Biology subjects

Legendre, R.

Publications and source records attributed to Legendre, R..

30 records · Page 2Linked to original sources

bioRxiv2021

Maternal age affects equine Day 8 embryo gene expression both in trophoblast and inner cell mass

BackgroundBreeding a mare until she is not fertile or even until her death is common in equine industry but the fertility decreases as the mare age increases. Embryo loss due to reduced embryo quality is partly accountable for this observation. Here, the effect of mares age on blastocysts gene expression was explored. Day 8 post-ovulation embryos were collected from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure or inner cell mass (ICM) enriched trophoblast, obtained by embryo bisection, were RNA sequenced. Deconvolution algorithm was used to discriminate gene expression in the ICM from that in the trophoblast. Differential expression was analyzed with embryo sex and diameter as cofactors. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed. ResultsMaternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity. In both compartments, the expression of genes involved in mitochondria and protein metabolism were disturbed by maternal age, although more genes were affected in the ICM. Mitosis, signaling and adhesion pathways and embryo development were decreased in the ICM of embryos from old mares. In trophoblast, ion movement pathways were affected. ConclusionsThis is the first study showing that maternal age affects gene expression in the equine blastocyst, demonstrating significant effects as early as 10 years of age. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.

physiology↗

bioRxiv2021

Post-transcriptional regulation of Leishmania fitness gain

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein abundance linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in abundance of flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

microbiology↗

bioRxiv2021

ChIPuana: from raw data to epigenomic dynamics

We present ChIPflow, a Snakemake-based pipeline for epigenomic data from the raw fastq files to the differential analysis. It can be applied to any chromatin factor, e.g. histone modification or transcription factor, which can be profiled with ChIP-seq. ChIPflow streamlines critical steps like the quality assessment of the immunoprecipitation using cross-correlation and the replicate comparison for both narrow and broad peaks. For the differential analysis ChIPflow provides linear and nonlinear methods for normalisation between samples as well as conservative and stringent models for estimating the variance and testing the significance of the observed binding/marking differences. ChIPflow can process in parallel multiple chromatin factors with different experimental designs, number of biological replicates and/or conditions. It also facilitates the specific parametrisation of each dataset allowing both narrow or broad peak calling, as well as comparisons between the conditions using multiple statistical settings. Finally, complete reports are produced at the end of the bioinformatic and the statistical part of the analysis, which facilitate the data quality control and the interpretation of the results. We explored the discriminative power of the statistical settings for the differential analysis, using a published dataset of three histone marks (H3K4me3, H3K27ac and H3K4me1) and two transcription factors (Oct4 and Klf4) profiled with ChIP-seq in two biological conditions (shControl and shUbc9). We show that distinct results are obtained depending on the sources of ChIP-seq variability and the dynamics of the chromatin factor under study. We propose that ChIPflow can be used to measure the richness of the epigenomic landscape underlying a biological process by identifying diverse regulatory regimes and the associated genes sets.

bioinformatics↗

bioRxiv2021

The CovR regulatory network drives the evolution of Group B Streptococcus virulence

Virulence of the neonatal pathogen Group B Streptococcus depends on the master regulator CovR. Inactivation of CovR leads to large-scale transcriptome remodeling and impairs almost every step of the interaction between the pathogen and the host. However, comparative analyses suggested a plasticity of the CovR signalling pathway in clinical isolates, probably due to the host selective pressure and leading to phenotypic heterogeneity in the bacterial population. Here, we characterize the CovR regulatory network in a strain representative of the hypervirulent lineage responsible of the majority of late-onset meningitidis. Genome-wide binding and transcriptome analysis demonstrated that CovR acts as a direct and global repressor of virulence genes, either as a primary regulator or with specialized co-regulators. Remarkably, CovR directly regulates genes of the pan-genome, including the two specific hypervirulent adhesins and horizontally acquired genes, as well as core-genes showing mutational biases in the population. Parallel analysis of the CovR network in a second isolate links strain-specificities to micro-evolutions in CovR-regulated promoters and to broad difference due to variability in CovR activation by phosphorylation. Our results highlight the direct, coordinated, and strain-specific regulation of virulence genes by CovR. This intra-species evolution of the signalling network reshapes bacterial-host interactions, increasing the potential for adaptation and the emergence of clone associated with specific diseases.

microbiology↗

bioRxiv2020

The oxidative stress response and virulence of pathogenic Leptospira are controlled by the interplay of two peroxide stress regulators

