resulted in increased success rate compared to traditional therapies without bismuth salt. H. pylori is a genetically variable bacteria with high rates of mutations and recombination. Interestingly till now, there is no report of Bismuth resistance in H. pylori clinical isolates. Very limited data is available about the mechanism of entry and the anti-bacterial action of bismuth in H. pylori. We selected laboratory strain of H. pylori for bismuth resistance in order to identify/determine its effect in H. pylori. The resistance strains exhibit different levels of susceptibility to bismuth compared to the parent strain. Comparative genomics will shed light on the mode of resistance(s) acquired in H. pylori strains. Identification of genes and pathway involved in bismuth resistance will be useful to determine the mechanistic details of bismuth's action.

Members of the genus Yersinia include environmental as well as pathogenic bacteria. Pathogenic species (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica) have historically been targets for research aimed at understanding how bacteria evolve into diverse mammalian pathogens. The advent of large-scale population genomic studies has greatly accelerated the progress in this field, revealing how gene gain, gene loss and genome rearrangement events have impacted evolution of enteropathogenic bacteria. In the context of the sequencing of many novel genomes of Yersinia spp., within this project we are interested in the generation of a bioinformatic tool that will allow us to compare and visualize single nucleotide polymorphisms as well as genome organization in members of the genus Yersinia, in order to better understand genomic events that drive evolution within this bacterial species.

Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica are major human pathogens. Bacnet is a Java based platform to develop websites for multi-omics analysis. Yersinomics allows comparative genomics and transcriptomics between pathogenic Yersinia species. In the tool a SynTView module is dedicated to study synteny profiles between several strains. We use complete genomic data from Yersinia bacterial strains obtained in the refseq ftp site to compute the orthologs between three pathogenic Yersinia species (Y. pestis, Y. pseudotuberculsis and Y. enterocolitica) with bi directional best hits strategy and use SyntView to do visual genomic comparison. Due to the discontinuation of the use of flash technology on some web-browser, SynTView must be rewrite in another language. Part of the application (snps visualization) is already developed in javascript and will serve as a starting point for the rewrite of SynTView local view.

The Yersinia pseudotuberculosis complex comprises the plague pathogen Yersinia pestis, the enteropathogen Yersinia pseudotuberculosis, as well as specific sub-group of Y. pseudotuberculosis strains responsible for the Far-East Scarlatine-Like Fever (FESLF). We have sequenced the genomes of FESLF strains and we are interested in investigating the genome organization of these bacteria.

Leishmania causes devastating human diseases – leishmaniases - representing an important public health problem in the Mediterranean basin and declared as emerging diseases in the EU due to climate change and population displacement. The LeiSHield-MATI consortium will for the first time investigate in an integrative fashion the complex parasite-vector-host interplay in cutaneous leishmaniasis affecting Morocco, Algeria, Tunisia, and Iran (MATI), using field isolates and human clinical samples. The ultimate goal of our project is to identify genetic factors selected during natural infection and to understand how the complex parasite-vector-animal interaction impacts clinical outcome in infected patients. This goal will be achieved through a highly ambitious secondment plan between all partners, and the organization of courses and workshops to train the next generation of scientists generating a long-term impact on the research capacities in endemic areas. Capitalizing on complementary infrastructures of its EU, African and Asian partners and their expertise in molecular parasitology, epidemiology, systems level analyses, bioinformatics, computational biology, immunology, dermatology, field studies, and public health, our project will drive important innovation in clinical research, strengthen capacities in disease endemic regions, inform authorities on control measures, and raise awareness in all partner countries on this emerging EU public health problem. The highly inter-disciplinary and inter-sectorial structure of LeiSHield-MATI, and its powerful integrative and comparative approach is novel in parasitic systems and will drive a unique bio-marker discovery pipeline for the future development of new prognostic and diagnostic tools, as well as novel preventive and therapeutic measures that will ensure long-term collaboration, promote scientific and commercial self-sustainability of its partners, and will have an important impact to improve public health.

Because of the fast-growing number of assembled genomes available in the public repositories (GenBank/RefSeq), it is now quite common to end up with many genome assemblies that belong to the same species. In this current context, determining the type strain/species of any new species/genus requires to identify the representive one(s) within a large collection of genomes. The medoid of a genome collection being the one(s) whose sum of distances to all other genomes is minimal, it is an excellent candidate to be the representative genome of a collection. We therefore aim at procuring a bioinformatic tool able to quickly and accurately identify the medoid of any genome collections.

Finely tuned sensory systems enable bacteria to sense and respond to fluctuating environments, coordinating adaptive changes in metabolic pathways and physiological outputs. For pathogenic Leptospira, signaling pathways allow a timely expression of virulence factors during the successive steps of infection of a mammal host. As the bacteria is excreted by its host, signaling pathways enable switching the expression towards factors promoting survival in the environment. A unifying theme across bacterial species is that biofilm formation coincides with the synthesis of the cellular signaling molecule bis-(3?-5?)-cyclic dimeric guanosine monophosphate (c-di-GMP) and this feature seems to be conserved in Leptospira. Our current work shows that the c-di-GMP regulation pathway is a major regulatory network involved in biofilm formation, virulence and motility in the pathogen Leptospira interrogans. Biofilm production and virulence expression is quite variable across the leptospira genus (highly virulent species, low virulent species and saprophytes species showing increase biofilm production). We would like to explore how the c-di-GMP metabolism, and the many genes associated with its synthesis, and degradation have evolved across the leptospira genus. We believe that understanding the evolutionary relationship of the c-di-GMP metabolism genes in the Leptospira genus would help us to understand the contribution of this second messenger to pathogenesis and biofilm formation in the Leptospira genus

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.

The mechanisms governing ageing, which is a multifactorial process, have not been resolved and constitute a fundamental open question in cell and organismal biology. Exceptionally, in rare genetic diseases like the Cockayne syndrome (CS), ageing is dramatically accelerated. We have recently identified a novel pathway that is altered in cells from CS patients and is not affected in a linked disease (UVSS) that is not associated with precocious ageing. This is a unique case in the literature of diseases due to mutations in the same proteins but only one resulting in precocious ageing. The defect linked to the progeroid phenotype consists in oxidative stress-dependent overexpression of a little known proteases, HTRA3, which degrades the key mitochondrial DNA polymerase and in turn affects mitochondrial function. We succeeded in rescuing the defective phenotype in patient cells using two independent strategies. We aim to understand the mechanism by which oxidative stress induces overexpression of HTRA3 and we have indications that this event may be linked to the methylation of target genes. Genome wide methylation will be analysed in these rare patient cells.

