The phylogenetic position and status of “giant viruses”, formerly called NucleoCytoplasmic Large DNA viruses (NCLDV) or putative order Megavirales, are controversial. Many preliminary phylogenetic analyses have been published, but their presentations are usually highly biased by the prejudice of the authors concerning the nature of giant viruses. Our own preliminary analyses suggest that giant viruses are indeed ancient (they predate the last universal eukaryotic ancestor) and have possibly provided important functions to emerging eukaryotic cells (e.g. DNA topoisomerase activities). The number of giant virus genomes has recently dramatically increased, opening new opportunity to study their position in the “universal tree of life” and their evolutionary relationships with eukaryotes. The aim of the project is to perform an exhaustive phylogenetic analysis of all giant virus proteins with eukaryotic (archaeal/bacterial) homologues to (i) test the monophyly of giant viruses, (ii) determine their contribution to early eukaryotic evolution, iii) determine if some giant virus proteins can be useful to root the eukaryotic tree. We need the help of a bioinformatics colleague with good expertise in building phylogenetic trees from large data sets using different methods of tree construction and robustness evaluation. This work will be complemented by the systematic search for significant indels (insertion/deletion) in the alignments obtained by two members of the BMGE team (Patrick Forterre and Morgan Gaia).

Background: The opportunistic pathogen Candida glabrata is a member of the Saccharomycetaceae yeasts. Like its close relative Saccharomyces cerevisiae, it underwent a whole-genome duplication followed by an extensive loss of genes. Its genome contains a large number of very long tandem repeats, called Megasatellites. In order to determine the whole replication program of C. glabrata genome and its general chromosomal organization, we used deep-sequencing and Chromosome Conformation Capture (3C) experiments. Results: We identified 253 replication fork origins, genomewide. Centromeres, HML and HMR loci and most histone genes are replicated early, whereas natural chromosomal breakpoints are located in late replicating regions. In addition, 275 Autonomously Replicating Sequences (ARS) were identified during ARS-capture experiments, and their relative fitness was determined during growth competition. Analysis of ARSs allowed to identify a 17 bp consensus, similar to the S. cerevisiae ARS Consensus Sequence (ACS) but slightly more constrained. Megasatellites are not in close proximity to replication origins or termini. Using chromosome conformation capture, we also show that early origins tend to cluster whereas non-subtelomeric megasatellites do not cluster in the yeast nucleus. Conclusions: Despite a shorter cell cycle, the C. glabrata replication program shares unexpected striking similarities to S. cerevisiae, in spite of their large evolutionary distance and the presence of highly repetitive large tandem repeats in C. glabrata. No correlation could be found between the replication program and megasatellites, suggesting that their formation and propagation might not be directly caused by replication fork initiation or termination.

We have recently identified around 500 long non-coding RNAs that follow a very precise expression pattern in undifferentiated mouse Embryonic Stem cells. In order to assess if any of those molecules are functionally relevant for the biology of ES cells we will perform a functional screening based on the CRISPR technology. We plan to use a modified version of the CRISPR-CAS9 technology, the CRISPRon system, in which (i) the guide-RNAs (gRNAs) will target the promoters of our candidate lncRNAs (at least 5 gRNAs per promoter) and (ii) an enzymatically-inactive CAS9 will be engineered to recrute 10 molecules of the potent VP64 transactivator. This will enable to upregulate the targeted lncRNA from its endogeneous locus.

The recent analysis of the Cryptococcus neoformans transcriptomes revealed the presence of thousands of lncRNAs. In these yeasts, different types of lncRNAs seem to exist. The ones that are antisense of coding genes (the NATs), the ones that are located between coding genes (the lincRNAs) and some others that seem to result from alternative transcription start site selection. We  identified growth conditions under which the expression of some of them is regulated. We have also identified some genes implicated in the regulation of some of these lncRNAs. This project deals with the characterisation of these lncRNAs, the analysis of their regulation and the study of their function in the biology and virulence of this pathogenic yeast.

