A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary

cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.Galupa et al. uncover elements important for Xist regulation in its neighboring TAD and reveal that these elements can influence gene regulation both within and between topological domains. These findings, in a context where dynamic, developmental expression is necessary, challenge current models for TAD-based gene-regulatory landscapes

Desinteresse sexual do panda-gigante : lenda ou fato?

O estudo traduz, para o grande público, a sofisticada natureza desse animal, de andar desengonçado, aparência infantil e inofensiva, com jeito de bicho de pelúcia e que atrai a simpatia de todos. Um carnívoro que só come bambu e que apresenta grandes dificuldades para se reproduzir, existindo atualmente apenas 1.600 exemplares da espécie, que vivem em reservas florestais na China ou em alguns poucos centros de reprodução no mundo. Panda, em chinês, significa comedor de bambu, vegetal que constitui 99% de sua alimentaçã

JC Polyomavirus whole genome sequencing at the single molecule level reveals emerging neurotropic populations in Progressive Multifocal Leucoencephalopathy

International audienceBackground JC polyomavirus (JCV) mostly causes asymptomatic persistent renal infections but may give rise in immunosuppressed patients to neurotropic variants which replicate in the brain causing progressive multifocal leukoencephalopathy (PML). Rearrangements in the JCV genome regulator non-coding control region (NCCR) and missense mutations in the viral capsid VP1 gene differentiate neurotropic variants from virus excreted in urine. Methods To investigate intra-host emergence of JCV neurotropic populations in PML, we deep sequenced JCV whole genome recovered from cerebrospinal fluid (CSF) and urine samples from 32 HIV- and non HIV-infected PML patients at the single-molecule level. Results JCV strains distributed among 6 out of 7 known genotypes. Common patterns of NCCR rearrangements included an initial deletion mostly located in a short 10-nucleotide sequence, followed by duplications/insertions. Multiple NCCR variants present in individual CSF samples shared at least one rearrangement suggesting they stemmed from a unique viral population. NCCR variants independently acquired single or double PML-specific adaptive VP1 mutations. NCCR variants recovered from urine and CSF displayed opposite deletion or duplication patterns in binding sites for transcription factors. Discussion Long read deep sequencing shed light on emergence of neurotropic JCV populations in PML

Core genome sequencing and genotyping of Leptospira interrogans in clinical samples by target capture sequencing

International audienceBackgroundThe life-threatening pathogen Leptospira interrogans is the most common agent of leptospirosis, an emerging zoonotic disease. However, little is known about the strains that are currently circulating worldwide due to the fastidious nature of the bacteria and the difficulty to isolate cultures. In addition, the paucity of bacteria in blood and other clinical samples has proven to be a considerable challenge for directly genotyping the agent of leptospirosis directly from patient material. Our understanding of the genetic diversity of strains during human infection is therefore limited.MethodsHere, we carried out hybridization capture followed by Illumina sequencing of the core genome directly from 20 clinical samples that were PCR positive for pathogenic Leptospira to elucidate the genetic diversity of currently circulating Leptospira strains in mainland France.ResultsCapture with RNA probes covering the L. interrogans core genome resulted in a 72 to 13,000-fold increase in pathogen reads relative to standard sequencing without capture. Variant analysis of the genomes sequenced from the biological samples using 273 Leptospira reference genomes was then carried out to determine the genotype of the infecting strain. For samples with sufficient coverage (19/20 samples with coverage > 8×), we could unambiguously identify L. interrogans serovars Icterohaemorrhagiae and Copenhageni (14 samples), L. kirschneri serovar Grippotyphosa (4 samples), and L. interrogans serovar Pyrogenes (1 sample) as the infecting strains.ConclusionsWe obtained high-quality genomic data with suitable coverage for confident core genome genotyping of the agent of leptospirosis for most of our clinical samples. The recovery of the genome of the serovars Icterohaemorrhagiae and Copenhageni directly from multiple clinical samples revealed low adaptive diversification of the core genes during human infection. The ability to generate culture-free genomic data opens new opportunities for better understanding of the epidemiology of this fastidious pathogen and pathogenesis of this neglected disease

Investigating the role of the carbon storage regulator A (CsrA) in Leptospira spp.

International audienceCarbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that plays a critical role in response to environmental changes in many bacteria. CsrA has been reported to regulate several metabolic pathways, motility, biofilm formation, and virulence-associated genes. The role of csrA in Leptospira spp., which are able to survive in different environmental niches and infect a wide variety of reservoir hosts, has not been characterized. To investigate the role of csrA as a gene regulator in Leptospira , we generated a L . biflexa csrA deletion mutant (Δ csrA ) and csrA overexpressing Leptospira strains. The Δ csrA L . biflexa displayed poor growth under starvation conditions. RNA sequencing revealed that in rich medium only a few genes, including the gene encoding the flagellar filament protein FlaB3, were differentially expressed in the Δ csrA mutant. In contrast, 575 transcripts were differentially expressed when csrA was overexpressed in L . biflexa . Electrophoretic mobility shift assay (EMSA) confirmed the RNA-seq data in the Δ csrA mutant, showing direct binding of recombinant CsrA to flaB3 mRNA. In the pathogen L . interrogans , we were not able to generate a csrA mutant. We therefore decided to overexpress csrA in L . interrogans . In contrast to the overexpressing strain of L . biflexa , the overexpressing L . interrogans strain had poor motility on soft agar. The overexpressing strain of L . interrogans also showed significant upregulation of the flagellin flaB1 , flaB2 , and flaB4 . The interaction of L . interrogans rCsrA and flaB4 was confirmed by EMSA. Our results demonstrated that CsrA may function as a global regulator in Leptospira spp. under certain conditions that cause csrA overexpression. Interestingly, the mechanisms of action and gene targets of CsrA may be different between non-pathogenic and pathogenic Leptospira strains

