Expertise

Hub members Have many expertise, covering most of the fields in bioinformatics and biostatistics. You'll find below a non-exhaustive list of these expertise

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Searched keyword : Bacteria

Related people (5)

Thomas BIGOT

Group : GIPhy - Embedded : Biology of Infection

I joined the C3BI Hub in 2016 after a curriculum widely dedicated to Bioinformatics studies, and more precisely to Phylogeny and Evolution, topics of my PhD thesis. At Institut Pasteur, I am involved in projects dealing with sequences homology : alignments, hmm profiles, making homologous family databases, kmers signatures. I am also a developer (Python / C++) with a solid interest in optimization as well as in developing usable tools for final user such as automated pipeline for metagenomics sequence analysis. I’m currently embedded in Marc Eloit’s team (80% of my work time). My main task in this team is to develop strategies to identify, in their metagenomics samples, new pathogens, or new combination pathogen / symptoms. The rest of my time, I manage small projects and participate to the Hub life. I am currently experimenting with functional programming (for now, using Python) and its applicability to bioinformatics issues.


Keywords
AlgorithmicsScientific computingSofware development and engineeringParallel computingGraph theory and analysis
Organisms
BacteriaFungiVirus
Projects (10)

Pascal CAMPAGNE

Group : Stats - Hub Core

Initially trained in evolutionary and environmental sciences, I studied population genetics and micro-evolutionary processes in a number of postdoctoral research projects. I recently joined the C3BI-Hub at the Institut Pasteur, where I work on various aspects involving Biostatistics and the analysis of genetic data.


Keywords
Association studiesGenomicsGenotypingBiostatisticsGeneticsEvolutionPopulation genetics
Organisms
BacteriaParasiteHumanInsect or arthropodOther animal
Projects (23)

Anđela DAVIDOVIĆ

Group : SINGLE - Embedded :

I have a joint MSc degree in Mathematical Modelling from three European universities: University of L’Aquila (Italy), University of Nice-Sophia Antipolis (France) and Autonomous University of Barcelona (Spain). I also hold a PhD degree in Applied Mathematics and Scientific Computing from University of Bordeaux, France. I have done my PhD and one year of post-doc at INRIA Bordeaux Sud-Ouest, and partially at IHU-Liryc. During this time I studied how electrical signals propagate through the cardiac tissue under certain diseased conditions. My model of interest was the bidomain model, which is a system of partial differential equations that takes into account physiological properties of the cardiac cells and the spatial organization of the cardiac tissue. I worked on the mathematical multiscale analysis and numerical simulations of the problem to understand how structural changes of the tissue affect the propagation of the signal on the heart level. I collaborated with biologists and engineers of the IHU-Liryc to apply my model on a rat heart using high-resolution MRI data. For this I also worked on image analysis and image processing. I’ve joined the Institute Pasteur in February 2018 as a member of the HUB in Bioinformatics and Biostatistics. Currently I am working on stochastic mathematical modeling and inference for systems biology, gene expression, RNA transcription, etc.


Keywords
ModelingScientific computingApplication of mathematics in sciencesGraphics and Image Processing
Organisms
BacteriaFungiInsect or arthropodEscherichia coliSaccharomyces cerevisiaeFly
Projects (3)

Julien GUGLIELMINI


After a PhD in Microbiology on bacterial toxin-antitoxin systems at the Free University of Brussels, I joined the Institut Pasteur for a 3 years postdoc in Eduardo Rocha’s lab. During this period, I performed comparative genomics and pylogenetic analysis on bacterial conjugation and type IV secretion systems. Then, I worked 2 years in Olivier Tenaillon’s team on the modelling and evolution of organismal complexity. I joined the HUB in 2015, and I am involved in phylogenetic and comparative genomics projects.


Keywords
GenomicsPhylogeneticsSequence analysisGenome analysisGeneticsEvolutionPopulation genetics
Organisms
ArchaeaBacteriaVirus
Projects (12)

Rachel LEGENDRE

Group : GORE - Hub Core

Rachel Legendre is a bioinformatics engineer. She completed her master degree in apprenticeship for two years at INRA in Jouy-en-Josas in the Genetic Animal department. She was involved in a project aiming at the detection and the expression analysis of micro-RNA involved in an equine disease. In 2012, she joined the Genomic, Structure and Translation Team at Paris-Sud (Paris XI) university. She worked principally on Ribosome Profiling data analysis, a new technique that allows to identify the position of the ribosome on the mRNA at the nucleotide level. Since november 2015, she worked at Institut Pasteur. During 4 years, she was detached to the Biomics Platform, where she was in charge of the bioinformatics analyses for transcriptomics and epigenomics projects. She was also involved in Long Reads (PacBio and Nanopore) developments with other bioinformaticians of Biomics. Since november 2019, she has joined the Hub of Bioinformatics and Biostatistics, et more precisely the Genome Organization Regulation and Expression group.


Keywords
AlgorithmicsChIP-seqEpigenomicsNon coding RNATranscriptomicsGenome analysisProgram developmentScientific computingSofware development and engineeringIllumina HiSeqRead mappingSequencingWorkflow and pipeline developmentChromatin accessibility assaysPac BioRibosome profiling
Organisms
BacteriaFungiParasiteHumanInsect or arthropodOther animal
Projects (24)

Related projects (154)

MicrocystOmics

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).



Project status : Closed

How ribosomal protein gene position impacts in the genome evolution during a long term evolution experiment.

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.



