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Searched keyword : Variant analysis
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Cryptococcus neoformans is a pathogenic yeasts that infect mostly immunocompromised people. The strain H99 belongs to the C. neoformans variety grubii group. This is the reference strain that have already been sequenced. A project initiated by the Broad Institute aims at analysing the genome of about 300 clinical isolates including 28 of our French isolates. Part of our French isolates have been screened in terms of interaction with macrophages (774). Huge variations in terms of phagocytosis and intracellular proliferation of the yeasts were observed between clinical isolates (Alanio et al. mBio 2011). Thirteen of these clinical isolates together with H99 were studied in terms of transcriptome (using RNASeq). This work was performed by MA Dillies at the PF2. We correlated the expression of about 150 genes with the phenotype of the 13 clinical isolates and H99.
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.
Dengue is an arthropod-borne viral disease caused by dengue virus (DENV), which is transmitted by Aedes mosquitoes. Recent studies estimate that 100 million cases occur annually worldwide, making dengue the most prevalent mosquito-borne viral disease of human beings. DENV transmission results from interactions between humans, mosquitoes, viruses and their environment. If the human immune response against DENV is well studied, there are knowledge gaps in understanding how mosquitoes and DENV interact with each other. Moreover, it was shown that virus transmission does not only depend on the viral strain and mosquito population but also on the specific interaction between the two partners, named genotype-by-genotype (GxG) interaction. One aim of the team is to highlight factors at the mosquito and virus levels that regulate vector competence, i.e. the ability for a mosquito to acquire viral infection and subsequently transmit the virus to a new host. To this aim, we use field-derived Aedes aegypti populations that we challenge in the lab with virus isolates from DENV infected patients. This allows us to refine our understanding of the basic biology of virus transmission by mosquitoes.
Candida albicans is responsible for the majority of life-threatening fungal infections occurring in hospitalized patients and is also the most frequently isolated fungal commensal of humans. The C. albicans population includes at least 18 phylogenetic groups (or clades). Specific phenotypes can distinguish isolates within a given clade from those in other clades and yet, the relationships between C. albicans natural genetic and phenotypic diversities have not been explored in depth. We have sequenced the diploid genomes of >150 C. albicans isolates selected from a collection of commensal/clinical isolates previously used to characterize the population structure and belonging to the 12 major C. albicans clades. The aim of this project is to develop the tools necessary for an in depth analysis of these genome sequences in order to allow us ask questions about the extent of C. albicans genetic diversity, the contribution of loss-of-heterozygosity to this diversity, and the history of C. albicans population.
Candida albicans is responsible for the majority of life-threatening fungal infections occurring in hospitalized patients and is also the most frequently isolated fungal commensal of humans. Microevolution of C. albicans isolates has been observed in a number of instances, being in particular characterized by loss-of-heterozygosity events. Yet, most studies that have investigated such microevolutions have not used whole-genome sequencing. In this project, we aim to characterize C. albicans microevolution at the genome-wide level. To this aim, we will take advantage of multiple isolates collected at the same time in healthy individuals and that share the same molecular type, thus providing information on the extent of genetic diversity of commensal isolates. We will also take advantage of series of isolates collected in patients with different forms of candidiasis and/or that have received antifungal therapy, thus providing information of the impact of pathogenic interaction and antifungal treatment on genome dynamics.
The 2013-2015 Ebola virus disease epidemic is the largest outbreak so far described with 27 305 cases and 11 169 deaths. The virus spread by human to human contact throughout Western Africa and never before has a variant been transmitted for such a sustained period of time. Ebola virus are RNA virus so as other RNA viruses they could accumulate mutations during evolution. Therefore it is an emergency to monitor viral changes and adaptation within and between individuals in order to help researchers to better understand susceptibility to Ebola infections, to guide research on therapeutic targets and to ensure accurate diagnosis. New technologies can provide information about pathogen’s evolution and in our lab we have access to an Ion PGMTM sequencer. Thanks to the national reference center for viral hemorrhagic fever (VHF) we have at our disposal a large number of samples collected from Ebola infected patients especially from Guinea. We have developed an Amplicon approach using sixteen couples of specific primers for Ebola viruses and a RNA sequencing method based on randomly primed cDNA synthesis to product our libraries. Ion PGMTM Hi-Q sequencing kit will be used to sequence up to 400 bp inserts loaded onto 316v2TM or 318v2TM chip. Through high depth sequencing we would like to follow up the profiling of intra and inter host viral quasispecies at different time of the epidemic in the geographic area of the Ebola Treatment Centre in Macenta. Thanks to the activities of national reference center for VHF and the Biomics Pole one aim of the project is also to occasionally compare viral quasispecies and consensus sequences between patients who get uncommon symptoms from those who get classical illness and to study intra host quasispecies in different biological fluids (cerebrospinal fluid, sperm, urine) to see if there are differences between persistent species and viral quasispecies found during symptomatic step.