HIV-1 replication requires the integration of the viral genome into the cell genome. A viral-encoded enzyme, integrase (IN), performs this critical step of infection and is a promising target for anti-viral therapeutics. If the catalytic properties of INs are well characterized, the mechanisms responsible for their site selectivity are still under investigation. Several cellular proteins, such as the LEDFGF/p75 transcription co-activator, the RNA polymerase II machinery, nuclear pore proteins and specific modified histones have been proposed to be involved in IN selectivity at a genomic level but the underlying molecular mechanisms remain to be demonstrated. In addition, structural parameters of the target DNA helix (curvature, flexibility, topology) are proposed to regulate IN selectivity at a local level. Our aims are to study the role of these different parameters of IN selectivity, using both in vitro and in vivo approaches. In vitro, we will map integration sites on various target DNA substrates (naked DNA or chromatin, minicircles, plasmids with different topologies, transcribed templates) and will test the effect of purified proteins suspected to regulate IN selectivity. In vivo, integration sites will be mapped in cells depleted of these suspected regulators or in cells incubated with drugs targeting enzymes involved in transcription, DNA topology or histone modifications. Integration sites will be mapped using published or “home-made” protocols and the sites will be compared with DNA structural parameters, nucleosome positions, histone modifications or transcriptional parameters (published maps). Bio-informatics tools are crucial for these correlative and statistical analyses of integration sites. Our project relies on complementary in vivo, in vitro and in silico approaches. It should establish molecular and mechanistic rules of HIV-1 integration selectivity that could serve in the development of new antiviral strategies and of safer gene therapy vectors.

Gli are transcriptional regulators involved in the Shh signalling pathway. Gli3 binding sites on DNA have been defined in the mouse genome by ChIP-chip experiments (Vokes et al., 2008). We are working on the Msx transcription factor family, and have strong evidence indicative of interactions between Msx1 and Gli3 at the protein level. This might reflect in Msx1 binding to DNA in the vicinity of Gli3 binding sites. However, Msx1 binding sites are ill-defined. One stategy to confirm this hypothesis is to compare sequences around gli3 binding sites (around 4200 such sequences in published data) and look for conserved stretches that might define Msx1 binding sites.

N/A

The Sudanese L. donovani strain Ld1SA is the most important experimental reference strain in our field and has been used for various systems level analysis (DNAseq, RNAseq, proteomics). However, all these analyses are strongly compromised by the fact that the current L. donovani genome reference strain LdBPK is from Nepal and very different from the Sudanese isolate (only 60% of DNAseq reads can be mapped).

Analyses fonctionnel des protéines humains de type ubiquitin qui interagir avec les virales protéines de la grippe

analyses et classification des échantillons grippales.

Collect statistics on variants found in different strains of influenza virus. Determine variant effects to the protein sequence.

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.    

Protein sequencing by MS: Automation of analysis

BioHub LeiSHield project This proposal summarizes the contribution of the BioHub to the LeiSHield action that may be carried out by a single BioHub Leishmania coordinator (Giovanni Bussotti). The coordinator will be implicated in the following actions: 1) Establish the link between LeiSHield members and the BioHub team for all questions regarding data analysis and interpretation. The coordinator will present to the BioHub the bio-informatics needs of the LeiSHield partners. Short (easy) tasks will be answered directly (following the BioHub open door strategy). For more involved tasks i twill be asked to deposit projects via the C3BI web site. 2) Coordinate the setup of an HTseq analysis pipeline, including quality control, read mapping, determination of CNV and SNPs, and data visualization using a combination of tools available at the BioHub, such as SyntView and Listeriomics. A link to Cedric Notredame will be established as scripts for Leishmania have been created there. 3) Oversee the submission of DNA from the different LeiSHield WPs to the IP HTseq facility, follow the progress, store the acquired data, and dispatch the datasets to the corresponding WP leaders. This will be coordinate with the Biomix infrastrcuture. 4) Apply the HTseq analysis pipeline (see point 1) on selected data sets for defined work packages, including WP4 (“Analysis of newly isolated anthroponotic L. donovani s.l. strains from Cyprus and correlation of genotypic profiles to tropism and drug resistance”), WP6 (“Population genetics of Brazilian L. infantum isolates from endemic areas presenting distinct transmission cycle”), WP7 (“Leishmania dovovani genome sequence diversity and disease tropism in the Sudan”), and WP9 (“Systems-wide analysis of Leishmania genomic and transcriptomic adaptation”). 5) Co-organize a course on HTseq data visualization (June 2016) with members of the BioHub team.