Thanks to RNA-Seq experiments we identified  a new orphan DNA methylase (not associated with a restriction enzyme) that is highly upregulated in the presence of low concentrations of aminoglycosides in V. cholerae. The results allow us to consider a differential DNA methylation profile depending on the environment (with or without antibiotics), which can possibly correspond to a novel epigenetic control of the response.

Enhancers of transcription are regulatory sequences enabling gene expression from a distance. The landscape of active enhancers is cell-type specific and provides extensive information on the transcription factors at play. Currently, enhancers are mostly mapped based on the histone modifications positioned on their DNA sequence. This type of data is abundantly available for tumor-derived tissue culture cells, but difficult to obtain when the biological material has a limited availability. As an alternative approach, it is possible to detect enhancers as sites of divergent transcription. The objective of this project will be to develop tools allowing detection of sites of divergent transcription in transcriptome and run-on data, to evaluate the quality of the prediction by comparing the outcome with existing enhancer maps, and then ultimately use this approach to identify changes in the enhancer landscape between patients with multiple sclerosis and healthy controls.

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.

It has been shown that methylation can act as a kind of memory of the immune system. For patients with co-infections, it is of particular importance to know when to begin an anti-retroviral therapy, especially if they are already infected with tuberculosis. The goal of this study is to find hyper or hypo methylated loci related to the reaction of HIV patients (co-infected or not) to different kind of treatments.

Cellular senescence is a complex stress response that durable (yet not irreversibly) arrests cell proliferation and is accompanied by widespread changes in chromatin structure, metabolism and gene expression including the production and secretion of a plethora of inflammatory factors. Cellular senescence plays beneficial roles during embryonic development, tissue regeneration, and tumor suppression. Paradoxically, it is also considered a major contributor to aging and age-related diseases, the latter mostly through its inflammatory phenotype, the so-called SASP (senescence-associated secretory phenotype). The proposed work aims at integrating time-resolved transcriptome, ChIP-seq, and ATAC-seq datasets into a comprehensive understanding of senescence-associated gene regulation.

We are in the process of publishing a paper showing that IRF8, a transcription factor, regulates MS4A1 expression. Indeed, we showed IRF8 was necessary for MS4A1 expression by knocking-out the gene. A reviewer is asking us to directly assess IRF8 binding on MS4A1 gene using already published CHIP-seq data (PMID:31000603).

In the past few years, evidence that Natural Killer cells can acquire memory properties has challenged the dogma that only adaptive immunity mediates memory responses. This new property of NK cells is defined by a specific enhanced response upon re-exposure linked to substantial chromatin remodeling during NK clonal expansion, and to the acquisition of stable transcriptional changes. However, NK cell memory is poorly understood at the molecular level and exclusively described in response to viral infection. Whether NK cells could acquire memory during bacterial infections and what chromatin remodeling mechanisms are involved remained uninvestigated. However, in the lab, we have shown that following LPS-induced endotoxemia, NK cells acquire long-lasting memory properties associated with specific epigenetic modifications. In this project, using S pneumo infection as a model, we will perform single cell ATAC-seq coupled with transcriptomics (Multiomics) to identify the transcriptional program of the emerging subpopulations of memory NK cells and define its epigenomic landscape. With these data, we aim to identify chromatin rearrangements and gene expression patterns that are induced and maintained in the sub-population of memory NK cells. We believe that this project will enlighten our understanding of how NK cell memory identity and function is encoded at the chromatin level, a wide-open field of study.

The aim of the project is to create a viewer that will help visualisation and correlation between genomic, transcriptomic, proteomic and metabolomic data generated by the comparison of amastigote and promastigote stages of the Leishmania donovani parasite.

Skeletal muscle stem cells constitute a population of cells with heterogeneous properties. Interestingly, muscle stem cells have a remarkable capacity to regenerate muscle fibres after regeneration. We are performing a molecular analysis of these stem cells.

Les cyanobactéries sont des microorganismes qui prolifèrent dans de nombreux plans d’eau et perturbent leurs fonctionnements et leurs usages car elles sont capables de produire des toxines dangereuses pour la santé humaine et animale. Si la réglementation sanitaire est basée, pour l’instant, sur la surveillance d’une seule toxine, il est désormais connu que ces microorganismes sont capables d’en synthétiser un grand nombre qu’il conviendrait de mieux prendre en compte dans le futur. C’est pourquoi, dans le but de mieux connaître le potentiel toxique des cyanobactéries, ma thèse s'applique, par des études sur leur génome et par une approche de chimie, à caractériser les gènes impliqués dans la synthèse de ces métabolites ainsi que les métabolites produits par ces gènes, à déterminer sur des souches de culture et dans des échantillons naturels provenant de plans d’eau d’Ile de France quel est le potentiel de production de ces métabolites et à mieux comprendre les facteurs environnementaux qui favorisent cette production. Deux équipes de Paris (Pasteur et iEES) sont associées sur ce travail qui implique également des collaborations étrangères. S'il est désormais bien connu qu'une part importante du métabolisme des cyanobactéries qui sont des microorganismes photosynthétiques, est régulée en fonction des phases de lumière et d'obscurité, les connaissances disponibles sur la synthèse des métabolites secondaires sont en revanche beaucoup plus limitées. Ces métabolites ont pourtant un double intérêt puisque certains sont toxiques pour l'Homme alors que d'autres ont un intérêt pharmaceutique potentiel. Leur synthèse repose sur l'expression de clusters de gènes pouvant être de très grande taille (jusqu’à 100 kb par région).

Human macrophages are the main cell target of Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis. Once phagocytosed, bacilli escape bactericidal functions of macrophages and they multiply inside the cell. Understanding interactions between bacilli and human phagocytes is primordial to develop new strategies to combat tuberculosis. We propose a project aiming at deciphering cell-cell communications between infected cells and non-infected cells. We will use the transcriptomic and the proteomic approaches to discriminate the global response of these differents sub-populations. To our knowledge, this work represents the first study that combines two different levels for the identification of a specific response of infected-cells and neighboring non-infected cells. This study would determine cellular markers of a Mtb signature.