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network. Author summaryLeptospirosis is a widespread infectious disease responsible for over one million of severe cases and 60 000 fatalities annually worldwide. This neglected and emerging disease has a worldwide distribution, but it mostly affects populations from developing countries in sub-tropical areas. The causative agents of leptospirosis are pathogenic bacterial Leptospira spp. There is a considerable deficit in our knowledge of these atypical bacteria, including their virulence mechanisms. In addition to the Leptospira PerRA regulator that represses defenses against peroxide, we have identified and characterized a second PerR regulator in pathogenic Leptospira species (PerRB) that participates in Leptospira tolerance to superoxide. Phenotypic and transcriptomic analyses of single PerRA and PerRB mutants suggest that the two PerRs fulfill distinct functions in the adaptation to oxidative stress. Concomitant inactivation of PerRA and PerRB resulted in a higher tolerance to both peroxide and superoxide. Moreover, the perRAperRB mutant lost its virulence. Major changes in gene expression, including a decreased expression of several virulence factors, were observed in the double perRAperRB mutant. Our study suggests that PerRA and PerRB cooperate to orchestrate a complex regulatory network involved in Leptospira virulence.

microbiology↗

bioRxiv2020

Queuine is a nutritional regulator of Entamoeba histolytica response to oxidative stress and a virulence attenuator

Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here we describe the effects of queuine on the physiology of the eukaryotic parasite, Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in tRNAAspGUC. Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in OS response, such as heat shock protein 70 (Hsp 70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in tRNAAspGUC, parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence. ImportanceEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of the bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces a mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.

microbiology↗

bioRxiv2020

Functionally distinct resident macrophage subsets differentially shape responses to infection in the bladder

Resident macrophages are abundant in the bladder, playing key roles in immunity to uropathogens. Yet, whether they are heterogeneous, where they come from, and how they respond to infection remain largely unknown. We identified two macrophage subsets in mouse bladders, MacM in the muscle and MacL in the lamina propria, with distinct protein expression and transcriptomes. Using a urinary tract infection model, we validated our transcriptomic analyses, finding that MacM macrophages phagocytosed more bacteria and polarized to a more anti-inflammatory profile, whereas the MacL subset died rapidly during infection. During resolution, monocyte-derived cells contributed to tissue-resident macrophage pools and both subsets acquired transcriptional profiles distinct from naive macrophages. Depletion of these altered macrophages resulted in the induction of a type 1 biased immune response to a second urinary tract infection, improving bacterial clearance. Our study uncovers the biology of resident macrophages and their response to an exceedingly common infection in a largely overlooked organ, the bladder.

immunology↗

bioRxiv2020

The adaptive transcriptional response of pathogenic Leptospira to peroxide reveals new defenses against infection-related oxidative stress

Pathogenic Leptospira spp. are the causative agents of the waterborne zoonotic disease leptospirosis. During infection, Leptospira are confronted with dramatic adverse environmental changes such as deadly reactive oxygen species (ROS). Withstanding ROS produced by the host innate immunity is an important strategy evolved by pathogenic Leptospira for persisting in and colonizing hosts. In L. interrogans, genes encoding defenses against ROS are repressed by the peroxide stress regulator, PerR. In this study, RNA sequencing was performed to characterize both the L. interrogans adaptive response to low and high concentrations of hydrogen peroxide and the PerR regulon. We showed that Leptospira solicit three main peroxidase machineries (catalase, cytochrome C peroxidase and peroxiredoxin) and heme to detoxify oxidants produced during a peroxide stress. In addition, canonical molecular chaperones of the heat shock response and DNA repair proteins from the SOS response were required for Leptospira recovering from oxidative damages. Determining the PerR regulon allowed to identify the PerR-dependent mechanisms of the peroxide adaptive response and has revealed a PerR-independent regulatory network involving other transcriptional regulators, two-component systems and sigma factors as well as non-coding RNAs that putatively orchestrate, in concert with PerR, this adaptive response. In addition, we have identified other PerR-regulated genes encoding a TonB-dependent transport system, a lipoprotein (LipL48) and a two-component system (VicKR) involved in Leptospira tolerance to superoxide and that could represent the first defense mechanism against superoxide in L. interrogans, a bacterium lacking canonical superoxide dismutase. Our findings provide a comprehensive insight into the mechanisms required by pathogenic Leptospira to overcome infection-related oxidants during the arm race with a host. This will participate in framing future hypothesis-driven studies to identify and decipher novel virulence mechanisms in this life-threatening pathogen. Author summaryLeptospirosis is a zoonotic infectious disease responsible for over one million of severe cases and 60 000 fatalities annually worldwide. This neglected and emerging disease has a worldwide distribution, but it mostly affects populations from developing countries in sub-tropical areas. The causative agents of leptospirosis are pathogenic bacterial Leptospira spp. There is a considerable deficit in our knowledge of these atypical bacteria, including their virulence mechanisms. During infection, Leptospira are confronted with the deadly oxidants produced by the host tissues and immune response. Here, we have identified the cellular factors necessary for Leptospira to overcome the oxidative stress response. We found that Leptospira solicit peroxidases to detoxify oxidants as well as chaperones of the heat shock response and DNA repair proteins of the SOS response to recover from oxidative damage. Moreover, our study indicates that adaptation to oxidative stress is orchestrated by a regulatory network involving PerR and other transcriptional regulators, sigma factors, two component systems, and putative non-coding RNAs. These findings provide a comprehensive insight into the mechanisms required by pathogenic Leptospira to tolerate infection-related oxidants, helping identify novel virulence factors, developing new therapeutic targets and vaccines against leptospirosis.