Chromosome amplification is commonly used by Leishmania during adaptation to environment.  In this context it is challenging to look for genes relevant for parasite virulence/attenuation/drug resistance... To restrict this chromosomal amplification, a cosmid approach (CoSeq) has been chosen to select for genes that provide fitness gain to  Leishmania donovani parasites in culture and in the animal. Therefore, a cosmid library has been generated with genomic DNA from the parasites which needs to be sequenced to control for genome coverage before transfection to the parasites. The transfected parasites will then be injected to animals or submitted to different culture conditions. Only those transfected with cosmids providing advantage under the studied conditions will be selected and will replicate. These cosmids will be extracted from the parasites and will be sequenced to reveal genes relevant for the parasite survival. The C3Bi would be implicated in the analyses of the sequencing data obtained from the PF1 (retrieve the data, mapping of the reads to Leishmania genome, estimation of the genome coverage, listing of genes selected for a given condition...).

The first two cases in France of botulism due to Clostridium baratii type F were identified in November 2014, in the same family. Both cases required prolonged respiratory assistance. One of the cases had extremely high toxin serum levels and remained paralysed for two weeks. Investigations strongly supported the hypothesis of a common exposure during a family meal with high level contamination of the source. However, all analyses of leftover food remained negative. Clostridium baratii was isolated from stool specimens from the two patients. A second cluster of three cases of food-borne botulism due to Clostridium baratii type F occurred in France in August 2015. All cases required respiratory assistance. Consumption of a Bolognese sauce at the same restaurant was the likely source of contamination. Clostridium baratii was isolated both from stool specimens from the three patients and ground meat used to prepare the sauce. This is the second episode reported in France caused by this rare pathogen. One strain from the first cluster and four from the second one have been sequenced (Illumina technology). A complete genome of a C. baratii strain (Sullivan) isolated from adult stool in 2007 in New York is available in Genbank (chromosome: CP006905; plasmid: CP006906).

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.

Integration of the viral reverse-transcribed genome into the genome of infected cells is an essential step of retroviral replication and is performed by a viral-encoded enzyme, named integrase (IN). In the case of HIV-1, IN is a new and efficient anti-viral target. The selectivity of this enzyme for its cellular genomic sites is also a major parameter of HIV replication and is regulated by several cellular parameters. One of them is chromatin, and different levels of this nucleoprotein complex are involved in the regulation of IN selectivity. Using in vitro integration assays, established by our team and collaborators, we have studied this regulation at two levels of chromatin architecture: large poly-nucleosome templates (Botbol et al., 2008; Lesbats et al., 2011; Benleulmi et al., 2015; Naughtin et al., 2015) or nucleosome-induced DNA curvature mimicked by DNA minicircles (Pasi et al., 2016). Our present project is to study IN selectivity into mononucleosomes (MN). These MNs will be used as target substrates of integration and the role of MN structure, histone modifications and IN cofactors will be studied. Results obtained in vitro, will be confronted to structural data obtained by molecular modeling and to integration sites observed in infected cells. This project will benefit from our expertise in integration in chromatin templates and a previous collaboration with the C3BI on the analysis of integration sites (Pasi, M., Mornico, D., S. Volant, S., et al., 2016). This project is funded by the ANRS.

Mycolactone, the lipid toxin produced by Mycobacterium ulcerans, has recently been shown to target the Sec61 transolcon, blocking protein translocation accross the ER membrane. As Sec61 has also been proposed as a critical mediator in cross-presentation in dendritic cells, this analysis aims to analyse mycolactone effects on the proteome of dendritic cells.

See below  

We previously showed that humanized immune system (HIS) mice generated in Balb/c Rag2^-/-γ_c^-/- SIRP^NOD (BRGS) recipients are susceptible to HIV-1 infection (X4 and R5 isolates) and maintain circulating HIV-1 in the plasma, resulting in a dramatic depletion of human CD4⁺ T cells. We also characterized features of HIV physiopathology in this model. Human thymocyte subsets developing in the thymus of HIS mice appear phenotypically normal, but in the periphery the T cell repertoire is restricted compared with that of human peripheral blood T cells. This negatively impacts on the ability of HIS mice to generate antigen-specific human immune responses when mice are vaccinated with protein antigens or following infection with lymphotropic viruses such as HIV. One likely explanation for these functional deficiencies involves the fact that human T cells are selected intrathymically by mouse MHC molecules and that naïve T cells in peripheral lymphoid organs interact primarily with mouse DC (as human DC development in HIS mice is limited). As a first line of improvement, we recently generated a novel mouse model by crossing our BRGS mice with the HLA-A*02-HHD class I transgenic mice and the HLA-DRB1*15 class II transgenic mice, resulting in BRGS-A2DR2 mice. Following intra-hepatic injection of these mice with MHC-matched CD34⁺ stem cells we observed increased engraftment, with faster kinetics. Moreover BRGS-A2DR2 HIS mice have an increased T cell development leading to a more equilibrated B/T and CD4/CD8 phenotype. We showed that BRGS-A2DR2 HIS mice were able to sustain replication of HIV R5 virus as the BRGS hosts. Viremia was similar in a first phase and then lower in a second phase in BRGS-A2DR2 compared to BRGS HIS mice, which could be a consequence of a better quality of the immune response. However, the viremia reached a similar plateau in the last phase. We propose to study the impact of the immune res