Currently there is increased focus for developing novel antibacterial strategies. The demand is driven by the rise in antibiotic resistance bacteria and many Gram-negative bacteria are on the list of increasingly drug resistant agents. A major target of successful antibiotics is the bacterial cell wall. The target of these drugs is often defined but what is much less understood is the off-target impact of these very important antibiotics. We designed and executed a mutli-omics strategy centered on the Gram-negative pathogen H. pylori. Our goal is to identify potential 'therapeutically susceptible' pathways associated with the physiological response to cell wall stress.

Over the past three decades Listeria has become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields including virulence-factor regulation, cell biology, and bacterial pathophysiology. More recently, the number of Listeria “omics” data produced has increased exponentially, not only in term of number, but also in term of heterogeneity of data. There are now more than 40 published Listeria genomes, around 400 different transcriptomics data and 10 proteomics studies available. The capacity to analyze these data through a systems biology approach and generate tools for biologists to analyze these data themselves is a challenge for bioinformaticians. To tackle these challenges we are developing a web-based platform named Listeriomics which integrates different type of tools for “omics” data manipulation, the two most important being: 1) a genome viewer for displaying gene expression array, tiling array, and RNASeq data along with proteomics and genomics data. 2) An expression atlas, which is a query based tool which connects every genomics elements (genes, smallRNAs, antisenseRNAs) to the most relevant “omics” data. Our platform integrates already all genomics, and transcriptomics data ever published on Listeria and will thus allow biologists to analyze dynamically all these data, and bioinformaticians to have a central database for network analysis. Finally, it has been used already several times in our laboratory for different types of studies, including transcriptomics analysis in different biological conditions, and whole genome analysis of Listeria proteins N-termini. This project is funded by an ANR Investissement d'avenir: BACNET  10-BINF-02-01

Malaria remains a major problem in many tropical countries with Plasmodium falciparum accounting for up to 1 million deaths, primarily in infants and children residing in endemic areas of sub-Saharan Africa. P. vivax, the other important species for human malaria is geographically more widespread and causes 80-100 million cases of malaria each year. All the pathology related to malaria is attributed to the blood stage of the parasite life cycle during which Plasmodium merozoites invade and multiply within host erythrocytes. We are interested in understanding the process of RBC invasion by malaria parasites at the molecular level with the goal of blocking their interaction with antibodies to inhibit invasion and kill the parasite. The first generation of recombinant blood stage malaria vaccine candidates based on antigens such as the merozoite surface protein (MSP1) and apical merozoite antigen-1 (AMA01) have been tested in field trials and failed to provide any efficacy against P. falciparum malaria. There is thus an urgent need to identify novel parasite antigens that play a role in invasion and can serve as vaccine candidates that elicit strong antibody responses that block blood stage parasite growth. In this project, we propose to mine the P. falciparum and P. vivax genome databases using bio-informatic tools to identify potential, novel blood stage invasion related parasite antigens that can serve as potential candidates for blood stage malara vaccines. Criteria such as expression profile, presence of conserved domains in orthologs from related plasmodium species, limited polymorphisms in field isolates, interaction with other invasion related proteins and localization to apical organelles or merozoite surface will be used to interrogate P. falciparum and P. vivax genome sequence data and identify potential invasion related antigens that will be selected for validation as vaccine candidates for malaria.

Detection of nucleic acids at the single cell level using microscopy has now reach high throughput levels which promise exciting discoveries concerning the functionning of the genomes. The HSC3D project funded by the CITECH in the Pasteur institute aims at multiplexing nucleic acid detection by Fluorescence In Situ Hybridisation (FISH). This necessitates libraries in the range of tens of thousands of  100mer DNA oligonucleotides which will be synthesised by digital lithography. Several constrains apply to these oligonucleotides which therefore require heavy duty bioinformatics for their design .

Insect vectors durably transmit many important human and animal diseases. Insects are mobile, adaptable and difficult to control, which makes them efficient vehicles for disease emergence, spread and maintenance. Genomic tools have been applied to the study of vectors and vector control, and some insects such as the African malaria vector Anopheles gambiae have now become new model organisms for natural host-pathogen interactions. We study mosquito vectors of malaria and arboviruses, which generates four main kinds of large-scale data that requires dedicated bioinformatics expertise: i) functional dissection of mosquito immune signaling pathways by RNAseq transcriptome profiling to detect responses to pathogen infection and/or silencing of target genes, including mRNA as well as small and other non-coding RNAs, ii) next-gen genetic linkage mapping by deep sequencing of index-tagged phenotyped individual mosquitoes, iii) population genomic analysis by whole-genome sequencing of hundreds of individual 250Mb mosquito genomes, iv) metagenomic analysis of the mosquito microbiome and pathogen susceptibility, and of ecological metagenomic communities in field samples of mosquito vectors.

Cryptococcus neoformans is a ubiquitous yeast present in the environment that is able to interact closely with numerous organisms including amoeba, paramecium or nematodes. The interaction with these organisms shaped its virulence with acquisition of infectious properties as a consequence especially in mammals . The ability to survive nutrient starvation, oxidative stress, desiccation, both in the environment and during infection, indicates a high level of physiological and metabolic plasticity of the yeast. In humans, after primary infection during childhood, the yeast is able to survive within the host for years before reactivation upon immunosuppression, leading to a life threatening  disseminated fungal infection. This phenomenon, called dormancy / quiescence is one of the main biological features of this fungus in relation with disease's pathogenesis. It is well known in bacteria (tuberculosis), parasites (Plasmodium, Toxoplasma). In C. neoformans, dormancy has only been demonstrated epidemiologically in our laboratory but not experimentally so far. We developed an assay where yeasts cells exhibiting characteristics of potentially dormant cells were generated. Indeed, dormant cells are characterized by a low metabolic activity sometimes undetectable under normal laboratory conditions, altered growth capacity, and the ability to resuscitate upon adequate stimulus. Dormant cells are known to have increased mitochondrial masse and activity justifying a screening strategy of a collection of KO mutants for mitochondrial proteins. In parallel the whole proteome, transcriptome and secretome will be obtain with the ambition to correlate these parameters. Our current project aims at exploring the metabolism of the dormant yeast to have a comprehensive picture of the pathways that are required for the maintenance of dormancy and fo exit from dormancy.  