microbiology↗

bioRxiv2019

Y-RNAs Lead an Endogenous Program of RIG-I Agonism Mobilized upon RNA Virus Infection and Targeted by HIV

Pattern recognition receptors (PRRs) protect against host invasion by detecting specific molecular patterns found in pathogens and initiating an immune response. While microbial-derived PRR ligands have been extensively characterized, the contribution and relevance of endogenous ligands to PRR activation during viral infection remain overlooked. In this work, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by a positive-sense RNA virus, a negative-sense RNA virus or a retrovirus. We found that several endogenous RNAs transcribed by RNA polymerase 3 (Pol3) specifically engage RLRs, and in particular the family of small non-coding repeats Y-RNAs, which presents the highest affinity as RIG-I ligands. We show that this recognition is dependent on Y-RNA mimicking viral secondary structure and its 5-triphosphate extremity. Further, we found that HIV-1 infection triggers a VPR-dependent downregulation of RNA triphosphatase DUSP11 in vitro and in vivo, leading to an increase of Y-RNA 5-triphosphorylation that enables their immunogenicity. Importantly, we show that altering DUSP11 expression is sufficient to induce a type-I interferon and T cell activation transcriptional program associated with HIV-1 infection. Overall, our work uncovers the critical contribution of endogenous repeat RNAs ligands to antiviral immunity and demonstrates the role of this pathway in HIV-1 infection.

immunology↗

bioRxiv2019

An impact of HP1γ on the fidelity of pre-mRNA splicing arises from its ability to bind RNA via intronic repeated sequences.

HP1 proteins are best known as markers of heterochromatin and gene silencing. Yet, they are also RNA-binding proteins and the HP1{gamma}/Cbx3 family member is present on transcribed genes together with RNA polymerase II, where it regulates co-transcriptional processes such as alternative splicing. To gain insight in the role of the RNA binding activity of HP1{gamma} in transcriptionally active chromatin, we have captured and analyzed RNAs associated with this protein. We find that HP1{gamma} specifically recognizes hexameric RNA motifs and coincidentally transposable elements of the SINE family. As these elements are abundant in introns, while essentially absent from exons, the HP1{gamma} RNA binding activity tethers unspliced pre-mRNA to chromatin via the intronic region and limits the usage of intronic cryptic splice sites. Thus, our data unveil novel determinants in the relationship between chromatin and co-transcriptional splicing.

molecular biology↗

bioRxiv2019

Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages

Free-living amoebae might represent an evolutionary niche. In order to get more insights into the potential amoebal training ground for Mycobacterium abscessus, we characterized its full transcriptome in amoeba (Ac) and macrophages (M{varphi}), as well as the Mycobacterium chelonae intra-Ac transcriptome for comparison. Up-regulated genes in Ac allowed M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing switch from carbohydrate carbon sources to fatty acid metabolism. Eleven genes implicated in the adaptation to intracellular stress, were mutated, with all but one confirmed to be involved in M. abscessus intra-M{varphi} survival. Cloning two of these genes in M. chelonae increased its intra-M{varphi} survival. One mutant was particularly attenuated in M{varphi} that corresponded to the deletion of an Eis N-acetyl transferase protein (MAB_4532c). Taken together, M. abscessus transcriptomes revealed the intracellular lifestyle of the mycobacteria, with Ac largely contributing to the enhancement of M. abscessus intra-M{varphi} survival.

microbiology↗

bioRxiv2017

Sequanix: A Dynamic Graphical Interface for Snakemake Workflows

SummaryWe designed a PyQt graphical user interface - Sequanix - aiming at democratizing the use of Snakemake pipelines. Although the primary goal of Sequanix was to facilitate the execution of NGS Snakemake pipelines available in the Sequana project (http://sequana.readthedocs.io), it can also handle any Snakemake pipelines. Therefore, Sequanix should be useful to all Snakemake developers willing to expose their pipelines to a wider audience.\n\nAvailabilitySource code available on http://github.com/sequana/sequana and standalone on http://bioconda.github.io (sequana package).

bioinformatics↗