In the context of the swiss consortion InfectX (www.infectx.ch), we performed siRNA and drug screens to investigate the invasion of host cells by the bacterial pathogen Listeria monocytogenes. As a primary readout for infection we detected by fluorescence microscopy the presence of the bacterially-secreted protein InlC, which accumulates in the cytoplasm of infected cells (Kühbacher et al. 2013). A first manuscript concerning the analysis of a kinome-wide siRNA library and a drug library has been published recently (Rämö et al. 2014) and the analysis of a genome-wide siRNA screen has been submitted for publication (Kühbacher et al. In Revision), in which major signaling pathways subverted by L. monocytogenes are identified. However, besides the presence of InlC, many other features have been measured in infected cells including InlC intensity, bacterial distribution in host cells, actin morphology and distribution, cellular replication state, cellular context of infected cells, etc. We intend to analyze the full set of measured parameters in order to identify global signaling cascades subverted by L. monocytogenes for infection or used by the cell to control bacterial proliferation.

Several mycobacterial species have been used for the screening of chemical libraries with the aim of avoiding natural resistance mechanisms developed by specific species. In this way, different compounds with antibacterial activity have been identified. Mutants resistant to each compound have been isolated and their genomes have been sequenced. The comparison of these sequences with the wild type strain genome will allow us to identify the targets for the antibacterial activity and the mechanism of action of the molecules. This will be instrumental for improving molecules for a better antibacterial activity with emphasis on tuberculosis.

Modification of MacSyFinder models and profiles data structure to facilitate models distribution, and integration in the Mobyle platform. This consists in organizing the way XML models and HMM profiles of given system or set of systems in order to allow running the detection of all the macromolecular systems in a given folder. This will also ensure that the profiles are always distributed with the appropriate definitions of the systems, and thus avoid collisions of profiles name.

CRISPR-Cas9 tools have recently emerged as a very powerful way to edit genomes and control gene expression. The Cas9 protein is an endonuclease that can be guided by a small guide RNA (gRNA) to any target DNA sequence of choice. This feature has enabled the construction of gRNA libraries to perform genome-wide CRISPR-Cas9 functional screens. Such screens have notably been used to identify factors involved in drug and toxin resistance in human cells. We are now in the process of developing such a screen for bacteria. In this screen, we use the catalytic null mutant of Cas9 known as dCas9 to block transcription at target positions and knockdown genes. The purpose of this project is to investigate the properties of this type of screen in bacteria.

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 PIBnet initiative is a joint effort by the above laboratories to modernize their activities, including collection management and microbial characterization approaches and technologies. Within this large concerted effort, a priority is to promote WGS as the major characterization approach of microbial agents for surveillance and outbreak investigation. Our ambition is to have shifted to WGS as a routine strain characterization method for epidemiological surveillance and outbreak investigation in the Institut Pasteur NRCs at the end of 2016. The target volume is 10 000 genomes a year. On the bioinformatics level, this requires implementing (1) fast data treatment tools and (2) Genotyping/classification schemes and methods to extract medically relevant information from genomic sequences (resistome, virulome).

Transfert des annotations d'un fichier genbank sur une séquence fasta ayant une région de 183 kb inversée par rapport à la séquence parentale

We are examining differences within the genomes of E. coli strains.

Improvement of two existing tools, COV2HTML (published in 2014) and SEQ2HTML (private), and transform two scripts into interactive web interfaces addressed to biologists.  

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.

We wish to investigate the extent of conservation and prevalence of small orfs of unknown function expressed in stationary phase of growth in Salmonella

To perform assignment of 16S sequences to bacterial genera and statistical analysis of variations of bacterial populations collected from mice feces

The colonisation of the murine gut by commensal bacteria has been shown to profoundly influence the host physiology. We would like to investigate if these effects are in part mediated by changes in epitranscriptomics, i.e. mRNA modifications influencing the stability and degradation of mRNA (Dominissini et al. Cell 2012, Meyer et al. Nature 2012). To this end, we aim to investigate the levels of the m6A modification of mRNA and differentially methylated targets in organs derived from mice with a conventional flora, germ-free mice and gnotobiotic mice colonized with a specific commensal, Akkermansia muciniphila, known for benificial effects on host metabolism (Everard et al. PNAS 2012, Shin et al. Gut 2014). Since also pathogenic bacteria have been shown to influence the host cells on many levels, including the dysregulation of transcription in the host, we would like to extend our study of host mRNA methylation patterns to mice that have been intra-gastrically infected with Listeria monocytogenes in the presence or absence of commensal bacteria.

The tool MacSyFinder and the viewer MacSyView provide a framework to model macromolecular systems in genomes, allow their precise detection, and their visualisation in genomic context. These tools were co-developped by Sophie Abby, Bertrand Néron, Hervé Ménager, Marie Touchon and Eduardo Rocha. MacSyFinder has been the subject of a demand for a US patent by the Institut Pasteur. The aim of the project is to increase the value and accessibility of these tools by designing a web interface, and giving the possibility to run MacSyFinder on a remote server in Pasteur, with a set of predefined model systems, or with user-supplied models. This will make these tools more easily accessible by a broad audience of biologists.

Leptospiral promoter regions are poorly characterized: experimentally proven transcription factor binding sites have not been described in the literature and promoter prediction algorithms and E. coli consensus sequences of DNA motifs are not appropriate for Leptospira. Identification of Transcription Start Sites (TSS) and promoters on a global scale will provide essential information on DNA motifs that are targets of RNA polymerases, sigma factors and transcription factors. RNA sequencing will also provide information on small regulatory RNAs. These small (~30-500 nt) non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators which play a variety of important roles in many biological processes. Studies on sRNA regulation of gene expression in Leptospira are currently in their infancy. Our results will provide new insights into the transcriptional landscape of L. interrogans, including the repertoire of sRNA, and it will establish the foundation for future experimental work on gene regulation.    

Integrons drive the spread of antibiotic resistance genes in bacterial populations. While their role is widely known (integron+antibiotic+resistance in PubMed gives > 1700 hits), there was no software available to identify integrons and their different components. We have made a script (in Python) which annotates DNA sequences (with prodigal), then identifies the integrase of the integron (using hmm protein profiles), searches attC sites (using covariance models from structurally annotated multiple sequence alignments of DNA sequences), searches certain types of promoters (string search using Python functions), and outputs all this and the genes in the cassettes (between attC sites). We would like the program to be available via Mobyle (or other convenient way) as a web server. Thus would facilitate its use by most biologists (which could still use the scripts if they wish to). It would also facilitate the publication of the methodological manuscript.

Assemblage de novo d'une souche bactérienne afin de pourvoir la soumettre dans une plateforme d'annotation.

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).

We have described recently a novel mechanism of regulation combining phase variation and attenuation of two pilus operons in Streptococcus gallolyticus . Phase variation occurs  by single-strand mispairing during replication due to the presence of repeats in the leader peptide located immediately upstream of the pilus operon. We wonder if the same mechanism is applicable to other bacterial genes.

Increasing evidence indicates that nucleoid spatiotemporal organization is crucial for bacterial physiology since these microorganism lack a compartmentalized nucleus. However, it is still unclear how gene order within the chromosome can influence cell physiology. In silico approaches have shown that genes involved in transcription and translation processes, in particular ribosomal protein (RP) genes, tend to be located near the replication origin (oriC) in fast-growing bacteria suggesting that such a positional bias might be an evolutionarily conserved growth-optimization strategy. Recently we systematically relocated a locus containing half of ribosomal protein genes (S10) to different genomic positions in Vibrio cholerae. These experiments revealed drastic differences in growth rate and infectivity within this isogenic strain set. We showed that genomic positioning of ribosomal protein genes is crucial for physiology by providing replication-dependent higher dosage in fast growing conditions. Therefore it might play a key role in genome evolution of bacterial species. We aim at observing how the genomic positioning of these genes would influence the evolution of Vibrio cholerae. To gain insight into the evolutionary consequences of relocating RP genes, we let evolve either the wild type or the most affected strains for 1000 generations in fast-growing conditions. NGS will be performed and analyzedon the evolved populations to understand the genetic changes responsible of the observed phenotypic changes.