Chromosomes are arranged non-randomly in the 3D space of cellular nuclei. This architecture is important to key biological processes including gene expression and DNA repair, but remains poorly understood in detail. Genome-wide chromosome conformation capture (Hi-C) is currently the most powerful experimental technique to probe chromosomal structures. However, this technique does not directly provide a description of high-resolution chromosomal configurations in individual cells and remains blind to the dynamics of chromatin in live cells. In this research, we developed experimental imaging and associated computational approaches to describe the structures of chromosomes at high resolution in individual cells.

The human gene ANOS1, responsible for the X-chromosome-linked form of Kallmann syndrome (a developmental disease affecting the olfactory system), has been identified in 1991 by positional cloning. It is located on the X chromosome short arm (at Xp22.3), close to the STS gene and close to the boundary of the pseudoautosomal region (common to the X and Y sex chromosomes). Since then, orthologous genes have been identified in all animal species (including invertebrates), except in the mouse, rat, and other rodents (an orthologue in the naked mole-rat Heterocephalus glaber is however present in the GeneBank data base). The orthologous STS in the mouse has been identified in the mouse, and is located in the XY pseudoautosomal region in this species. The sequence of the mouse STS is unusually GC-rich and has markedly diverged from the human orthologous sequence, even though the amino acid sequence of the protein is highly conserved. The ANOS1 orthologous genes have not yet been identified in the mouse/rat, although we have been able to detect the encoded protein anosmin-1 in these species, with antibodies directed against the human protein (the orthologous proteins in the mouse and rat have the expected size in western blot analysis, i.e. about 690 amino-acids).

It has been found that specific fragments of the peptidoglycan (PG), namely the Muramyl-triDAP (MTP) and Muramyl-dipeptide (MDP) are specifically recognized by the innate immune receptors Nod1 and Nod2, respectively (Girardin, 2003). However, it is still not known how these fragments reach the cytosol after being shed by bacteria. Therefore, the near-haploid human cell line HAP-1 (horizondiscovery;HAPLOGEN) has been transducted with a retroviral construct containing a promoterless GFP that will fuse with whatever gene after the construct has randomly inserted into the genome. It is possible to have several insertions in one cell. The cells will be stimulated with fluorescently labelled PG, afterwards interesting samples will be sorted by FACS sorter and the DNA will be prepared and sequenced by NGS.

Aedes albopictus is an important vector for transmitting arboviruses, such as Dengue, Chikungunya, West Nile or Zika viruses. Its worldwide distribution due to its high ability to adapt to variable environments makes this species a serious threat. This mosquito also better transmits Chikungunya than Dengue virus, and many studies are still trying to understand the deep relationships between these viruses and their vectors in order to develop new control strategies. Moreover, it is now well known that retroviruses partially integrate into host's genomes since it is mandatory for their replication cycle. Interestingly, Retroviridae are not the only viral family capable of host integration. Indeed, in the last decade, owing to new molecular technologies such as next-generation sequencing and bioinformatic tools, non-retroviral integrated RNA virus sequences (NIRVS) have been found into many animal genomes, including Aedes mosquitoes. This came as a surprise since RNA viruses do not have DNA intermediate in their replication cycle. However, little is known about these integrations and many questions remained unsolved. The aim of this project is to assess the production of NIRVS in persistently-infected cells from Ae. albopictus and Ae. aegypti. It is divided in several critical points : the first one is to know how fast the virus can integrate into the cell genome. The second one is to determine which part of the viral genome can integrate and finally, where can it integrate in the genome. This will allow us to understand the virus/host interaction and if NIRVS formation is a mechanism that vectors/hosts evolved as a general response to RNA viruses.

Many neurodegenerative disorders are caused by the large expansion of a repeated sequence, called a "trinucleotide repeat". Our laboratory is using the CRISPR-Cas family of endonucleases in order to shorten the repeat tract below the length that is known to be pathological in humans. Some of the nucleases tested are very efficient at cutting the repeat tract, but in order to make this approach a viable gene therapy strategy, we must ensure that the nuclease is not inducing mutations in other parts of the genome (so-called "off-targets"). The present project aims at analyzing all possible off-targets of these nucleases on the different repeated sequences involved in neurodegenerative disorders, in order to validate (or invalidate) this approach.

In the TSARA project, we aim at developing and validating a molecular platform (Dual Index Targeted Amplicon Deep Sequencing on Illumina iSeq) and a dedicated bioinformatics pipeline for targeted high throughput sequencing of genetic loci related to antimalarial drug resistance. The iSeq Illumina platform, proposed here, is designed for dual indexing of samples, consisting of a 3’ and 5’ individual barcode, which allow the user to connect every sequence generated to a specific sample (up to 384 samples). The development and the validation of this novel molecular tool will be performed at IP Paris first by using DNA extracted from P. falciparum reference strains (3D7, Dd2 and culture-adapted Cambodian strains) (WP1) and second, by using P. falciparum DBS samples collected from two malaria endemic areas (Central African Republic and Cameroon) (WP2). In the WP3, we will host and train staff from IP Bangui and CP Cameroon to this new approach. Our final objective is that Dual Index Targeted Amplicon Deep Sequencing on iSeq along with minimal bioinformatics infrastructure operated in countries that are endemic for malaria to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

Bacteria of Shigella species are responsible for bacillary dysentery (shigellosis) in humans. A characteristic feature of these bacteria, used for identification purposes, is their inability to use lactose as a carbon source, in contrast to the closely related species Escherichia coli. We recently isolated clones of Shigella exhibiting a Lac+ phenotype on Drigalski medium and we are attempting to identify the gene(s) involved in this phenotype.

The mechanisms underlying Anopheles mosquito susceptibility to malaria parasite infection in nature are not understood. We infected wild Anopheles pedigrees in West Africa to map loci for susceptibility to the human malaria parasite Plasmodium falciparum. Individuals from phenotyped pedigrees were entirely sequenced in order to identify and fine-map segregating loci.

Antibiotic resistance development is a public health liability and treatens treatment of bacterial infections. We are inetersed in genetic determinant of antibiotic resistance in Gram negative bacteria. We have identified V. cholerae mutants which are more tolerant to aminoglycosdies. Our aim is to identify cellular processes involved and characterize molecular mechanisms.