Whole-genome sequencing of microbial agents for disease surveillance, outbreak investigation, epidemiology and population biology
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).
The goal of this project is to set sequence analysis tools to evaluate the frequency of mutations within a pool of bacteriophage genomes sequenced by NGS.
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.
Genomic DNA is hierarchically packed within the living cells and genome duplication requires the concerted effort of many thousands of individual replication units. As such, to ensure the integrity of transmission of the genetic information, both eukaryotes and prokaryotes have evolved sophisticated mechanisms to monitor DNA replication. Some of these mechanisms aim to maintain both a temporal and a spatial organization of the replication program, leading to multiple replication time regions and the compartmentalization into replication foci, subnuclear sites which accumulate numerous DNA replication factors. It should be noted that Saccharomyces cerevisiae represents an exception to the standard eukaryotic strategy for genome duplication. Similar to bacteria, S. cerevisiae possess well-defined replication origin sequences that can fire at a very efficient rate during S phase, leading to a very homogenous pattern of DNA replication. A common mo del suggests that, once replication starts dynamic events take place since co-regulated replication forks, having similar replication timing, cluster within a discrete number of foci that show distinct patterns of nuclear localization over the S-phase. Once initiated, the DNA synthesis might be compromised if the replication fork encounters an RFB (Replication Fork Barrier) such as DNA lesions, tightly bound protein-DNA complexes etc. The RFBs are considered a potential source of genetic instability and may lead to many chromosomal rearrangements. As a consequence, eukaryotes employ a complex DNA damage response against RFBs, which aims to maintain the stability of the stalled forks and provides the time required to repair and resume replication. Recent observations suggest that the non-random organization of the nucleus affects where repair occurs. The aim of this project is to reach a better understanding of the influence of the nuclear spatial architecture and organization at replication fork blocks.
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.
Pasteur International Bioresources Network (PIBnet) bioinformatics: whole-genome sequencing of microbial agents for disease surveillance, outbreak investigation, epidemiology and population biology
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).
A major program of evolutionary and comparative genomics of yeasts has been in progress in my laboratory for many years (see publications). In the next few months (before summer 2015) I need to finish a few comparisons about a new clade to publish as soon as possible.
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.
The goal of this project is to set sequence analysis tools to evaluate the frequency of mutations within a pool of bacteriophage genomes sequenced by NGS.
Collect statistics on variants found in different strains of influenza virus. Determine variant effects to the protein sequence.
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.
Massive amplification at an unselected locus accompanies complex chromosomal rearrangements in yeast
Gene amplification has been observed in different organisms in response to environmental constraints, such as limited nutrients or exposure to a variety of toxic compounds, conferring them specific phenotypic adaptations by increasing expression levels. But the presence of multiple gene copies has generally not been found in natural genomes in absence of specific functional selection. Here we show that the massive amplification of a chromosomal locus (up to 880 copies per cell) occurs in absence of any direct selection, associated with low-order amplifications of flanking segments in complex chromosomal alterations. These results were obtained in the mutants with restored phenotypes that spontaneously appear from genetically engineered strains of the yeast Saccharomyces cerevisiae with severe fitness reduction. Grossly extended chromosomes (macrotene) were formed, with complex structural alterations but sufficient stability to propagate unchanged over successive generations. Their detailed molecular analysis, including complete genome sequencing, identification of sequence breakpoints and comparisons between mutants reveals novel mechanisms to their formation whose combined action underlies the astonishing dynamics of eukaryotic chromosomes and its consequences.
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.
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.
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.
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).