The PIBnet initiative is a joint effort by 15 National Reference Centers (NRC), 8 Collaborative Centers of World Health Organization, the Collection de l’Institut Pasteur & Cyanobacteria collection and the CIBU to modernize their activities, including collection management and microbial characterization approaches and technologies. Within this large concerted effort, a priority is to promote whole genome sequencing (WGS) as the major characterization approach of microbial agents for surveillance and outbreak investigation.   Our ambition is to have shifted to WGS as a routine strain characterization method for epidemiological surveillance and outbreak investigation in the Institut Pasteur at the end of 2016. The target volume is 10,000 genomes a year. On the bioinformatics level, this requires implementing fast data treatment tools, databases, genotyping schemes and methods to extract medically relevant information from genomic sequences (resistome, virulome).

Horizontal gene transfer (HGT) is a major driving force of bacterial diversification. For mycobacteria, a special type of HGT was described in Mycobacterium smegmatis which is linked to distributive conjugal transfer (Gray et al., PLoS Biology, 2013). In the current project we are trying to reproduce the results and explore the process.

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

The 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.

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.

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.

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.

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.

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

An experiment of RNAseq was performed in triplicate with the strain A of Clostridium tetani in TGY (low production of toxin) and MS (high production of toxin) at 24 and 48 h of culture.  This RNAseq was performed in the Pasteur platform PF2. Different comparisons were performed: MS-48h vs MS-24h, TGY-48h vs TGY-24h, TGY-24h vs MS-24h and TGY-48h vs MS-48h. Excel files with differentially expressed genes were provided. For each comparison there are three excel files: complete.xls (contains results for all features), up.xls (contain results for significantly up-regulated features. Features are ordered from de most significant adjusted p-value to the less significant one) and down.xls  (contain results for significantly down-regulated features. Features are ordered from de most significant adjusted p-value to the less significant one). We would like to perform GO term enrichment analysis on the set of genes showing differential expression patterns in the considered contrasts. State of art tools to identify functional enrichments (e.g. DAVID knowledge) do not recognize Clostridium tetani protein and gene identifiers. One possibility is that such tools do not include Clostridium tetani entries in their databases.  The aim of this project is to develop an alternative approach to carry out the functional enrichment analysis on the differentially expressed genes.

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.

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.

Recently 6 strains of Leptospira kirschneri ser grippotyphosa have been sequenced, assembled and annotated. These strains possess 99% genome similarity, but their provenance, virulence and growth characteristics remains different. We would like analyze the SNP of each strain using the SynTView/SNPView tool.  

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

In the context of the Swiss consortium InfectX (www.infectx.ch), Javier PIZARRO-CERDA previously performed siRNA, microRNA, drug screens and proteomic analyses to investigate signaling pathways modulating invasion of host cells by the bacterial pathogen Listeria monocytogenes. In a first consortium study, based on results from drug and siRNA screens targeting the human kinome, we identified major kinases which up- or down-regulate cell invasion by L. monocytogenes and by 7 additional bacterial and viral pathogens (Rämö et al. 2014). Subsequently, a siRNA genome-wide screen allowed us to revisit and redefine the role of cytoskeletal complexes required for L. monocytogenes cellular invasion and actin-based motility (Kühbacher et al. 2015). Applying a proteomic ‘surfaceome’ analysis, we also revealed that late endosomal compartments are recruited to L. monocytogenes infection foci to promote invasion (Kühbacher et al. Submitted). More recently, we have started the analysis of a microRNA screen which highlights novel gene clusters associated to regulation of phosphoinositide metabolism during L. monocytogenes cell entry (Kühbacher et al. Unpublished Results). These different projects have generated vast amounts of data that have been until now only independently analyzed. However, this information can now be exploited from a systems biology perspective to identify hidden connections between relevant signaling cascades and gene networks which may highlight novel cellular functions exploited by pathogens in the context of infection. The team of Benno SCHWIKOWSKI will perform two types of analysis on the data generated by Javier PIZARRO-CERDA. In both cases, p-values will be aggregated across gene sets using suitable statistical approaches. We will then

• Pathway-based analysis. This type of analysis considers genes in sets that have been recognized to operate together to perform certain biological functions (e.g.,

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.  

  Shigella is an enteroinvasive bacterium that induces bacillary dysentery. It invades human intestinal epithelial cells causing the inflammatory destruction of the colonic epithelium. Our previous work reported that Shigella inhibits the secretion of its host cell (Mounier et al Cell Host Microbes 2012) via several virulent proteins injected by the bacterium such as IpaJ and VirA. These two effectors may have redundant action. Thus, to address the effect of Shigella on the secretome of its host cell and in particular define the precise action of IpaJ and VirA, we conducted a mass spectrometry (MS) analysis on apical (A) and basal (B) secretion of polarized infected human intestinal cells using several strains: wild type (WT), avirulent strain (mxiD) and mutated strains either on virA, ipaJ or both (virAipaJ) and compared to non-infected cells (NI). We have in total 6 infection conditions and we collected apical and basal secretion medium (total 12 MS data). Most of the conditions were done in triplicate. We have now these quantitative MS data (excel file) that gave between 300 (basal secretion) to 1000 proteins (apical secretion) secreted differently among the different conditions. We need your collaboration to analyze our data to: Check whether our protein candidates are in databases of secreted factors Obtain protein clusters of our hits among the 6 conditions used (12 datasets)

We're interested in the production of a toxin by Klebsiella pneumoniae. We have a mutant unable to produce this toxin, and would like to identify the mutation responsible of this inability.

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.

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

Mycobacterium tuberculosis is an extremely successful, aerosol-transmitted human pathogen, thought to have evolved from a Mycobacterium canettii-like progenitor. In contrast, extant M. canettii strains are rare, genetically diverse and geographically restricted mycobacteria of only marginal epidemiological importance. Comparative genome studies of several Mycobacterium canettii strains previously identified genetic events that contribute to the contrasting evolutionary success observed between M. canettii and M. tuberculosis strains. In this project we now focus on the genetic factors required for the emergence of highly specialised, virulent tuberculosis bacilli.

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.

Label free quantification of proteins after the infection with M. tuberculosis. Macrophages isolated from seven patients were used in this study. Four conditions were compared.

Context. Bacteria of the genus Shigella and strains of entero-invasive Escherichia coli (EIEC) are responsible of bacillary dysentery (shigellosis) in humans. Although (very) closely related to E. coli, the genus Shigella is divided in four "species": S. boydii, S. dysenteriae, S. flexneri and S. sonnei. Most virulence determinants enabling these bacteria to enter into and disseminate within epithelial cells are encoded by a 200-kb virulence plasmid (VP). The first complete sequence of a VP (pWR100 from a S. flexneri strain of serotype 5a) was determined by our laboratory in 2000. The VP contains genes of different origins, as attested by their G+C content ranging from 30 to 60%, traces of four plasmids and a large numbers of various insertions sequences (IS) representing 30-40% of the total sequence (Buchrieser et al., 2000). In addition to IS sequences, the VP carries members of several multigene families (exhibiting over 90% identity). Such repeated sequences are potentially prone to recombination (allelic exchange, gene conversion) and deletion. Based on the analysis of three genes carried by the VP, it has been proposed that, depending of the species / phylogenetic group, there are two forms of the VP (pInvA & pInvB) that were acquired independently in different original E. coli strains. General questions. What are the architectures of the VP from different phylogenetic groups and how different are pInvA and pInvB ? Which genes are conserved in all VP and which genes are unique to some VP ? Did recombinations occur and, if so, where and when ? To answer these questions, a comparative analysis of the genetic organization and gene conservation among the VP from different phylogenetic groups of Shigella/EIEC has been undertaken using the available complete (or presented as such) sequences of 15 VP, including three members for each of five phylogenetic groups (S. boydii, S. dysenteriae 1, S. flexneri, S. sonnei and EIEC).

Listeria monocytogenes is a gram-positive bacterium responsible for the food-borne disease listeriosis. This pathogen can invade and replicate in the cytoplasm of both macrophages and non-professional phagocytes. In order to better characterize the host response to Listeria, we are using microarrays to identify genes and cytokines up or downregulated during infection.

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

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

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.

This project is intended to explore the novel protein interactions that are involved in the protein folding in cyanobacteria. Slr0286 was previously identified as a protein interacting with the D2 protein of photosystem 2 and affecting its functional assembly and stability in Synechocystis sp. PCC6803. The gene that encodes this protein appears to be in one operon with slr0285 in all strains where these two genes are found. This may suggest the functional relationship between the products of these genes. We would like to explore the question of whether these genes co-evolved and whether there could be an evidence of interaction between Slr0285 and Slr0286.