In the TSARA project, we aim at developing and validating a molecular platform (Dual Index Targeted Amplicon Deep Sequencing on Illumina iSeq) and a dedicated bioinformatics pipeline for targeted high throughput sequencing of genetic loci related to antimalarial drug resistance. The iSeq Illumina platform, proposed here, is designed for dual indexing of samples, consisting of a 3’ and 5’ individual barcode, which allows the user to connect every sequence generated to a specific sample (up to 384 samples). The development and the validation of this novel molecular tool will be performed at IP Paris first by using DNA extracted from P. falciparum reference strains (3D7, Dd2 and culture-adapted Cambodian strains) (WP1) and second, by using P. falciparum DBS samples collected from two malaria-endemic areas (The central African Republic and Cameroon) (WP2). In the WP3, we will host and train staff from IP Bangui and CP Cameroon to this new approach. Our final objective is that Dual Index Targeted Amplicon Deep Sequencing on iSeq along with minimal bioinformatics infrastructure operated in countries that are endemic for malaria to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure the continued success of the malaria treatment policy

Historically circulating in Africa, Rift Valley fever virus (RVFV) was firstly identified in Kenya in 1930, and responsible for dozens of outbreaks in Africa in the past decades. As an arbovirus, RVFV is mainly transmitted by mosquito vector, and needs to be altered between mammalian host and mosquito vector for maintaining the genome stability. In this project, we are analysing the mosquito vector competence for several RVFV variants obtained from different host cells, to better interpret the risk factors of transmission.

Our project aims to study the consequences of the systemic inflammation on muscle stem cells (MuSCs). To address this question, we use two in vivo mouse models associated with high systemic inflammation, an acute viral respiratory infection, with the influenza virus, and a solid tumor growth. Both paradigms are associated with muscle catabolism, named cachexia, which is intensively studied in the cancer field. However, most studies are focusing on the muscle fibres, the contractile apparatus of the muscle. Our project is mainly focused on the study of the MuSCs, cells which give rise to skeletal muscle. We aim to characterize the molecular and functional modifications occurring in MuSCs when exposed to high inflammatory stress. RNAseq was performed for both paradigms to gain information on gene expression.

Myotonic dystrophy type 1 (DM1 or Steinert disease) is a monogenic neurodegenerative disorder due to the expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene on chromosome 19. We developed a strategy to induce a DNA double-strand break (DSB) within the expanded repeat in order to contract it below the pathological range in humans. This approach was successful in yeast and mouse cells and we are now trying to apply it to human cells from a patient affected by DM1. This patient was diagnosed with a large CTG repeat expansion (≈ 2000 triplets) and we estimated the size of the repeat tract by Southern blot to be 1980 triplets. These cells (ASA cells) were immortalized by overexpression of the telomerase catalytic unit, theoretically allowing to grow them for 100 generations (instead of ca. 20 generations for primary cells). A TALEN directed to the expanded CTG repeat was expressed in ASA cells for several weeks and the population was subcloned. Trinucleotide repeat length was analyzed in many clones, by Southern blotting. This showed that the repeat was partially contracted in most clones, the others showing no size change (or a small expansion). Ten clones were chosen to be sequenced by PacBio, in addition to a non-treated ASA clone that will be used as the reference for future work on ASA cells.

Advances in high-throughput sequencing may allow more rapid characterisation of viral diversity in samples. However, although there are many tools to perform taxonimuqye assignment of the reads obtained, there is no automated pipeline to obtain the different sequences by species and genus, and when the depth is sufficient to apply correction algorithms. In the end, obtaining these sequences will allow us to better understand the diversity and prevalence of each of the species highlighted in our samples.

Leishmania causes devastating human diseases – leishmaniases - representing an important public health problem in the Mediterranean basin and declared as emerging diseases in the EU due to climate change and population displacement. The LeiSHield-MATI consortium will for the first time investigate in an integrative fashion the complex parasite-vector-host interplay in cutaneous leishmaniasis affecting Morocco, Algeria, Tunisia, and Iran (MATI), using field isolates and human clinical samples. The ultimate goal of our project is to identify genetic factors selected during natural infection and to understand how the complex parasite-vector-animal interaction impacts clinical outcome in infected patients. This goal will be achieved through a highly ambitious secondment plan between all partners, and the organization of courses and workshops to train the next generation of scientists generating a long-term impact on the research capacities in endemic areas. Capitalizing on complementary infrastructures of its EU, African and Asian partners and their expertise in molecular parasitology, epidemiology, systems level analyses, bioinformatics, computational biology, immunology, dermatology, field studies, and public health, our project will drive important innovation in clinical research, strengthen capacities in disease endemic regions, inform authorities on control measures, and raise awareness in all partner countries on this emerging EU public health problem. The highly inter-disciplinary and inter-sectorial structure of LeiSHield-MATI, and its powerful integrative and comparative approach is novel in parasitic systems and will drive a unique bio-marker discovery pipeline for the future development of new prognostic and diagnostic tools, as well as novel preventive and therapeutic measures that will ensure long-term collaboration, promote scientific and commercial self-sustainability of its partners, and will have an important impact to improve public health.

Myotonic dystrophy type 1 (DM1 or Steinert disease) is a monogenic neurodegenerative disorder due to the expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene on chromosome 19. We developed a strategy to induce a DNA double-strand break (DSB) within the expanded repeat in order to contract it below the pathological range in humans. This approach was successful in yeast and mouse cells and we are now trying to apply it to human cells from a patient affected by DM1. This patient was diagnosed with a large CTG repeat expansion (? 2000 triplets) and we estimated the size of the repeat tract by Southern blot to be 1980 triplets. These cells (ASA cells) were immortalized by overexpression of the telomerase catalytic unit, theoretically allowing to grow them for 100 generations (instead of ca. 20 generations for primary cells). A TALEN directed to the expanded CTG repeat was expressed in ASA cells for several weeks and the population was subcloned. Trinucleotide repeat length was analyzed in many clones, by Southern blotting. This showed that the repeat was partially contracted in most clones, the others showing no size change (or a small expansion). Ten clones were chosen to be sequenced by PacBio, in addition to a non-treated ASA clone that will be used as the reference for future work on ASA cells.