Identification of genes involved in the attenuation of virulence of rough M. abscessus Tn5 mutants in zebrafish
Mycobacterium abscessus is the fast growing mycobacterial species the most frequently associated with lung infection, characterized by severe and very inflammatory cases, after a slow and chronic infectious process. The Cystic Fibrosis patients are particularly susceptible to this bacteria. M. abscessus can exist in a smooth (S) or rough (R) shape depending respectively on the presence or absence of glycopeptidolipids (GPL) associated with the wall of the bacillus. These GPL are involved in motility and biofilm formation, two important features of bacterial colonization. A correlation between the morphotype R, capable of producing morphological structures called "cording" visible in microscopy, and virulence was established. Natural switching from a phenotype S to R allow the transition from a colonizing status to an invasive and pathogenic form. Our project is focusing on deciphering specific mechanisms and analysis of benefits from this transition S/R in vivo. On the basis of transcriptomic data and RNA-Seq previously obtained and/or by screening a transpositional library of colonies deficient in the cording formation, several genes were selected and their corresponding mutants were generated. Since are analyzed : i) the (glyco)lipid composition of their wall; ii) their respective virulence in zebrafish and mouse models and iii) the involvement/recruitment of macrophages and neutrophils and their role in inflammation in response to infection. Our aims are to identify and describe new virulence determinants specific to the establishment of chronic stages of M. abscessus and identification of molecular locks of the S/R switch to understand how an environmental mycobacterium evolves into a pathogen in the host.
Our recent analyses suggest that the genetic determinants of human neuroanatomical diversity are massively polygenic. Like other quantitative traits such as height – but also IQ or ASD risk – neuroanatomical diversity seems to result from the aggregated effect of thousands of frequent variants, each of small effect. GWAS should then require populations of hundreds of thousands of individuals to start to detect the individual variants. GCTA (genomic complex trait analysis) offers an alternative approach to obtain valuable neurogenetic information despite the current impossibility to detect enough individual variants to explaining any substantial part of the variability. We are currently pooling together neuroimaging genomics data from multiple international projects (in particular, IMAGEN, ENIGMA, UK Biobank) to replicate and extend our earlier analyses. We aim to: (1) Compute the amount of variance captured by genome-wide SNPs (SNP-heritability) for the several brain regions: ICV, BV, Hip, Th, Ca, Pa, Pu, Amy and Acc, (2) Compute the matrix of SNP-based genetic correlation among structures, (3) Partition the variance captured by SNPs among structural and functional sets: per chromosome, genic vs non-genic, low/medium/high minor-allele frequency, positive/negative selection, involved or not in neurodevelopment, etc. (4) Compare our results with those obtained using GWAS-based estimations (for example, those used in ENIGMA2). GCTA requires the computation of matrices of genetic relationship among all individuals, and thus, direct access to the genotyping data. Once the matrices are computed, the genotyping data is no longer required, and it is not possible to reconstruct an individual's genome from the matrices. Our analysis of the IMAGEN cohort was based on 1,765 Individuals, which gave us sufficient statistical power (80%) to detect only strong heritabilities (h2~45%), and the estimations had very large standard errors (~20%). A cohort of 4,000 subjects should allow us to decrease the standard error to ~8% (80% power to detect h2=22%), and a cohort of 8,000 subjects should decrease it to ~4% (80% power to detect h2=11%). In this way, we could obtain more accurate estimates, but also detect eventually more subtle effects related to functional genomic partitions. References Yang et al (2010) Common SNPs explain a large proportion of heritability for human height. Nature Genetics, doi: 10.1038/ng.608 Davies et al (2011) Genome-wide association studies establish that human intelligence is highly heritable and polygenic. Molecular Psychiatry, doi: 10.1038/mp.2011.85 Gaugler et al (2014) Most genetic risk for autism resides with common variation. Nature Genetics, doi: 10.1038/ng.3039 Wood et al (2014) Defining the role of common variation in the genomic and biological architecture of adult human height, doi: 10.1038/ng.3097
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.
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.
Research of SNPs to explain the non virulent phenotype of a mutant of L. interrogans serovar Manilae L495 affecting the expression of a protein not involved in virulence
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.
Vérification et cartographie de génomes de corynebacteries modifiés. Recherche de suppresseurs à partir de délétions de gènes de kinase (pknA; pknB ...)
Trichosporon asahii is a yeast responsible of human invasive infection worldwide. Actually, no genotyping method is available to determine relationship between clinical isolates. At the NRCMA we have more than 40 clinical isolates and 2 collection strains associated with clinical data. Thanks to P2M facility, whole genome for 33 isolates was sequenced. The aim of this project is to study the genetic diversity of Trichosporon asahii and the potential relationship with clinical and/or phenotypic data and finally propose a new genotyping method that could be usefull for clinician in case of local or national outbreak.