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.

One of the best models for the study of bacteria-host interactions is Listeria monocytogenes. One interesting facet of this bacterium is its ability to modify host chromatin. Recently, we have shown that Listeria causes a drastic deacetylation of histone H3 on lysine 18 (H3K18dc). Interestingly, to impose this modification, Listeria highjacks previously undescribed host machinery: the host protein sirtuin 2 (SIRT2) is relocalized to the nucleus where it causes genes repression during infection. SIRT2 is a NAD-dependent deacetylase that has been implicated in the regulation of complex processes such as aging and cancer. This protein had been mainly studied in the cytoplasm, and although SIRT2 had been show to shuttle between the cytoplasm and the nucleus, the mechanism and its biological role remained unknown.        In order to further characterize the role of SIRT2 during infection and identify all the genes at which it is recruited, we are performing ChIP-seq analysis. These studies are expected to bring new insights into the function and regulation of SIRT2 by characterizing this new phosphorylation site.

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

Bacterial strains used for quality control (e. g. fertility and viability controls) are recommended in different ISO norms. Each strain is at least deposited in two different collections. So, equivalent strains are found in different collections. Their preservation is generally performed by freeze-drying. As control strains, they are much distributed, thereby, new freeze-dried batches have been done in the course of time. The submitted project aims to see what impact has the long-term strain preservation at the genomic level and if control strains preserved in two different collections can always be considered as equivalent.

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.

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.

Rapid and accurate identification of microorganisms is a prerequisite for appropriate patient care and infection control. In the last decade, Mass Spectrometry (MS) has revolutionized the field of clinical microbiology with the introduction of MALDI-TOF for rapid microbial identification. However, MALDI-TOF MS suffers from important limitations. Some bacteria remain difficult to identify, either because they do not give a specific profile or because the database lacks the appropriate reference. In addition, the discriminatory power of the technique is often insufficient for reliably differentiating sub-species within species or clones within sub-species. More importantly, virulence or resistance determinants cannot be characterized, which is a severe obstacle for appropriate patient care and antibiotics prescription in hospitals. In recent years, proteomics approaches have been increasingly used to study host-pathogen interactions. State-of-the-art bottom-up approaches rely on the enzymatic digestion of proteins and LC-MS/MS analysis of peptides. In contrast, top-down proteomics is an emerging technology based on the analysis of intact proteins by high-resolution mass spectrometry. The major advantage of top-down proteomics is its ability to address protein variations and characterize proteoforms arising from alternative splicing, allelic variation, or post-translational modification. We have recently set-up a robust top-down proteomics platform for the analysis of intact bacterial proteomes. Our final objective is to use this platform to better characterize bacterial pathogens in a clinical context, but a major requirement to achieve this goal is to build up accurate bacterial proteoform databases.  

Integrons drive the spread of antibiotic resistance genes in bacterial populations. While their role is widely known (integron+antibiotic+resistance in PubMed gives > 1700 hits), there was no software available to identify integrons and their different components. We have made integronFinder (with Bertrand Néron) for this (Cury, NAR, 16). We would like to make improvements to IntegronFinder:

• Unit tests to facilitate the development of the tool. Especially since Jean Cury (the main author) will leave the lab in the summer of 2017.
• Add the possibility of using multi-fasta as input.

Extracellular capsules constitute the outermost layer of some bacteria and establish the first contact between the cell and its environment. They are major virulence factors involved, amongst other, in antibiotic tolerance and immune escape. Bacterial capsules are also known to mediate unspecific cell-to-cell interactions between and across species and affect population structure and dynamics. Despite the great number of studies characterizing capsule diversity and their role during infection in a few model species, most evolutionary and ecological questions have yet to be adressed. Furthermore, there is no tool to identify and annotate capsule systems across all prokaryotic genomes. We have built such a tool and would like to make it available.

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.

Shigella species and E. coli are very closely related bacteria belonging to the Enterobacteriaceae family. Phenotypically they are very similar and genotypically they could be considered the same species. The differentiation of Shigella species from E. coli is a significant diagnostic challenge for the clinical microbiology laboratories. They increasingly use maldi-tof mass spectrometry-based microbial identification systems but the latter are currently not able to distinguish these species. As the National Reference Center of E. coli and Shigella, we possess an exhaustive and extensive collection of strains (all serotypes and a wide range of phenotypic profils). We want to use our unique strain collection to propose a highly performant approach for differentiating these bacterial species by MALDI-TOF MS. Success of this study would be incredibly valuable for making an immediate impact on the clinical microbiology diagnosis.

Nous avons pu montrer grâce au projet de recherche clinique VOYAG’R (PHRC AOR 11101- IP Pr S. Matheron) que les voyages dans une zone tropicale constituaient un risque d’acquérir une Entérobactérie multi-résistante (EMR). Pendant 15 mois, la présence d’EMR a été recherchée chez 574 sujets avant et après leur voyage en zone tropicale. Les voyageurs porteurs au retour étaient suivis sur une période de 12 mois (1, 2, 3, 6 et 12 mois ou jusqu’à négativation). Au cours de leur séjour, la moitié des voyageurs ont acquis une EMR. Nous avons également observé que trois mois après leur retour, 95% des voyageurs avaient éliminé leur EMR. Cependant 11 sujets ont conservé leur(s) souche(s) d’EMR pendant au moins 6 mois. Ces souches persistantes pourraient avoir des capacités pour perdurer au sein du tube digestif

Pathogen leptospires are responsible for the zoonotic disease leptospirosis. This neglected but emerging infectious disease has a worldwide distribution and affects people from developing countries, mostly under tropical areas. The clinical manifestations of this infection range from a febrile state to a severe life-threatening form characterized by multiple organ hemorrhages. More than one million cases of leptospirosis are currently reported annually in the word, with 10% of mortality. During infection, Leptospira are confronted with dramatic adverse environmental changes such as deadly reactive oxygen species (ROS). Defenses against ROS, e.g. peroxidase activity, are crucial for Leptospira virulence. These defense mechanisms are controlled by PerR (Peroxide stress Regulator), which represses peroxidase-encoding genes. We study the role of PerR regulators in Leptospira virulence and adaptation to oxidative stress.

We are studying mutants of L. interrogans obtained after random mutagenesis (M58) along with complemented mutant strain (C5M58), constructed using a secondary random mutagenesis. The M58 mutant was found non virulent in gerbils and mice, but the C5M58 strain showing restored expression of the protein, is also non virulent. We have another mutant strain (M1901) in the same gene that retained its virulence, which is the expected phenotype. We are interested in findings SNPs present in both mutant M58 and complemented  C5M58 strains that are absent from the parental L495 and M1901. This approach  could give hints about the gene(s) involved in the loss of virulence.

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 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.

Extracellular capsules constitute the outermost layer of some bacteria and establish the first contact between the cell and its environment. They are major virulence factors involved, amongst other, in antibiotic tolerance and immune escape. Bacterial capsules are also known to mediate unspecific cell-to-cell interactions between and across species and affect population structure and dynamics. Despite the great number of studies characterizing capsule diversity and their role during infection in a few model species, most evolutionary and ecological questions have yet to be adressed. Furthermore, there is no tool to identify and annotate capsule systems across all prokaryotic genomes. Our previous project led to the installation of our tool on the Galaxy Server. The goal now is to put the data of our survey (>2000 genomes) for the capsule available to the community.

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

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

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.