The objective of this project is to study the virome present in noctule bats from East Europe, in order to identify potential (re) emerging zoonotic viruses.

Research of homopolymers in the integron integrase genes

Persistent viral infections in mosquitoes require equilibrium between viral replication and efficient antiviral defenses. Arboviruses are able to counteract immune defenses and especially, the RNAi system. Production of DNA forms from several non-retroviral RNA viruses (vDNA) has been found to promote persistent infections and prevent lethal acute infections in mosquitoes and cell cultures. In vitro, vDNA was detected at the early stage of infection (24 hours post-infection for dengue virus). vDNA was suggested to be produced from defective viral genomes (DVGs) rather than from a full genome. NIRVs are non-retroviral integrated RNA virus sequences (NIRVS) present in host genomes. Most of the NIRVS found in Aedes mosquitoes and cell lines were from insect specific viruses (ISVs) belonging to Rhabdoviridae and Flaviviridae families. Among NIRVS originating from flaviviruses, most of them displayed similarities to sequences encoding non-structural proteins. These NIRVS are more often embedded in regions rich in retrotransposons and piRNA clusters. It is likely that according to their position in the mosquito genome, NIRVS functions (if any) would be different. We aim to characterize NIRVS production in in vitro models by establishing persistent infections. We selected five different viruses: two arboviruses causing important public health issues (chikungunya and dengue), two ISFs displaying high prevalence in Aedes mosquitoes (CFAV and KRV) and a rhabdovirus (vesicular stomatitis virus (VSV) which is also an arbovirus). Infections were performed in two different cell lines that are both RNAi-proficient and isolated from embryonic tissues: Ae. aegypti Aag2 and Ae. albopictus U4.4 cell lines. This experiment will help to characterize regions of the mosquito genome that are suitable for viral integration, but also parts of the viral genome that are more prone to integrate.

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 estimated annually in the word, with 10% of mortality. Pathogenic Leptospira are well-equipped to sustain the oxidative stress encountered when infecting their hosts. Surprisingly, unlike most aerobic bacteria, they lack classical enzymes that detoxify superoxide, i. e. superoxide dismutase or reductase. We have explored whether pathogenic Leptospira can genetically adapt to the presence of superoxide. Mutants exhibiting a higher resistance to superoxide have been isolated and we aim at identifying genetic variations responsible for the higher resistance to superoxide.

The gastrointestinal tract of humans is colonized by hundreds of microbial species, - bacteria, archaebacterial, fungi, protozoa and viruses -, collectively named the gut microbiome. The intestinal commensal bacteria have an important role in metabolic processes and contribute to colonization resistance against intestinal pathogens. Fungi are usually considered to be a minor component of the global microbiome. However, the mycobiome (fungal component of the entire microbiome) has been in fact little studied particularly with regards to its relationships with the other components of the microbiome. Fungi could be important players of the microbiome because some fungal species are able to proliferate in response to diet or during dysbiosis due to antibiotic treatment or gut inflammation. The aim of this project is to investigate the effect of specific antibiotics (primarily anti-Gram negative bacteria antibiotic) on the gut mycobiome. More specifically, we will examine the impact of cefotaxime and ceftriaxone, 2 antibiotics with same antimicrobial spectra but different rates of biliary elimination, on the changes in fungal communities.

Bacteria analysis of fecal samples by 16S sequencing has provided a wealth of information for the distribution of bacterial species in both health and disease conditions. When looking at bacterial composition, the focus in generally put on the presence or absence of the most abundant species; yet, even low abundant species may have significant effects on the host when present at even low abundance. To assess the possible contribution of low abundance bacterial species, we aim to undertake a targeted search of publicly available 16S databases for a restricted number of commensals to assess their possible role in the various conditions tested.

Our hypothesis is that gut microbiota could define predictive markers of response and tolerance to biologics. A. Preliminary results: gut bacteria predicting response to TNF blockers. A proof-of-concept study has been performed on 58 patients who were recruited according to the following criteria: active disease despite NSAIDs intake; no history of inflammatory bowel disease; no antibiotics intake within 3 months prior recruitment. Bacterial 16S rRNA gene sequencing region was performed on stools samples before and after TNF-blocker treatment. Diversity metrics and custom LefSe were used to explore the relationship between the composition of the intestinal microbiota and the efficacy of TNF-blockers. A lower alpha diversity at baseline was unexpectedly associated with better treatment response, HLA-B27 genotype and smoking behavior. Meanwhile, beta diversity was associated with smoking behavior and HLA-B27 genotype before and after treatment. Beta diversity at baseline was associated with the BASDAI index after treatment, and the response to the treatment. These results indicate a potential regulatory role for the gut microbiota on the underlying mechanisms involved in the response to TNF-blockers. Moreover, a LefSe-like approach identified 6 bacterial species as potential biomarkers for the treatment response, despite the absence of global changes (beta diversity) in the microbiota composition following a 3-month TNF-blockers intake. B. Current project: ITS2 fungal rDNA sequencing analyses In order to establish a causal link between host-microbe interactions and clinical efficacy of anti-TNF, we expect the following research endpoints from our experimental and translation approach (cohorts/clinical trials): 1. Defining the impact of anti-TNF on fungal microbiota and on the relative representation of fungal/bacterial components 2. Defining correlations between gut fungal composition and clinical outcome, with the aim of identifying stool microbial fingerprint of durable responses and/or primary resistance to anti-TNFα in SpA patients. The analyses will be performed on the same 58 SpA patients that were previously analyzed for their 16S bacterial component. These patients perfectly described regarding their disease characteristics, demographics, ongoing treatments, response to anti-TNF after a 3-month treatment period.

Cepia mass produces two species of Anopheles mosquitoes for research teams studying malaria. The aim of this project is to assess 1) whether or not the compostion of the bacterial microbiota fluctuates over time and if so to what extent ; 2) if there are significant variations can these be correlated with the degree of infectability by plasmodium parasites and/or the fitness of the mosquitoes (measured by their survival rate).

Previous animal models of Primary Antibody Deficiency (PAD) display severe dysbiosis and gut inflammation, which makes them incompatible to study a wide spectrum of PAD (e.g., most IgAD subjects display only mild dysbiosis and are asymptomatic). We would like to evaluate a novel mouse model of PAD and then perform microbiome transplant and IgA supplementation. The first objective is to evaluate the causal role of the microbiome in mediating PAD-associated disease. Our second objective is to apply an IgA supplement to reverse gut dysbiosis in symptomatic PAD and look at the microbiome changes caused by supplementation.