Samples were collected in the field from infected patients admited in Ebola treatment Center of Macenta (ETC Guinea). Deep sequencing was performed in order to look for majority and minority variants. The aim of this study is to compare quasispecies of patients who survived and those who died and also to get detailed description of EBOV evolution in an ETC to identify phylogenetic clusters or transmission chain.
Yellow fever virus (YFV), a Flavivirus transmitted by mosquitoes causes a severe hemorrhagic fever in humans. Despite the availability of a safe and effective vaccine (17D), YFV is still a public health problem in tropical Africa and South America. In the Americas, the massive campaign of mosquito control during the first half of the 20th century led to the eradication of Aedes aegypti from most American countries, and as a consequence, urban outbreaks of YF were no longer observed. However, the relaxation of vector control led to the reinfestation of urban areas by Ae. aegypti and the subsequent establishment of the Asian tiger mosquito Aedes albopictus. In Brazil, while human cases are sporadically detected in the Amazonian basin where sylvatic YFV strains circulate between non-human primates and arboreal canopy-dwelling mosquitoes (Haemagogus sp.), they are increasingly reported outside the jungle moving towards the Atlantic coast, the most populated area. In the absence of routine immunization programs, YF may come back in the American towns as it was in the past. The causes leading to the current YF resurgence are multifactorial. From a mosquito vector viewpoint, changes in vector densities, distribution, vector competence or vector as a site of selection for epidemic YFV strains, can be regarded as critical factors. Our project aims to address the contribution of the invasive mosquito Ae. albopictus as a missing link to allow a selvatic YF strain (1D) to become adapted for a transmission in urban areas by the human-biting mosquito, Ae. aegypti. It will be done through three specific objectives: (i) identify Ae. albopictus-adaptive mutations after serial cycling of the selvatic YFV-1D on Brazilian Ae. albopictus mosquitoes, (ii) evaluate their potential to be transmitted to a vertebrate host, and (iii) deepen the transmission of the experimentally selected viruses by field-collected mosquito populations.
Antimalarial drug resistance in Africa: A comprehensive molecular analysis of the emergence of artemisinin resistant parasites in Africa
We are involved, in collboration with the WHO, in the SaMARA which aims at detecting the emergence of antimalarial drug resistance in Africa. Samples (dried blood spots) are collected from the Nantional Malaria Control Programmes in Africa during clinical efficacy studies. My group at the Institut Pasteur in collaboration with the Institut Cochin (Frédéric Ariey) tested these samples and assessed the proportion of parasites harboring SNPs or CNV associated to antimalarial drug resistance. We have previously demonstrated that mutations in the propeller dommain of a Kelch gene located on the chromosome 13 are strongly predictive of artemsinin resistance. Until last year, artemisinin resistance was confined in South east Asia. Recently, we have detected in African samples a high proportion of mutant parasites. This mutation has never been observed before in South east Asia. To perform comprenhensive genomic analysis, we have sequenced (Illumina) all mutants (=24) and paired wild type samples. Analysis of these sequences (and 400 whole genome sequences from parasites collected in Asia, Africa and America, upload on a dedicated website) should allow to: - define if the mutants have emerged locally or have spread from Asia - define the genetic background of these mutants (are specific alleles facilitated the emergence of K13 mutantions) - describe the genomic profile(s) of these mutants (especially the haplotypes associated to multidrug resistance)
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.
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.
Genetic and statistical analysis of data produced with the Collaborative Cross at the Institut Pasteur
The project aims at developing (1) a database dedicated to the storage and statistical analysis of Collaborative Cross based data and (2) implement genetic analysis tools based on rqtl2 for the identification of QTL on this data.
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.
The chikungunya virus (CHIKV) is an emerging mosquito-borne virus which has widely spread around the world in the last two decades. The virus is transmitted between human hosts by Aedes mosquitoes, including Aedes albopictus which is now established in more than 51 departments in France. The transmissibility of CHIKV can be affected by a combination of both intrinsic and extrinsic factors. Since mosquitoes are poikilothermic insects, environmental temperature is of particular importance. Indeed, temperature can shape transmission of pathogens through direct effects on viral replication and/or indirect effects via the vector immune responses. The relation between temperature and virus transmission is very complex and remains poorly defined. The aim of the project is to better understand how temperature affects mosquito-virus interactions. To meet this objective, our studies will focus on (i) how CHIKV genetically evolves under low temperature pressure (in vitro and in vivo), (ii) mosquito molecular responses to a low temperature exposure, (iii) and ultimately, how mosquito microbiota is affected by a low temperature. Our study will help in identifying the determinants that facilitate CHIKV transmission by Ae. albopictus in temperate conditions.