Linked with the project #7283 managed by Thomas Bigot, this proposal aims to detect antibiotic resistance genes in whole-genome sequence data of the bacteria deposited and stored at CIP. The CARD library (http://arpcard.mcmaster.ca) would be used to screen for common acquired resistance genes. Detection of point mutations in house-keeping genes and/or transporters would require specific queries from a fasta file continuously implemented by myself.

The adenylate cyclase (CyaA) produced by B. pertussis, the causative agent of whooping cough, is one of the major virulence factors of this organism. CyaA plays an important role in the early stages of respiratory tract colonization by B. pertussis. This toxin uses an original intoxication mechanism: secreted by the virulent bacteria, it is able to invade eukaryotic target cells through a unique but poorly understood mechanism that involves a direct translocation of the catalytic domain across the plasma membrane. CyaA is a 1706-residue long protein organized in a modular fashion. The ATP-cyclizing, calmodulin-activated, catalytic domain (ACD) is located in the 400 amino-terminal residues. Once secreted by the bacteria, the toxin binds calcium in the extracellular milieu and refolds into a functional state. Then, CyaA translocates its catalytic domain directly across the plasma membrane from the extracellular medium to the host cell cytoplasm where, upon activation by endogenous calmodulin, it increases the concentration of cAMP to supraphysiological levels that ultimately leads to the cell death. Recently, we succeeded to refold CyaA in a stable and monomeric form that is fully folded and functional (at variance with all prior procedures in which the polypeptides were largely aggregated upon urea removal). Both calcium and molecular confinement are mandatory to produce the monomeric state and CyaA acylation also strongly contributes to the refolding process. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. Hence, despite recent advances in the understanding of CyaA, its mechanisms of cell intoxication process, in particular the membrane translocation step, remains poorly understood from a fundamental perspective. The description of the molecular events occurring prior to and during the translocation of the catalytic domain across the lipi

I would like to retrieve all HMT and HDM in Leishmania

Our lab is interested in the assembly of the inner viral membrane of Vaccinia virus and what are the molecular regulators (proteins and lipids) playing a role in this complex and unique process of virion morphogenesis.

Vérification et cartographie de génomes de corynebacteries modifiés. Recherche de suppresseurs à partir de délétions de gènes de kinase (pknA; pknB ...)

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.

Phylogenetic analysis of protein families to find mutations that increase their thermal stability and expression levels in a cost- and labor-effective way.

Harmonin Homology Domains (HHD) are protein-protein interaction domains primarily identified in a few proteins involved in earing and vision. Only six HHD-containing proteins encompassing 9 HHD domains have been identified so far in mammals. Four of these proteins are neuronal and also contain PDZ domains, exhibiting different HHD-PDZ modular organisations. This project aims at 1) searching orthologs with HHD domains, 2) establishing the phylogenetic tree of HHD-containing proteins and 3) understanding how various patterns of HHD-PDZ modules from the previsouly mentioned neuronal proteins originated.

phage phiVC8 is a lytic phage for Vibrio cholerae 01 biotype classic. This phage has a narrow host range and we are interested to undersatand why. We have selected several mutants of the Vibrio strain resistant to the phage. Resistance could be due to several mechanisms that we want to uunderstand

Membrane fusion is an essential process in all forms of life, and fusion proteins are responsible of catalysing this reaction by forcing the two membranes against each other by undergoing a fusogenic

Alpacas belong to the family of camelids, which has the peculiarity to produce two kinds of antibodies: conventional antibodies made of heavy and light chains and particular single chain antibodies ma