Insect vectors durably transmit many important human and animal diseases. Insects are mobile, adaptable and difficult to control, which makes them efficient vehicles for disease emergence, spread and maintenance. Genomic tools have been applied to the study of vectors and vector control, and some insects such as the African malaria vector Anopheles gambiae have now become new model organisms for natural host-pathogen interactions. We study mosquito vectors of malaria and arboviruses, which generates four main kinds of large-scale data that requires dedicated bioinformatics expertise: i) functional dissection of mosquito immune signaling pathways by RNAseq transcriptome profiling to detect responses to pathogen infection and/or silencing of target genes, including mRNA as well as small and other non-coding RNAs, ii) next-gen genetic linkage mapping by deep sequencing of index-tagged phenotyped individual mosquitoes, iii) population genomic analysis by whole-genome sequencing of hundreds of individual 250Mb mosquito genomes, iv) metagenomic analysis of the mosquito microbiome and pathogen susceptibility, and of ecological metagenomic communities in field samples of mosquito vectors.

The gut microbiota is instrumental in stimulating, shaping and educating the host immune system, both in and outside of the gut. One of the most immunostimulatory commensals identified to date is the unusual bacterium Segmented Filamentous Bacteria (SFB). SFB intimately attaches to absorptive epithelial cells in the ileum after weaning in a host-specific manner. We aim to do compare the genomes of SFB from a range of vertebrate species to identify shared and strain-specific differences that may bring insights into their niche adaptations.

The objective of this project is to study the virome present in specific bat colonies in China and their associated ectoparasites, in order to identify potential (re) emerging zoonotic viruses and to elucidate their transmission route, especially as putative arboviruses.

With an estimated 1031 particles on earth, bacteriophages are the most abundant genomic entities across all habitats and important drivers of microbial communities. Growing evidence suggest that they play roles in intestinal human microbiota homeostasis, and recent metagenomics studies on the viral fraction of this ecosystem have provided crucial information about their diversity and specificity. However, the bacterial hosts of this viral fraction, a necessary information to characterize further the balance of these ecosystems, remain poorly characterized. Here we unveil, using an enhanced metagenomic Hi-C approach, a large network of 6,651 host-phage relationships in the healthy human gut allowing to study in situ phage-host ratio. We notably found that half of these contigs appear to be sleeping prophages whereas ¼ exhibit a higher coverage than their associated MAG representing potentially active phages impacting the ecosystem. We also detect different candidate members of the crAss-like phage family as well as their bacterial hosts showing that these elusive phages infect different genus of Bacteroidetes. This work opens the door to single sample analysis and concomitant study of phages and bacteria in complex communities.

Immune responses are conditioned by host genetics as well as environmental stimuli that include disease-causing pathogens but also a diverse community of commensal micro-organisms (bacteria, fungi, viruses). However, we still have little information on how immune reactivity is regulated in normal healthy humans. Knowledge in this arena is critical since it could lead to experimental approaches that might impact on the amplitude and duration of immune responses against pathogenic infections. A better understanding of the regulation of mucosal responsiveness can have a major impact on the development and implementation of future vaccine candidates as well as the regulation of systemic diseases, including metabolic, allergic, autoimmune, and psychiatric disorders, which are now suggested to be associated with the perturbation of the normal commensal microbiota.

Leishmania genomic adaptation during sand fly infection is only poorly understood. In particular, the possibility of allelic selection in a given parasite population inside the insect vector has not been investigated, even though sexual recombination and the possibly of self-sex (selfing) can change allele frequencies and thus may be relevant for parasite transmission and virulence. We investigate this important open question conducting experimental sand fly infection with bona fide amastigotes isolated from infected hamster spleen, and derived promastigotes with different karyotypic profiles. Applying our genome instability pipeline (GIP) on sand fly-recovered parasites revealed a novel form of Leishmania genome instability inside the insect vector relying on gene conversion, which causes haplotype shuffling and likely allows for the selection of beneficial alleles.

Listeria monocytogenes is a gram positive facultative intracellular foodborne bacterium responsible for serious clinical manifestations including febrile gastroenteritis, meningitis, encephalitis and maternofetal infections in humans and livestock. Intestinal microbiota plays fundamental roles in the resistance to foodborne infections. Intestinal microbiota commensals protect against pathogens by direct antimicrobial activity through production of bacteriocins, competition for nutrients or binding sites, stimulation of epithelial barrier function, immunomodulation and inhibition of virulence factors expression in gastrointestinal pathogens. On the other side, pathogens have developed tools to avoid commensal-mediated resistance to colonization. Thus, the interplay between gut commensals and L. monocytogenes is critical for the infection and the development of the disease. We have identified a Listeria monocytogenes toxin which modifies the intestinal host microbiota and allows Listeria survival in the intestinal content to later invade the intestine and deeper organs.

The dramatic increase in bacterial resistance to antibiotics, particularly of Gram-negative bacteria, poses an immediate threat to our health system. The main driver of this trend is the impact that antibiotics, including b-lactams, exercise on intestinal commensal flora of humans and animals by selecting resistant strains. The intestinal microbiota, with its large spectrum of bacterial populations, density and diversity, can be considered the epicentre of the emergence of resistance. The presence of a large community of phages, bacteria’s viruses, called phageome, able of performing gene transfer between bacteria, raised the question of their role as a reservoir of genes entailing resistance. This project has two objectives (i) to study the role of human stools’s phages in the spread of antibiotic resistant genes with a selection pressure and (ii) to gain insights into the fundamental ecology and biology of phages in the human gut over a time series analysis.

We are comparing the bacterial communities of domestic and sylvatic  breeding sites and midguts of Aedes aegypti collected in Gabon.

