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.

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.

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

In wild life, yeast cells are able to survive in severe conditions, without nutriment for a long period of time because the cell is able to enter in stationary phase. During this phase, the cell can transform varied sources of energy and pause its growth to preserve the cells from death. It is known that most of genes are downregulated in stationary phase and the cell activity is globally reduced to its strict minimum, while a subset of “specialized” genes is induced to promote survival in extreme conditions. We are analysing the transcriptome of exponentially grown or stationary phase yeasts, investigating different level of regulation.

The increased incidences of invasive fungal infections coupled with the paucity of available antifungal drugs for treatment have driven the search for novel agents with unique fungal targets. Studies

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

Le génome de Cryptococcus neoformans a été réannoté grâce à des données RNA-seq. Ces nouvelles annotations doivent être soumises dans les banques publiques.

Meiosis is the specialized, highly regulated process at the basis of the sexual reproduction of eukaryotes. During this process, a diploid cell undergoes a single round of DNA replication and two successive rounds of chromosome segregation, halving the chromosome set to generate four haploid products (gametes). Homologous (i.e. paternal and maternal) chromosomes during the prophase of the first division of meiosis undergo a highly regulated succession of events that include recognition, pairing, and synapsis along their length. An important aspect of chromosome organization, besides pairing and synapsis, consist in the condensation step: axial elements form between sister chromatids, bridging distant axis sites comprising cohesins, and resulting in the extrusion of chromatin loops away from the axis. The precise organization of these chromatin loops remain unclear, and is notably impaired by the sequence similarities of the two homologs. Our aim is to characterize the fine organization of chromatin on both homologs during meiotic prophase, and how this organization is functionally related to a recombination event.

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.

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.

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.

Cryptococcus neoformans is a sugar-coated yeast that is able to interact closely with numerous organisms in the environment including amebae, paramecium of nematodes. The interaction with these organisms probably shaped its virulence. The ability to survive nutrient starvation, oxidative stress, desiccation, both in the environment and in humans, indicates a high level of physiological and metabolic plasticity of the yeast. In humans, after primary infection during childhood, the yeast is able to survive within the host for years before reactivation, leading to a deadly disseminated fungal infection. This phenomenon, called dormancy / quiescence is one of the main biological features of this fungus in relation with disease pathogenesis. It is known in bacteria, parasites and other fungi. There is no consensus on the definition of dormancy. Most often, dormant cells are characterized by a low metabolic activity sometimes undetectable under normal laboratory conditions and the ability to be resuscitated by adequate stimuli. In C. neoformans, dormancy has only been demonstrated epidemiologically in our laboratory but not experimentally so far. We developed an assay where yeasts cells exhibiting characteristics of potentially dormant cells were generated. Our current project aims at exploring the conditions leading to, the biology of the entry in and the mechanisms sustaining dormancy.

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.

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

Project context and summary : Cryptococcus neoformans is a sugar-coated yeast that is able to interact closely with numerous organisms in the environment including amebae, paramecium of nematodes. The interaction with these organisms probably shaped its virulence. The ability to survive nutrient starvation, oxidative stress, desiccation, both in the environment and in humans, indicates a high level of physiological and metabolic plasticity of the yeast. In humans, after primary infection during childhood, the yeast is able to survive within the host for years before reactivation, leading to a deadly disseminated fungal infection. This phenomenon, called dormancy / quiescence is one of the main biological features of this fungus in relation with disease pathogenesis. It is known in bacteria, parasites and other fungi. There is no consensus on the definition of dormancy. Most often, dormant cells are characterized by a low metabolic activity sometimes undetectable under normal laboratory conditions and the ability to be resuscitated by adequate stimuli. In C. neoformans, dormancy has only been demonstrated epidemiologically in our laboratory but not experimentally so far. We developed an assay where yeasts cells exhibiting characteristics of potentially dormant cells were generated. Our current project aims at exploring the conditions leading to, the biology of the entry in and the mechanisms sustaining dormancy.

Our aim is to characterize the fine organization of chromatin on homologs during meiotic prophase, and how this organization is functionally related to recombination.

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.