Project status : Awaiting Publication

Mise a disposition d'un(e) bioinformaticien(ne) du hub pour les analyses bioinformatiques du transcriptome et de l epigenome

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.



Project status : Closed

RNAseq analysis-gene ontology enrichment Clostridium tetani



Project status : Closed

SNP based analysis of French Bordetella pertussis isolates: comparison of isolates producing all the vaccine antigens to isolates producing only some of them.

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.



Project status : Closed

Interactions and dynamics of fungal and bacterial microbiome in healthy people



Project status : Closed

Listeriomics - Development of a web platform for visualization and analysis of Listeria omics data

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



Project status : Closed

Systems Biology of Cell Infection by the Bacterial Pathogen Listeria monocytogenes

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.,



Project status : Closed

secretome analysis of human intestinal cells during shigella invasion



Project status : Closed

Comparative genomic and phylogenetic analysis of Clostridium baratii strains



Project status : Closed

Comparative analysis of the virulence plasmids of Shigella Spp. and entero-invasive Escherichia coli

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).



Project status : Closed

Nanotherapeutics for multidrug-resistant tuberculosis

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.



Project status : Closed

Identification of new or unexpected pathogens, including viruses, bacteria, fungi and parasites associated with acute or progressive diseases

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.



Project status : In Progress

Development of top-down proteomics for clinical microbiology

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.  



Project status : Closed

Bioinformatic analysis of the adenylate cyclase CyaA toxin

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



Project status : Closed

Genomic analysis of catheter-related Escherichia coli infection



Project status : Closed

Infection of Ixodes ricinus by Borrelia burgdorferi sensu lato by in peri-urban forests of France

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.



Project status : Closed

Relationships between ESBL-producing Escherichia coli from food and healthy mothers in Phnom Penh, Cambodia

Extended spectrum β-lactamase (ESBL) genes encode resistance to penicillins and cephalosporins and can be horizontally transferred among Enterobacteriaceae. More than 60% of healthy humans living in southeast Asia are faecal carriers of ESBL-producing Enterobacteriaceae (ESBL-PE), compared to <10% in Western Europe, suggesting diverse exposure routes. In Cambodia, meat and fish consumption is high and food safety is poorly enforced. Thus, we hypothesized that meat and fish could be a source of exposure to ESBL-PE. From Sept-Nov 2016, we conducted a meat sampling study in Phnom Penh in collaboration with the BIRDY program (http://www.birdyprogram.org/), an ongoing study of neonatal health in low-income countries. We evaluated ESBL-PE contamination among pork, fish, and chicken from two markets and collected survey data from BIRDY mothers. We performed whole genome sequencing on 87 ESBL-producing E. coli recovered from meat and fish and from 91 BIRDY mothers who provided faecal swabs less than one year prior. Now, we will conduct a two-step exploratory analysis of these WGS data:

  • First, we will estimate the pairwise evolutionary distance between E. coli isolates, in order to infer a phylogenetic tree. We will use this tree to investigate such epidemiological questions as “Do E. coli sequences from women who reported eating poultry 3+ times/week cluster more closely with E. coli recovered from poultry samples, compared to women who reported never eating poultry?” This phylogenetic tree may be re-constructed using a finer resolution, as needed.
  • Second, we will annotate all E. coli genomes in order to identify genomic islands related to antibiotic resistance. We will subsequently use logistic regression to model associations between BIRDY mothers’ reported dietary patterns (i.e. exposure) and the presence or absence of these genomic islands among the ESBL-E. coli they were colonized with (i.e., outcome).



Project status : Closed

Microbiota dysbiosis in human colon cancer



Project status : Closed

ChIP-seq analysis of the majour regulator of GBS virulence

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.



Project status : Closed

Implémentation d’un algorithme rapide de génotypage cgMLST

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.



Project status : Closed

Role of small non coding RNAs in the adaptive response to oxidative stress in pathogenic Leptospira

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.



Project status : Closed

Determination of host response elicited by different Salmonella lifestyles

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.



Project status : Declined

Training project for bacterial ChIP-seq Analysis on Streptococcus agalactiae

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.



Project status : Closed

Genomic DNA sequencing of Burkholderia ambifaria Q53 strain isolated from peanut rizospheric soil

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.



Project status : Closed

Identifying subpopulation-specific signatures of tuberculosis persistence for enhanced diagnostics.

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.



Project status : Awaiting Publication

Determination of the transcriptome controlled by the two-component system BvrR/BvrS using dominant positive and negative BvrR mutants



Project status : Awaiting Publication

Hamper cell-to-cell variation to enhance drug-mediated killing

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.



Project status : Pending

Uncovering diversity and improving gene annotation of Leptospira sppo

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.



Project status : Closed

Proteomic analysis of the intracellular compartments containing Brucella abortus

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.



Project status : In Progress

Functinal analysis of proteome and phopshoproteome changes in murin intestine – mechanism of Streptococcus gallolyticus-induced carcinogenesis



Project status : Declined

An integrated software having a graphical user interface for the analysis of time-lapse images of bacterial microcolonies



Project status : Closed

Phages - bacteria interactions network of the healthy human gut



Project status : Closed

A pipeline to detect correlated evolution on phylogenetic trees



Project status : In Progress

Comparative genomics analysis of cyclic-di-GMP metabolism across the Leptospira genus

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



Project status : In Progress

Etude de la réponse immunitaire néonatale dans la coqueluche maligne : approche transcriptomique



Project status : Pending

MicrocystOmics

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).



Project status : Closed