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

Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. In Europe, it is transmitted by Ixodes ticks that carries bacteria belonging to the Borrelia burgdorferi sensu lato complex. Our study was focused on peri-urban forests of Île-de-France. These forests are frequented by many visitors and the risk of exposure to tick bites is high. One of them, the Sénart forest, is located 30 km south of Paris (in the Île-de-France region) and has a large number of visitors (3 million per year in the late 1990s). This forest has the characteristics of being partly invaded by chipmunks (Tamias sibiricus). The chipmunk has been introduced from Eurasia, particularly Siberia, China and Korea. The first individuals were released by their owners at the western end of the Sénart forest, in the 1970s. The northeastern part of the forest was colonized recently. Our current study aims to evaluate the evolution of the infection of Ixodes ricinus by Borrelia burgdorferi sl. by comparing the results obtained during 3 years and to determine the consequences of the proliferation of this non-native rodent species, Tamias sibiricus, on the risk of transmission of Lyme borreliosis. For this purpose, we analyzed the rate of infection and the density of infected ticks during 2008, 2009 and 2011 in several locations of the Sénart forest. These results were compared to those obtained for ticks collected in 2009 in two other peri-urban forests of Île-de-France (Rambouillet and Notre-Dame) that have not yet been colonized by these rodents. The density of nymphs, adults as well as the infected density of nymphs and adults were compared according to several factors: location of tick collection in the forest,  presence or absence of chipmunks, type of vegetation, temperature and humidity.

The present work is to systematically investigate the role of TLRs and NODs in the host defense during C. trachomatis infection using KO animals. The inflammatory cytokines and bacterial burden will be measured using Bio-Plex ELISA kit. C3BI will provide help in the data analysis.

Colon cancer (CRC) is frequent, of bad prognosis, whilst the cost continues to increase. Environmental factors play a role in the majority of sporadic CRC cases. All members of a family share common environment factors and gene determinants. In about 5% of CRC, dominant role of gene is identified as Lynch Syndrome (LS) with mutation in DNA mismatch repair (MMR) genes. Considering the high lifetime risk of developing carcinomas in these individuals, it is relevant to study whether modifiable lifestyle factors can affect the cancer risk. Faecal microbiota that (all bacteria present in the colon) is specific to each individual and highly influenced by environment factors. It boosts CRC development in animal, exerts modulation on inflammation and immunity in the tumour microenvironment and its changes (dysbiosis) is associated with higher CRC risk. It therefore is considered as a mirror of individual “environment-host” interactions. PI of the present project initiated the APHP and EMBL scientific collaboration in 2009 that yielded these results. Fusobacteria (Fu) and particularly, nucelatum animalis and vicentii (FuN) species, could be considered as “bad” bacteria because appeared more prevalent in CRC. We presently whish identifying and quantifying all significant bacteria associated to CRC as community for evaluating predictive values at baseline up to a subsequent occurrence (longitudinal analysis) of high grade dysplasia (HGD), polyps and/or CRC.

Quantitatively understanding the stochastic dynamics of gene expression requires measurements at the level of single cells. A common approach to follow the expression of genes in single cells and in real time is to make use of fluorescent reporter proteins and to record the cells' fluorescence by microscopy. However, this provides only an indirect readout of the biological processes that are of interest such as the regulation mechanisms at the promoter. A possible way to uncover the unobservable biological processes is to infer the hidden dynamics from the available data through the use of mechanistic models of gene expression. The goal of this project is to develop methods for state estimation and parameter inference for such models and to test these methods on real data.

Whole genome sequencing is revolutionizing the surveillance of foodborne and waterborne bacterial pathogens. The speed with which public health laboratories obtain information after the onset of symptoms and the regular sharing of this information between public health laboratories and epidemiologists are critical for the successful use of information to detect outbreaks early and to identify their source. For this purpose, this project aims at providing the most relevant bacterial genomic information in a timely-manner by integrating different validated in silico tools (core genome MLST, CRISPR, O and H molecular serotype, ....) into a single automated analysis pipeline.

Streptococcus agalactiae (GBS) is a gram positive-bacteria which asymptomatically colonize the genital and intestinal tract of healthy women, although the leading cause of bacterial invasive infections in newborns in developed countries. The ability of GBS to succeed both as a commensal and a pathogen can be linked to its capacity to efficiently colonize the host, while still retaining its toxic capacities for the invasive phase of the infectious process. This highly dynamic regulation relies on the major regulator of virulence gene expression, the two-component system CovSR (Control of Virulence Sensor and Regulator). The transcription of almost 15% of the genome is dependent on CovR but up to now only three genes or operons have been identified as directly CovR-regulated by a DNAse I footprint low throughput approach. To characterize the genome-wide CovR binding site, we performed chromatin immunoprecipitation and sequencing (ChIP-Seq) with an epitope-tagged and functional form of CovR expressed in a ∆covR mutant. Quantitative PCR on ChIP samples (ChIP-qPCR) revealed an enrichment of binding regions on the promoters of known target genes. Sequencing of enriched regions has been done to characterize the landscape of CovR binding sites along the chromosome and to reveal the mechanism of regulation and the function of genes directly regulated by CovR. Since CovR phosphorylation state has an important role on DNA binding affinity, defined levels of CovR expression and phosphorylation will be tested to point out low and high affinity targets and the study of different GBS genetic backgrounds will help us elucidate strain-specificities associated with bacterial meningitides in neonates.

Le génotypage MLST (Multi-Locus Sequence Typing) est une technique standard qui permet une caractérisation génotypique précise et reproductible des souches bactériennes. Elle consiste à déterminer la séquence nucléotidique de différents gènes répartis dans le génome (généralement entre 5 et 10). L’Institut Pasteur développe depuis de nombreuses années des schémas MLST pour différentes souches d’intérêt biomédical (e.g. Bordetella, Klebsiella, Listeria, Escherichia, Salmonella). Ces schémas consistent en la définition des différents loci et, pour chacun d’entre eux, en l’identification des allèles observés dans les différentes souches isolées (cf. bigsdb.pasteur.fr). Ainsi, en pratique, le génotypage d’une nouvelle souche s’effectue en déterminant le numéro de l’allèle observé au sein de son génome pour chaque locus du schéma MLST associé. Plus récemment, cette approche de classification de souches a été étendue à l’ensemble des gènes communs aux différents génomes d’une espèce donnée (i.e. core-gene) afin d’observer une meilleure discrimination entre souches proches (e.g. issues d’un même foyer épidémiologique). Ce nouveau système de typage cgMLST (core-gene MLST) s’articule ainsi sur un nombre beaucoup plus important de loci que l’approche MLST standard (e.g. plusieurs centaines ou milliers de loci, chacun contenant entre une dizaine et une centaine d’allèles). L’apparition des nouveaux schémas cgMLST implique en pratique des temps calculs relativement importants lorsque plusieurs centaines de génomes doivent être génotypés en même temps. Malheureusement, les solutions bioinformatiques actuellement disponibles pour déterminer l’ensemble des allèles à partir d’un génome assemblé s’articulent uniquement sur des recherches de type BLAST (e.g. LOCUST ; mlst), alors que de nouveaux algorithmes rapides sont actuellement développés mais uniquement pour effectuer cette tâche à partir de fichiers de reads séquencés (e.g. MentaLIST ; stringMLST). Or, l’utilisation de recherches BLAST pour déterminer les occurrences exactes d’un ensemble de séquences nucléotidiques pré-déterminées n’est trivialement pas la solution la plus optimale. Ainsi, dans le contexte actuel où le séquençage et l’assemblage de centaines de génomes bactériens est devenu routinier, il serait utile et pertinent de disposer de l’implémentation d’un algorithme très rapide de recherche des occurrences exactes d’un très grand nombre de séquences alléliques au sein d’un génome. Un tel logiciel permettrait d’accélérer significativement les missions de surveillance épidémiologique (Bordetella pertussis, mais également Klebsiella pneumoniae et Corynebacterium diphteriae) au sein de l’unité BEBP (Biodiversité et Epidémiologie des Bactéries Pathogènes), mais permettrait également de faciliter certaines analyses bioinformatiques basées sur la recherche exacte d’un grand nombres de motifs nucléotidiques au sein d’un génome.

Taking advantage of a murine model of controlled microbiota composed of 12 strains, we evaluated the activity of a cocktail of three virulent bacteriophages to target a murine Escherichia coli strain in the mammalian gut.

In this project we study ecological diversification of Klebsiella pneumoniae and closely related species. Using comparative genomics we want to identify the pattern of genome adaptation to different environments

We want to automatize the merging of CSV document for data analysis purposes. We've already discussed it with Gael Millot.