We are interested in the cytoplasmic quality control of gene expression and more especially into the behavior of aberrant peptides which could be generated from non-conform translation events. We are now investigating the role of a Saccharomyces cerevisiae RNA helicase protein that we named Tac4 (for Translation associated Component 4). We showed that this protein is involved in translation. We demonstrated, by sucrose gradient and affinity purification that Tac4 interacts with the ribosome. A first UV cross-linking and cDNA analysis (CRAC) experiment clearly revealed that Tac4 interacts with the 18S rRNA of the 40S ribosomal subunit and we precisely defined the crosslink point. These preliminary results also suggested an enrichment of the 3’-end regions of mRNAs. This implies that Tac4 could not only interact with the small ribosomal subunit but also directly with mRNA. Tac4 is conserved through the evolution and its mammalian homologue is involved in initiation of translation. Therefore, we thought that Tac4 could be associated with the 5’-end rather than with the 3’-end. However, a recent paper from the Rachel Green’s lab showed that translation reinitiation into the 3’-UTR region may occurs when translation termination is affected (Young et al., Cell 2015). The factors and molecular mechanisms implicated in these events are not known. Altogether, our preliminary results suggest that Tac4 is an excellent candidate participating to the unwinding of RNA structure or to the release of some RNA-binding proteins into the 3’-end mRNA. We now would like to 1) confirm that Tac4 preferentially interacts with the 3’-end of mRNA, 2) determine whether Tac4 interacts with a region upstream the Stop codon or in the 3’-UTR of the mRNA, 3) identify the mRNA targets to determine whether Tac4 could have a general role in translation or could only be involved in translation of some specific mRNA.

We are interested in the cytoplasmic quality control of gene expression and more especially into the behavior of aberrant peptides which could be generated from non-conform translation events. We are now investigating the role of a Saccharomyces cerevisiae RNA helicase protein that we named Tac4 (for Translation Associated Component 4). We showed that this protein is involved in translation. We demonstrated, by sucrose gradient and affinity purification that Tac4 interacts with the ribosome. A first UV cross-linking and cDNA analysis (CRAC) experiment clearly revealed that Tac4 interacts with the 18S rRNA of the 40S ribosomal subunit and we precisely defined the crosslink point. These preliminary results also suggested an enrichment of the 3’-end regions of mRNAs. This implies that Tac4 could not only interact with the small ribosomal subunit but also directly with mRNA. Tac4 is conserved through the evolution and its mammalian homologue is involved in initiation of translation. Therefore, we thought that Tac4 could be associated with the 5’-end rather than with the 3’-end. However, recent data showed that translation reinitiation into the 3’-UTR region may occurs when translation termination is affected. The factors and molecular mechanisms implicated in these events are not known. Altogether, our preliminary results suggest that Tac4 is an excellent candidate participating to the unwinding of RNA structure or to the release of some RNA-binding proteins into the 3’-end mRNA. We now would like to 1) confirm that Tac4 preferentially interacts with the 3’-end of mRNA, 2) determine whether Tac4 interacts with a region upstream the Stop codon or in the 3’-UTR of the mRNA, 3) determine whether Tac4 could also interact with other mRNA region, such as the 5'-UTR region, 4) identify the mRNA targets to determine whether Tac4 could have a general role in translation or could only be involved in tra

Analysing the transcriptome of exponentially grown or stationary phase yeasts in a genetic background that stabilises pervasive transcipts, we identified a first subset of ≈ 140 antisense transcripts anti-correlated with gene transcripts that are specifically expressed in quiescence. We are further investigating whether these genes are subject to a transcriptional interference and what are the mechanisms underlying this regulation. More in detail, we would like to analyse the loci where an antisense ncRNA are detected.

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

Invasion of epithelial cells by the obligate intracellular bacterium Chlamydia trachomatis results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have isolated a mutant strain that shows several developmental defects. The C3BI will contribute to the statistical analysis of the data.

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

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.

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

The canonical (“textbook”) process of DNA homology search and recognition is initiated by DNA double-strand breaks and is mediated by the universally conserved recombinases of the RecA family. Using t