Our research team recently identified an Aedes aegypti mosquito population that is partially resistant to dengue virus infection. We also have candidate genes that potentially innerly this phenotype. In this project, we would like to test wether specific SNPs are associated with virus infection in those mosquitoes. Sequencing data has already been generated with the Omics platform and handled by C3BI.
We are currently characterising a set of genes that are involved in the yellow fever mosquito, Aedes aegypti, vector competence. We would like to investigate the polymorphisms of those genes using exome sequencing data.
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.
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.
Yellow fever (YF) is a fatal hemorrhagic disease caused by an arbovirus, Yellow Fever Virus (YFV) transmitted by the Aedes aegypti mosquito vector. YFV is currently endemic in Africa (5 strains) and South America (2 strains), and although an effective vaccine is available, YFV remains a major public health issue. Native from Africa, YFV was transported to South America and the Caribbean during the slave trade (15th to 19th century) and caused devastating outbreaks. After two centuries of outbreaks, YF is no longer reported in the Caribbean thanks to the successful mosquito control program implemented at the beginning of the 20th century. The virus disappeared from the Caribbean and Martinique experienced its last outbreak in 1908. However, the relaxation of anti-vectorial control programs contributed to the reintroduction of Ae. aegypti in most Caribbean islands. We evaluated the vector competence to YFV of different populations of Ae. aegypti from Martinique and other mosquito populations of the Caribbean. Our results showed that Ae. aegypti from Martinique were competent to African as well as to American YFV genotypes with however differences of viral dissemination and transmission. In this project, we aim at determining the diversity of viral populations at the crossing of two anatomical barriers in mosquitoes: midgut and salivary glands. The virus should enter into the midgut epithelial cells, replicate and be released into the hemocel. The final step will be the infection of the salivary glands from where the virus is excreted with the saliva. At each step, a selection of viral populations operates with only a fraction of the viral population transmitted between different tissues within a same host. The virus diversification promoted by virus replication in mosquitoes depends on the mosquito species. Such vector-specific conditions may have great impacts on emergence processes.
Several arboviruses have emerged and/or reemerged in the New World in the past decades. While yellow fever and dengue are historical diseases which continue to cause deadly epidemics, Zika and chikungunya have recently invaded the South American continent, causing great concern. In Colombia, Aedes aegypti is the vector of most of human arboviruses. We collected Ae. aegypti eggs in Medellin in Colombia in 2020 and infected adults with dengue (DENV), chikungunya (CHIKV), yellow fever (YFV) and Zika virus (ZIKV). We show that Ae. aegypti Nor Oriental was more prone to become infected, to disseminate and transmit CHIKV and ZIKV than DENV and YFV. In this project, we aim at determining the diversity of viral populations at the crossing of two anatomical barriers in mosquitoes: midgut and salivary glands.
According to the WHO, the Hepatitis B virus (HBV) infects chronically 295 million persons. After several decades, persistently infected patients become at risk to develop liver cirrhosis and/or liver cancer (hepatocellular carcinoma, HCC). Each year around one million persons die from these two complications directly triggered by HBV. In the course of infection, the HBV DNA genome accumulates mutations that become progressively dominant. Some of the recurrent mutations are suspected to play a significant role in the pathophysiology of liver disease by changing the biological activity of HBV-encoded genes or proteins. However, the virus is rarely acting alone to promote terminal liver diseases, and its deleterious activity is generally potentialized by other risk factors concomitant to viral presence. It is the case of alcohol or tobacco consumption, metabolism-associated fatty liver disease, herbal medicine containing aristolochic acid, or mycotoxin-contaminated food. Most of these agents are known to leave mutation fingerprints on the human genome. However, we do not currently know whether these mutagens contribute or not to the mutational burden observed on HBV DNA after decades of infection. We hypothesize that it could be the case. The characterization of the full mutation spectrum of the HBV genome including the application of tools initially proposed to analyze human mutations is the aim of this research program.
We are working on malaria drug discovery. In an attempt to understand the mode of action of epigenetic inhibitors active against Plasmodium falciparum, we culture the parasite under drug pressure to induce drug resistance, along with a control treated in the same conditions with the solvent only. We then clone resistant and control parasite lines and sequence their genomes to compare them to try to understand which gene is responsible for the resistance and thus may constitute the inhibitor's target (through SNPs, amplification of gene copies).