The CRBIP, Centre de Ressources Biologiques de l’Institut Pasteur, is a structure created in 2001 that encompasses the Pasteurian culture collections: the CIP (bacteria collection), the PCC (cyanobacteria collection), the ICAReB (human samples collection) and the Eukaryotic virus collection. Since January 2016, the CIP, has sequenced more than a thousand bacterial genomes and assembled them using the P2M pipeline. An initiative has been proposed by the CRBIP, in line with its new strategy of valorization, to annotate all genomes and make publicly available those from type strains with a private section for non-type strains that will be available to users on demand. To attain this objective, a long-term collaboration will be established between the CIP, the C3BI (Natalia Pietrosemoli's team) and the CRBIP that will start with a small project to do the draft annotation of the available bacterial genomes using the open source software, Prokka.

This project is integrated in the analysis of the gut ecosystem of children implicated in the AFRIBIOTA project, a translational project performed within a consortium of researchers and medical doctors from the Central African Republic, Madagascar, France and Canada (see https://research.pasteur.fr/fr/program_project/the-afribiota-project/). AFRIBIOTA aims at understanding the risk factors and pathophysiological changes underlying chronic child malnutrition as manifested through delayed growth. Within the project submitted, we aim at assessing a possible link between asymptomatic pathogen carriage, gut inflammation and stunted growth of children included in the study site in Antananarivo, Madagascar.

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

Nous souhaitons analyser les séquences de sept mutant de Mycobacterium marinum générées par l’utilisation de concentrations croissante d’un antibiotique candidat dont nous ne connaissons pas la cible. Avec ces mutants, nous essayons de connaître la cible de ce composé.

Bacteriophages infect bacteria. One bacteriophage can infect several strains. Around the world, many labs have performed spot tests to determine the host range of bacteriophages but this information is not accessible because there is no tool to process and store it. Developing such a tool will be useful for the phage community but also for the entire community of virologists.

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.

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

Streptococcus agalactiae (GBS) is a commensal gram-positive bacteria which asymptomatically colonize the genital and intestinal tract of healthy women. However, GBS is the leading cause of bacterial invasive infections in newborns in developed countries. The ability of GBS to succeed both as a commensal and a pathogen relies on a highly dynamic regulation of colonization and virulence related genes. The major regulator identified to date is the two-component system CovSR (Control of Virulence Sensor and Regulator). The transcription of almost 15% of the genome is dependent on CovR, but the genes directly regulated by CovR and the regulation of CovR-DNA binding by CovR-phosphorylation are ill-defined. To characterize the genome-wide CovR binding sites, we performed chromatin immunoprecipitation and sequencing (ChIP-Seq). Technically, we developed an epitope-tagged and functional form of CovR expressed under an inducible promoter. Quantitative PCR on ChIP samples (ChIP-qPCR) and small-scale footprint experiments revealed an enrichment of binding regions on specific promoters whose transcription are CovR-dependent. Sequencing (ChIP-seq) has been done to 1) characterize the landscape of CovR binding sites along the chromosome and to reveal the function of genes directly regulated by CovR; 2) to decipher the mechanism of regulation by performing the same experiment in strains with different level of CovR phosphorylation; and 3) to unravel an evolutionary strategy of genetic rewiring leading to the emergence of hypervirulent GBS strain by comparing the CovR direct regulon and the evolution of promoter sequences in different clinical stains.

There exists a broad biodiversity inside the Listeria monocytogenes species, which can be summarized by the existence of evolutionary lineages and more than 100 clonal complexes (CCs or clones) based on core genome multilocus sequence typing (cgMLST), which are geographically and temporally widespread. We aim to link genomic markers to temporal, geographical and sampling origin in order to better understand the ecology and evolution of Listeria monocytogenes.

crispr.pasteur.fr is a website providing resources to visualize CRISPR screen data and design CRISPR experiments in bacteria

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.

Burkholderia ambifaria bacteria are uiquituos microorganisms present in different environmental sources. Particularly these bacteria have been found to be frequently isolated from rhizospheric soils of a wide variety of plants. Burkholderia bacteria have been proved to be benificial to the plants by improving their growth trough different mechanisms. The B. ambifaria Q53 strain was isolated from the rizosphere of peanut (Arachis hypogaea L.). This leguminous plant is a very important crop in many countries around the world. The searching for clean environmental strategies to improve the productivity of the crops becomes an important issue of study for plant in general and peanut in particular. B. ambifaria Q53 strain has been tested in lab assays to have several mechanism of plant growth promotion including the increasing and availability of nutrients (phosphorus; nitrogen; ferrum) as well as the protection against fungus pathogenic agents. To achieve these mechanisms bacteria have to efficiently colonize the plant rizosphere. To do that bacteria communicate with each other by a mechanism dependent on the production and self detection of an autoinducer signal. In this way bacteria population is able to collectivelly regulate a subset of particular genes driving to global patterns of bacterial behaviour. Knowing and the genome sequence of this bacterium represents a key for studying and understanding the role of environmental burkholderias in the process of interaction with the plants.

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

Our project is focused on the intracellular life of Brucella abortus, the causative agent of bovine brucellosis. B. abortus is a facultative extracellular-intracellular pathogen that upon infection of its mammalian host, invades professional phagocytes in which the bacterium transits into the Brucella-containing vacuole and reaches its replicative niche, the endoplasmic reticulum (ER). A key element required for B. abortus intracellular replication is the BvrR/BvrS two-component system. Previous results support a model where B. abortus, upon entrance to host cells, senses the transition from the extracellular to the intracellular milieu through BvrR/BvrS (Altamirano-Silva et al, 2018). Intracellularly, the regulatory protein BvrR is activated by phosphorylation in response to intracellular cues found in early compartments, such as low pH and nutrient deprivation. BvrR activation triggers the expression of virulence factors as the Type Four Secretion System, VirB, essential for trafficking to the ER. We intend to analyse the transcriptome of BvrR dominant positive and negative BvrR mutants (by comparing to the wild type transcriptome) in order to understand the set of factors regulated by modulating the expression of this system.

Without new treatment development tuberculosis could cause about 70 million deaths by 2050, mostly due to the spread of multidrug-resistant strains. The standard drug regimen still builds on the first drugs introduced decades ago, and takes 6 months in the case of drug-sensitive tuberculosis, and up to 2 years in the case of drug-resistant tuberculosis, with heavy side effects. This long therapeutic regimen often results in patients not being able to follow it or complete it correctly, which promotes the chronicity of the infection and ultimately the onset of drug resistance. Although a few new molecules have been discovered, improving both the quality and the duration of tuberculosis chemotherapy remain pressing needs. Furthermore, the failure of chemotherapy is not only due to genetic resistance, which takes relatively long to occur, but also to the intrinsic ability of mycobacteria to diversify in discrete phenotypic states, which can endure drugs even in the absence of genetic mutations. This phenomenon, known as persistence, can eventually favor the onset of resistance, with major repercussions on disease control. In sum, tuberculosis therapy presents many challenges and in our view it is critical to study the ability of a drug or a drug combination to sterilize discrete subpopulations, which may either pre-exist in the population or result from adaptive processes. We found that, prior to drug exposure, phenotypically distinct subpopulations exist that display different drug susceptibility. In light of this, we hypothesized that phenotypic variation from cell to cell favors persistence and can consequently bring about treatment failure. Here we aim to identify molecules that reduce phenotypic variation, making the population more uniformly and rapidly susceptible to standard treatment. To this end, we developed a microfluidic system that allows us to track single cells by live imaging and to carry out a screening at the single-cell level, looking for molecules that homogenize the bacterial population and enhance the effectiveness of the standard treatment. Our approach could ultimately offer original therapeutic strategies towards better control of tuberculosis.

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.

The servers like LegioList are used by my group and many groups around the world working on Legionella pathogenesis since its establishment in 2004. This server has provided us with an important reserach tool and valuable information for research and is providing Institut Pasteur with international visibility

Intégration d'outils bio-informatique dans Galaxy pour identification bactérienne

mise à jour suite à modification du lien du catalogue CRBIP

E. coli sequence type 131 is primarily responsible for 40-80% of extended-spectrum beta-lactamase (ESBL) infections causing million antimicrobial resistant infections annually. Population genetic analyses indicate that ST131 consists of three different sublineages, clade A, B and C, genetically defined by polymorphisms in the gene fimH (fimH41, H22 and H30, respectively). The Cytotoxic necrotizing factor 1 (CNF1) is one major Uropathogenic E. coli (UPEC) virulence factor that catalyzes a pro-oncogenic mutation on Rho GTPases and triggers pejorative inflammatory responses. E. coli ST131 were first reported to lack some UPEC key virulence factors among which CNF1 but recently we and other groups have noticed the presence of the cnf1 gene in some ST131 isolates but the occurrence of CNF1 among E. coli ST131 is still unknown and in order to better define the success of pandemic MDR E. coli the benefit of cnf1 carriage must be elucidated. To address this question, by using bio-informatics approaches the present study analyzed the distribution and the evolution of CNF1 in E. coli ST131 isolates and also examined the association between the presence of CNF1 and antibiotic resistance genes.