The gastrointestinal tract of humans is colonized by hundreds of microbial species, - bacteria, archaebacterial, fungi, protozoa and viruses -, collectively named the gut microbiome. The intestinal commensal bacteria have an important role in metabolic processes and contribute to colonization resistance against intestinal pathogens. Fungi are usually considered to be a minor component of the global microbiome. However, the mycobiome (fungal component of the entire microbiome) has been in fact little studied particularly with regards to its relationships with the other components of the microbiome. It seems however that fungi can be important players of the microbiome because some fungal species are able to proliferate in response to diet or during dysbiosis due to antibiotic treatment or gut inflammation. Consensus approaches to explore mycobiome together with other components of the mycobiome are still lacking. In this context the primary goal of our project will be to determine the best means to analyze bacterial and fungal microbiome concomitantly, using an identical technical procedure. We will evaluate the effectiveness of different methods for: 1) sample conservation; 2) DNA extraction; 3) mapping fungal and bacterial databases. The best procedure then will be used on samples from different studies to analyze interactions and respective dynamics of fungal and bacterial microbiome in different clinical settings.

A large fraction of diversity of the Archaea is still poorly explored. We are targeting environmental samples to extract genomic data of archaeal lineages of specific interest from an evolutionary point of view. We will probe available sequence data from public environmental metagenomics databases to identify specific lineages. In parallel, we will produce our own data from selected environmental samples containing these lineages. These data will allow filling up poorly explored branches of the tree of Archaea and bring important information on the metabolic diversity and adaptation to various environmental conditions, including the human microbiome.  

The aim of this project is to implement, at the level of the research units, a bioinformatics workflow dedicated to the analysis of the microbiome, and of the virome in particular, based on Next Generation Sequencing (NGS) data (Illumina technology). This workflow will be applied in different contexts, from the identification of nucleotide sequences belonging to a novel or emerging pathogen (mainly viruses) presente in a clinical sample (such as serum or cerebrospinal fluid), to the determination of the whole sequence genome of isolated and uncharacterized viruses (such as bacteriophages of Leptospira).  

Poliomyelitis has been a major public health concern and currently, efforts are being made towards eradicating wild poliovirus type 2 (WPV2). A global switch from trivalent oral poliovirus vaccine (tOPV) to bivalent oral poliovirus vaccine (bOPV without PV2) is planned to take place this year in countries, like Madagascar, where epidemics of type 2 pathogenic circulating vaccine-derived polioviruses (cVDPVs) occur when low polio vaccine coverage allows the circulation and genetic drift of vaccine attenuated PV2 that can thus become pathogenic. In an attempt to monitor the presence and eventually absence of wild poliovirus 2, environmental and human surveillance will be conducted before, during, and after the switch in two regions in Madagascar. PV2, in particular cVDPVs and other enteroviruses will be genetically characterized. These data will support monitoring efforts and confirm the elimination of type 2 PV strains that have been implicated in outbreaks in this country.

16S OTU assignment and statistical analysis of zebrafish microbiota in different experimental conditions.

Microbial discovery remains a challenging task for which there are a lot of unmet medical and public health needs. Deep sequencing has profoundly modified this field, which can be summarized in two questions : i) which pathogens or association of pathogens are associated with diseases of unknown etiology and ii) among microbes infecting animal (including arthropod) reservoirs, which ones are able to infect large vertebrates, including humans. We are currently addressing these two questions and our current request comes with the willingness for Institut Pasteur to increase its contribution and visibility of this thematic, in particular in relation with hospitals and the Institut Pasteur International network (IPIN).  We expect to identify new microbes associated with human diseases, and this is expected to pave the way for basic research programs focusing on virulence mechanisms and host specificity, and will also lead to phylogenetic and epidemiological studies (frequency of host infection, mode of transmission etc...), as well as the development of improved diagnostic tests for human infections. Our objective is also to contribute to the efforts of Institut Pasteur in the field of infectious diseases, by building a pipeline, from sample to microbial identification, able to manage large cohorts of samples. This project is currently supported by the LABEX IBEID and the CITECH, and critically requires a bioIT support, justifying this application. Partners include different hospitals including Necker-Enfants malades University Hospital regarding patients with progressive disease, different IPIN laboratories, as well as INRA and CIRAD regarding animal/arthropod reservoirs.

As a part of BIRDY program (http://www.charliproject.org/), this project aims to study the microbiome composition and its dynamics in early infancy in Madagascar. It will provide genomic knowledge on antibiotic resistant strains and resistance genes circulating in this island. It will identify factors influencing the outgrowth of pathogenic bacteria, including ESBL-PE and decipher the role of the gut microbiome. The developed comprehensive modeling framework will be easily adaptable to other epidemiological questions related to the impact of any human intervention on the gut microbiome and of other environments such as skin, nose or urogenital tract. This project will contribute to the transfer of expertise in genomics and bioinformatics to the IP Madagascar partners.

The aim of the project is to search for the effects of hepatic inflammation on microbiota.

Anopheles mosquitoes are the vectors of Plasmodium parasites, the etiological agents of malaria in humans. In Anopheles gambiae, a major vector in Africa, parasite transmission is largely under genetic control. We have previously shown that a gene family is implicated in the immune control of the parasite in this vector. The response of the family members is pathogen-specific, with one controlling P. falciparum infection and the other controlling rodent parasites. Recently, the genome of Anopheles stephensi, the major Asian vector, has been sequenced and, in this species, there is only one gene. Knocking down this gene reduced lifespan of A. stephensi and antibiotic treatment restores a normal longevity, suggesting the implication of the gut microbiota. Therefore, we conducted a metagenomic analysis in order to identify the bacteria responsible for the shorten of the mosquito lifespan.

Although the microbiota of mosquitoes is known to play an important role in their vectorial capacity for human pathogens, most earlier studies have focused on mosquito-bacteria interactions at the adult stage. Mosquitoes are holometabolous insects whose larvae develop in aquatic habitats, whereas the adults are terrestrial. Larval and adult stages are not independent from each other because the larval environment can influence adult life-history traits through carry-over effects. We recently provided experimental evidence for such carry-over effects in the mosquito Aedes aegypti, the main vector of dengue, yellow fever and Zika viruses. Using gnotobiotic mosquitoes, we demonstrated that larval exposure to different bacteria can cause variation in Ae. aegypti adult traits underlying vectorial capacity (Dickson et al. 2017). This proof of principle is an important first step toward a more comprehensive understanding of how the environment shapes the risk of vector-borne disease. However, the larval microbiota has not been thoroughly characterized across ecologically diverse breeding sites in the field. The aim of this project is to characterize the bacterial and fungal microbiota of Ae. aegypti breeding sites and larvae in Gabon using targeted metagenomics and metatranscriptomics.