Lyme borreliosis (LB) is an important tick-borne disease which can cause a broad range of symptoms mainly affecting the skin, the nervous system and the joints. It is caused by bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex. In Europe, at least five genospecies are known to be pathogenic, namely Borrelia afzelii, Borrelia garinii, B. burgdorferi sensu stricto, Borrelia spielmanii and Borrelia bavariensis. In order to assess the health burden, data on the incidence of all the different clinical manifestations are required. The National Reference Centre for Borrelia collected epdiemiological, clinical and biological data from general practioners and hospitals of patients bitten by Ixodes ticks from 2003 to 2011. This large amount of data needs statistical analyses both univariate and univariate in order to obtain a global and precise vision of all the elements allowing us to understand this disease, both in the complexity of its symptoms and its management but also in the context of its prevention.

Leptospirosis is an emerging zoonotic disease, with high prevalence in tropical regions. It affects both wild and domestic animals, as well as humans, and it is disseminated by asymptomatic carriers, such as rats and mice, that contaminate water with urine. Its etiological agents are some species of the genus Leptospira, member of the phylum Spirochaetes, which are able to colonize hosts through skin injuries or mucous membranes. Once inside the host, the bacteria is able to disseminate quickly through blood and replicate inside the kidneys, provoking renal failure, hemorrhages, liver damage, among other severe potential complications. High-throughput sequencing technologies have allowed to reconstruct the genomes of hundreds of Leptospira strains, enabling comparative analyses which in turn have accelerated the understanding of the its biology. However, leptospirosis is still classified as a neglected disease and many aspects of Leptospira pathogenesis remain unknown. This project aims to strengthen and establish new collaborations between members of the Réseau International des Instituts Pasteur (RIIP) (the Institut Pasteur, France, and the Institut Pasteur Montevideo, Uruguay), in the context of an ongoing, more extensive PTR project entitled “Global diversity, genomic epidemiology and pathogen evolution of Leptospira spp.” (coordinator: Mathieu Picardeau). In the context of this PTR project, we have proposed that a PhD student from Montevideo (Ignacio Ferrés) spend a couple of months in Paris to develop some specific project goals. This internship will be directed by Dr. Mathieu Picardeau and will be performed under the supervision of Dr. Amine Ghozlane at the C3BI. Specifically, the goals of the internship are: 1) to uncover hidden Leptospira's genome diversity by analyzing public environmental metagenomics databases; and 2) to improve functional annotation of Leptospira genomes by applying comparative molecular modeling techniques developed by Dr. Ghozlane and collaborators.

The litterature examine mostly the basic metabolism of this photosynthetic organism that forms toxic bloom worldwide. Previously, Straub et al (2011) demonstrated the differential expression of the natural products of Microcystis in function of the day/night rythm, and more recently, we observed even changes in the metabolic profile of several Microcystis involved in intraspecific interactions (Briand et al., 2016). Here, we explore the expression of the genes dedicated to natural products in the context of transcriptomics for selected Microcystis strains.

MacSyFinder is a framework to model and detect macromolecular systems in genomes using decision rules (gene content and architecture) and similarity searches (protein profiles). It was initially developed at GEM by Sophie Abby with the help of Bertrand Néron and Hervé Ménager. The generic program - MacSyFinder - and its associated tools developed subsequently (i.e., CapsuleFinder, CasFinder, CONJscan, TXSScan) were pivotal in several recent analyses performed and published by our unit (Perrin, Nat Commun, 17;; Cury, NAR, 17;; Oliveira, Nat Commun, 17;; Rendueles, Plos Path, 17;; Bernheim, Nat Commun, in press). These resources have been recently integrated in Galaxy and are significantly used (~3000 jobs on Galaxy this year). To ensure its continuous success and usefulness, MacSyFinder needs to be adapted to new technologies and to be improved with some novel features. In this project, we propose to develop MacSyFinder to make it more portable and lasting, implement novel important features, modernize its distribution and deployment, and improve its visualization tool (MacSyView).

Brucella abortus is a zoonotic pathogen that affects cattle by inducing abortion. Humans are accidental hosts that acquire the infection through contact with animal fluids from infected animals. The pathogenesis of brucellosis relies in the ability of B. abortus to survive intracellularly. This bacterium enters host cells, evades the lysosomal route and re directs its traffic to the endoplasmic reticulum. The type IV secretion system VirB has been shown to be crucial for the intracellular fate of B. abortus. It has been postulated that through the secretion of bacterial effectors, this system modifies the compartment used by the bacterium to reach the endoplasmic reticulum. We have undertaken a comprehensive proteomic approach to understand the molecular changes induced by B. abortus in this intracellular compartment. Murine macrophages were infected with wild type B. abortus or an isogenic mutant in the type IV secretion system VirB. At 1 and 6 h post infection the cells were disrupted, the compartments were purified using sucrose gradients and after resuspension in triton-containing buffer the bacteria removed by centrifugation. Thus, we obtained fractions representing the compartments and secreted bacterial proteins. These fractions have been analyzed by comprehensive proteomic methods and preliminar analysis indicate that there are consistent differences between the compartments derived from wild type and VirB mutants to believe that we could understand the modulation exerted by the Type IV secretion system. In this project we intend to apply state of the art bioinformatics methods to analyze the already existing proteomic data. We hope to understand in detail the differences in prokaryotic and eukaryotic proteins between compartments derived from wild type and VirB mutants and how the function of the differential proteins might impact the route of the Brucella-containing compartment. These results will allow us to understand how B. abortus directs its intracellular traffic to the endoplasmic reticulum.

Ensuring good storage conditions is fundamental for a long-term conservation of bacterial strains. Freeze-drying, freezing, and liquid nitrogen are methods the most frequently used for long term storage. The effectiveness of these different methods to ensure the stability of the genetic characteristics of the bacterial strains over the long term preservation needs to be evaluated. The submitted project aims to see what impact has the long term preservation at the genomic level and if the different batches (frozen, in liquid nitrogen or freeze-dried) of the strain Campylobacter fetus CIP 53.96) are the same.

Evaluation of the mutation rate per site and dN/dS of the genomes of 4 Yersinia enterocolitica strains isolated from the same patient during 14 years.

We aim to establish a comparison of the genetic diversity of different Yersinia spp. genomes and their geographic coordinates in order to characterize the possible associations of genotypes and spatial distances from an epidemiological point of view

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

Dans le cadre d'une étude de souches de Klebsiella pneumoniae multirésistantes, nous avons besoin de comparer nos génomes avec ceux présents dans la base de données ncbi. Dans ce cadre, nous avons téléchargé l’ensemble des gènome de K. Pneumonie disponible fin novembre 2019. Nous utilisons des outils comme kleborate pour obtenir le Sequence Type, le résistome et des informations sur des mutations dans les gènes de la capsule et les porines. Afin de lier les caractéristiques génétiques et phénotypiques des souches que nous étudions avec les données épidémiologiques (la date, le lieu d’isolement, la source, l’origine, l’hôte) et techniques (séquençage, assemblage, couverture), nous avons besoin de récupérer ces information. La demande porte sur une méthode permettant à partir d’une liste d’identifiants d’extraire ces informations.

Salmonella enterica serovar Typhimurium (S. Tm) and Shigella flexineri actively invade non-phagocytic human epithelial cells in a similar fashion. Both pathogens use bacterial effectors injected through a Type III Secretion System to interfere with host pathways for invasion, survival and proliferation within host cells. Our team has performed different screening approaches to identify subverted host pathways during infection. The wide range of obtained data includes functional data from loss-of-function screens, proteomics datasets of the involved cellular compartments, and transcriptomics data on pathogen-localization dependent gene expression responses. In this project, we aim to implement an analytical bioinformatics approach that integrates the data from our different screen datasets for the identification of the most relevant host pathways implicated in Shigella and S. Tm lifestyles in human epithelial cells.

Oral administration is a preferred model for studying infection by bacterial enteropathogens such as Yersinia. In the mouse model, the most frequent method for oral infection consists of oral gavage with a feeding needle directly introduced in the animal stomach via the esophagus. In this study, we compared needle gavage to bread feeding as an alternative mode of bacterial administration.

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

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

We wish to study the genome-wide integration preferences of integron cassettes in the MG1655 Escherichia coli genome.

Parvimonas micra is a gram+ anaerobic coccus bacteria associated with colorectal cancer (CRC). The stool of patients with CRC has higher Parvimonas micra concentration compared to healthy individuals. It is also present in the colonic mucosa of CRC patients. Even if we find it in the adjacent healthy tissue, it is predominantly found in the tumor. Furthermore, it is correlated with the CpG island methylator phenotype (CIMP), a particular subtype of CRC. More specifically, it is associated with the hyper-methylation of tumor suppressor gene promoters. To find out whether this bacteria is involved in the initiation of CRC, we have developed a model of a differentiated epithelial monolayer of human colonic primary cells. After co-culture with this microorganism, we found a significant increase in the global DNA methylation level of the cell genome compared to the bacteria-free control. Now we would like to determine whether this increase in DNA methylation is specific to certain regions of the genome. Therefore, we will use Illumina's Infinium MethylationEPIC BeadChip Kit.

We want to look at the prevalence of protein composed of an orphan LytTR DNA-binding domain in diverse bacteria.

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

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.

In our laboratory we focus on the single-cell biology of tuberculosis. Phenotypic variation helps bacterial cells to endure stressful environmental conditions, and is one of the possible causes of antibiotic persistence and chronic infections. We have recently demonstrated that phenotypic variation is indeed associated with a different response to a class of antimicrobials, in particular in subpopulations of mycobacteria experiencing different levels of DNA damage. Therefore, targeting phenotypic variation may prove to be a successful strategy to weaken the population and to make it more susceptible to antimicrobials. To probe and target phenotypic variation in mycobacteria, we use time-lapse microfluidic microscopy and spatiotemporal analysis of individual cells and microcolonies. We have recently completed a screening for compounds that target phenotypic variation, generating a large dataset of image sequences. Here we aim to develop an automated and user-friendly software for the analysis of image sequences of bacterial microcolonies. This analysis platform will serve to study not only the physiology of mycobacteria but also of other bacterial species. In conclusion, this analytical tool could be very useful for the microbiology community dealing with live single-cell imaging.

We studied the impact of deletion of different component of a regulatory system on the transcription of specific genes.

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.

Bacteria exposed to bacteriophages deploy several defence systems that we wished to identify by genomes sequencing of bacteriophage-resistant isolates

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.

Paleo(meta)genomics is an emerging and rapidly growing field where most is yet to be done. In most cases, it consists in the analysis of ancient DNA high-throughput sequencing data obtained from archaeological material or historical samples, and the goal is to retrieve and interpret the genomic information from species that from the past (microbial, eukaryotic, etc.) It combines tools borrowed from different fields, such as genomics, computational biology, microbial ecology, phylogenetics, population genetics, etc. However, at the moment there are no well-established tools for the analysis of this type of data. Hence, each lab must develop custom solutions, combining existing tools or developing new ones to meet the goals of their research programs. As a recently established lab, the microbial paleogenomics unit will spend the upcoming months setting up diverse pipelines and analysis tools for the different projects that will be developed in the coming years, many of which have been already used but need to be re-written in an understandable languaje and structure.

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.

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

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.

Identification of a secondary mutation which likely appeared during the construction of a mutant strain of Salmonella serotype Typhimurium

Ndd expression in E.coli induces nuclear disruption where the nucleoid is swiftly relocated from a central position in the cell to a submembrane location. RNAseq analysis showed Ndd expression induces the differential expression of hundreds of E.coli genes. We would like to see if some of those genes are related to regulation networks of several nucleoid associated proteins.

Klebsiella pathogens affect human and animal health and are widely distributed in the environment. Among these, the Klebsiella pneumoniae species complex, which includes seven phylogroups, is an important cause of community and hospital infections. The Klebsiella oxytoca species complex also causes hospital infections and antibiotic-associated haemorrhagic colitis. The unsuitability of currently used clinical microbiology methods to distinguish species within each of these species’ complexes leads to high rates of misidentifications that are masking the true clinical significance and potential epidemiological specificities of individual species. Through this project, we will propose a single website for Klebsiella species identification by the mean of MALDI-TOF mass spectrometry. This website will host two tools. One is Klebsiella MALDI TypeR, which was built at Pasteur based on the automatic detection of defined biomarkers. The other one is based on machine learning, with a classifier based on a random forest algorithm trained on hundreds to thousands of spectra. The latter was developed in Melbourne University.

The aim of this study is to shed light into novel mechanisms which increase antimicrobial resistance (AMR) in Gram negative bacteria. In order to gain insight into new mechanisms involved in development of AMR, we study the strategies undertaken by Gram negative bacteria in response to low antibiotic stress, and particularly the role codon bias. Our preliminary results point to a role of codon usage in the survival to low antibiotics targeting various functions. Differences in codon decoding may mpact adaptation and survival to changing environments. We will explore the proteome of V. cholerae to search for protein groups with particular codon biases, expected to impact their translation in the presence of antibiotics.

Correlated evolution is the process by which the evolution of a trait (e.g., a genetic mutation) influences the evolution of other traits. Notably, when genetic mutations evolve together, the fitness effect resulting from the combination can be more or less important than the sum of the independent fitness effects. The disposition of mutations on a phylogenetic tree reflects to some extent the relationships that may exist between different mutations. One example is the evolution of co-occurrent mutations, i.e., mutations that evolve on the same branch of the phylogenetic tree. In other words, one mutation cannot evolve without the other. We developed recently two tools to infer the correlation between evolutionary events mapped on a phylogenetic tree. Notably, our tools were able to retrieve the correlation between residues in contact in the ribosomal RNA three-dimensional structure. We further assemble our tools in a pipeline with a graphical interface to share the tool to as many relevant scientists that would implement the detection of correlated evolution in their projects. Examples of applications are the dynamics of gene gains and losses, the correlation between amino acid residues that are in interaction within the same protein or the evolution of compensatory mutations following the evolution of antibiotic resistance in bacteria.

The general objective of the course (October 4th -15th) will be to obtain and characterize mutations leading to colistin resistance arising spontaneously during K. pneumoniae growth under laboratory conditions. During one week, the student will learn how to use complete genome sequence data to characterize mutations. Like last year, due to the SARS epidemic, bioinformatics analysis will be given to 2 groups of 10 students, each time for one week on the campus. If distance learning is required, each student will use the toolkits through the VPN and a VM with all the software required.

This project addresses two main questions: does Chlamydia trachomatis modify glycolysis and/or oxidative phosphorylation in the host, and do the bacteria need these metabolic pathways to complete their developmental cycle.

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

Vibrio cholerae has a multipartite genome. This project aims to analyze the codon usage bias of each replicon to see if there is a difference.

Leptospirosis is a re-emerging zoonosis that affects more than one million people and causes nearly 60,000 deaths per year worldwide. This disease transmitted to humans via an environment contaminated by bacteria of the genus Leptospira has a record incidence in Oceania. Pathogenic leptospires are able to survive for several weeks in the environment. The production of a biofilm allows them to efficiently resist to hostile conditions and would explain their persistence in the environment. Our recent work has shown that the regulation of biofilm formation is under the control of cyclic di-GMP, an intracellular second messenger recognized as a signaling molecule coordinating the transition between motile (planktonic) and sessile (biofilm) life styles. This project aims to determine the role of cyclic di-GMP in the regulation of pathogenesis and biofilm formation in Leptospira interrogans in order to better understand the environmental survival of this pathogenic bacterium.

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. We aim at exploring the regulatory and genetic network allowing pathogenic Leptospira to adapt to superoxide, one of the deadly infection-related oxidants. We have obtained RNASeq data of Leptospira in the presence of superoxide and we aim at mapping the non-coding RNA that participate in the adaptive response to superoxide.

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.

The fliC locus codes for the FliC protein, the flagellin molecule that constitutes the flagellar filament of motile bacteria, like Y. pseudotuberculosis. Until now, it was commonly accepted that transcription of this locus was completely repressed in the non-motile Yersinia pestis (a recent clone of Y. pseudotuberculosis, which still harbors flagellar genes), but by transcriptomics analysis we observed that it was actually active. Even more, we showed that deletion of the fliC locus in Y. pestis was associated to an attenuation of virulence in the mouse model of bubonic plague. However, for the time being we have no proof that FliC protein is indeed synthesized. Recently, a more detailed analysis of the transcriptomics data showed that a great part of the transcripts detected was actually coming from transcription of the fliC locus in the ‘anti-sense’. Therefore, we would like to explore the hypothesis that the actual molecule playing a role in the phenotype observed is an anti-sense ARN with regulatory functions, instead of the genic product of the fliC ORF, i.e. the protein FliC.

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

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.

La coqueluche est une infection respiratoire communautaire causée par la bactérie Bordetella pertussis (Bp). Elle est à l’origine d’un nombre important d’hospitalisations malgré une couverture vaccinale élevée, et est responsable d’une mortalité conséquente. La forme la plus sévère de cette maladie, la coqueluche maligne, est observée chez des nourrissons âgés de moins de 3 mois et est caractérisée par une détresse respiratoire aigüe, une défaillance multiviscérale avec une atteinte neurologique manifestée par des convulsions voire un coma, ainsi qu’une hyperleucocytose majeure, responsable d’un taux de mortalité particulièrement élevé. La physiopathologie de la coqueluche notamment celle de sa forme maligne demeure indéterminée. En effet, le type de cellules immunitaires et les gènes impliqués dans la réponse immunitaire à Bp restent encore inconnus. Nous émettons l’hypothèse que la forme maligne de la coqueluche est due à la combinaison de facteurs de virulence spécifiques et une réponse immunitaire « immature » du jeune nourrisson. Ainsi, l’objectif du travail est de déterminer l’implication de la réponse immunitaire systémique néonatale dans la sévérité de la coqueluche, à travers une approche transcriptomique.

RepABC replicons consist one of the most important groups of extrachromosomal replicons in Alphaproteobacteria, which includes numerous plasmids (e.g. pTi of Agrobacterium), megaplasmids (e.g. pSymA of Sinorhizobium) and chromids (e.g. chromosome 2 of Brucella). Recently, we have experimentally demonstrated that within this family some replicons replicate unidirectionally and others bidirectionally. This project aims to identification of phylogenetical patterns of distribution of these two replication modes among RepABC replicons. We would like to follow the evolution of the replication mode in this group of replicons and to indicate mechanims which could be responsible for switch between uni- and bidirectional replication.

The genus Paracoccus (Alphaproteobacteria) includes bacteria carrying genomes of very different structure - from unichromosomal strains containing no plasmids (P. suum), through strains possesing multiple extrachromosomal replicons of medium size (hundreds kb) and different nature (megaplasmids and chromids) (e.g. P. aminophilus, P. yeei or P. marcusii), to strains having a large well-constituted chromosome 2 (a chromid of over 1.5 Mb with high density of housekeeping genes), which are found in one sub-clade of the genus (including P. denitrificans, P. versutus and P. pantotrophus). In this project, we would like to follow the distribution of the core genes in all available Paracoccus genomes to uncover the events that led to the formation of chromosome 2 in this group of bacteria. At the same time, we would like to check if the core genes are preferentially found in certain localization in the genome (relatively to ori and ter of the chromosomes), possibly indicating the advantages of the bipartite genomes found in some Paracoccus species.

Based on the analysis of two bacterial strains isolated from the sea cucumber Holothuria forskali, we are describing a novel species in the genus Pseudoalteromonas. Vibrionaceae and Pseudoalteromadaceae represent the only two families from the gamma-proteobacteria class with multipartite genomes. Whereas the Vibrionaceae genome is well studied, the information on Pseudoalteromadaceae is scarce. In addition, the taxonomy of the genus Pseudoalteromonas needs to be greatly clarified.

Severe bacterial infections are a leading cause of neonatal deaths, with low income countries (LICs) bearing the highest burden. In LICs, neonatal bacterial infections are mainly caused by Enterobacteriaceae. One important driver of unfavorable outcome in infections caused by these bacteria is multidrug resistance. Of particular concern, extended-spectrum beta-lactamase-producing enterobacteriaceae (ESBL-PE) are resistant to most penicillins and especially cephalosporins (3rd-4th generation). Enterobacteriaceae are known to colonize the digestive tract, which represents the first step for potential neonatal infections. The newborn is exposed at birth and during the first weeks of life to several possible sources of ESBL-PE acquisition, e.g. maternal, health-care facilities, and outside hospitals: parents, others relatives, food and environment. However, the routes of ESBL-PE acquisition in neonates are not well defined. Also, local environments and practices influence strongly the interactions of the infant with its environment. However, practices contributing to the transmission of ESBL-PE to neonates are not well characterized in LICs. The objectives of the project are: a better knowledge of the role of different routes of ESBL-PE transmissions to newborns in the community, and more specifically within a household, to understand local environments and practices that facilitate ESBL-PE transmission in Madagascar In Madagascar, 60 newborns and members of their households will be follow-up for one month. ESBL-PE will be characterized with last generation DNA sequencing methods. To integrate all these data globally, we will develop novel sophisticated analytical approaches combining mathematical modeling and statistics. Also, in-depth interviews will be conducted and mother-newborn pairs will be followed with a participants-observations methodology. Our study aims at identifying and quantifying the role of the different routes of transmission for newborns in the community. With a multidisciplinary approach, it should allow identifying the most acceptable interventions for local population to prevent ESBL-PE acquisition in newborns in Madagascar, where the burden of neonatal infection is huge. Partner : Institut Pasteur in Madagascar

Archaea and Bacteria have adapted multiple times independently to the gut environment, but the general mechanisms of adaptation to this environment are not very well know or it as been studied on very specific models. This project is focused on the multiple adaptations of Archaea to the gut environment, with a comparison to selected lineages of Bacteria.

Gastrointestinal (GI) cancers are due to over time accumulation of genetic and epigenetic alterations resulting from complex interplay between host, bacterial and environmental factors. Several bacteria have been associated with GI cancers development. Our hypothesis is that GI tumorigenesis likely involves synergistic or sequential impacts of several pathobionts. Based on molecular tracks establishing links between bacteria and host pathways, we aim to understand how several pathobionts co-inhabitation in the same host can modulate their respective pro-tumoral potency. To investigate this, we developed in vivo and in vitro co-infection models to study underlying mechanisms that could promote tumorigenesis.

We want to add/create a similar website to CIRPSR.pasteur.fr to use CRISPR-Cas9 as an antimicrobial.

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.

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.

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).

mise à jour suite à modification du lien du catalogue CRBIP

The litterature examine mostly the basic metabolism of this photosynthetic organism that forms toxic bloom worldwide. Previously, Straub et al (2011) demonstrated the differential expression of the natural products of Microcystis in function of the day/night rythm, and more recently, we observed even changes in the metabolic profile of several Microcystis involved in intraspecific interactions (Briand et al., 2016). Here, we explore the expression of the genes dedicated to natural products in the context of transcriptomics for selected Microcystis strains.

E. coli sequence type 131 is primarily responsible for 40-80% of extended-spectrum beta-lactamase (ESBL) infections causing million antimicrobial resistant infections annually. Population genetic analyses indicate that ST131 consists of three different sublineages, clade A, B and C, genetically defined by polymorphisms in the gene fimH (fimH41, H22 and H30, respectively). The Cytotoxic necrotizing factor 1 (CNF1) is one major Uropathogenic E. coli (UPEC) virulence factor that catalyzes a pro-oncogenic mutation on Rho GTPases and triggers pejorative inflammatory responses. E. coli ST131 were first reported to lack some UPEC key virulence factors among which CNF1 but recently we and other groups have noticed the presence of the cnf1 gene in some ST131 isolates but the occurrence of CNF1 among E. coli ST131 is still unknown and in order to better define the success of pandemic MDR E. coli the benefit of cnf1 carriage must be elucidated. To address this question, by using bio-informatics approaches the present study analyzed the distribution and the evolution of CNF1 in E. coli ST131 isolates and also examined the association between the presence of CNF1 and antibiotic resistance genes.