Capturing SARS-CoV-2 from patient samples with low viral abundance: a comparative analysis

International audienceSince the beginning of the SARS-CoV-2 coronavirus pandemic, genome sequencing is essential to monitor viral mutations over time and by territory. This need for complete genetic information is further reinforced by the rapid spread of variants of concern. In this paper, we assess the ability of the hybridization technique, Capture-Seq, to detect the SARS-CoV-2 genome, either partially or in its integrity on patients samples. We studied 20 patient nasal swab samples broken down into five series of four samples of equivalent viral load from CT25 to CT36+ . For this, we tested 3 multi-virus panel as well as 2 SARS-CoV-2 only panels. The panels were chosen based on their specificity, global or specific, as well as their technological difference in the composition of the probes: ssRNA, ssDNA and dsDNA. The multi-virus panels are able to capture high-abundance targets but fail to capture the lowest-abundance targets, with a high percentage of off-target reads corresponding to the abundance of the host sequences. Both SARS-CoV-2-only panels were very effective, with high percentage of reads corresponding to the target. Overall, capture followed by sequencing is very effective for the study of SARS-CoV-2 in low-abundance patient samples and is suitable for samples with CT values up to 35

The cell wall lipoprotein CD1687 acts as a DNA binding protein during deoxycholate-induced biofilm formation in Clostridioides difficile

Abstract The ability of bacterial pathogens to establish recurrent and persistent infections is frequently associated with their ability to form biofilms. Clostridioides difficile infections have a high rate of recurrence and relapses and it is hypothesized that biofilms are involved in its pathogenicity and persistence. Biofilm formation by C. difficile is still poorly understood. It has been shown that specific molecules such as deoxycholate (DCA) or metronidazole induce biofilm formation, but the mechanisms involved remain elusive. In this study, we describe the role of the C. difficile lipoprotein CD1687 during DCA-induced biofilm formation. We showed that the expression of CD1687, which is part of an operon within the CD1685-CD1689 gene cluster, is controlled by multiple transcription starting sites and some are induced in response to DCA. Only CD1687 is required for biofilm formation and the overexpression of CD1687 is sufficient to induce biofilm formation. Using RNAseq analysis, we showed that CD1687 affects the expression of transporters and metabolic pathways and we identified several potential binding partners by pull-down assay, including transport-associated extracellular proteins. We then demonstrated that CD1687 is surface exposed in C. difficile, and that this localization is required for DCA-induced biofilm formation. Given this localization and the fact that C. difficile forms eDNA-rich biofilms, we confirmed that CD1687 binds DNA in a non-specific manner. We thus hypothesize that CD1687 is a component of the downstream response to DCA leading to biofilm formation by promoting interaction between the cells and the biofilm matrix by binding eDNA

Broad spectrum compounds targeting early stages of rabies virus (RABV) infection

International audienceABMA and its analogue DABMA are two molecules of the adamantane family known to perturbate the endosomal pathway and to inhibit cell infection of several RNA and DNA viruses. Their activity against Rabies Virus (RABV) infection has already been demonstrated in vitro. (Wu et al., 2017, 2019). Here, we describe in more details their mechanism of action by comparison to Arbidol (umifenovir) and Ribavirin, two broad spectrum antivirals against emerging viruses such as Lassa, Ebola, influenza and Hantaan viruses. ABMA and DABMA, delivered 2 h pre-infection, inhibit RABV infection in vitro with an EC50 of 7.8 μM and 14 μM, respectively. They act at post-entry, by causing RABV accumulation within the endosomal compartment and DABMA specifically diminishes the expression of the GTPase Rab7a controlling the fusion of early endosomes to late endosomes or lysosomes. This may suggest that ABMA and DABMA act at different stages of the late endosomal pathway as supported by their different profile of synergy/antagonism with the fusion inhibitor Arbidol. This difference is further confirmed by the RABV mutants induced by successive passages under increasing selective pressure showing a particular involvement of the viral G protein in the DABMA inhibition while ABMA inhibition induces less mutations dispersed in the M, G and L viral proteins. These results suggest new therapeutic perspectives against rabies

Molecular and epidemiological characterization of recurrent Mycobacterium ulcerans infections in Benin

International audienceBackground: Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans, an environmental mycobacterium. Although transmission of M. ulcerans remains poorly understood, the main identified risk factor for acquiring Buruli ulcer is living in proximity of potentially contaminated water sources. Knowledge about the clinical features of Buruli ulcer and its physiopathology is increasing, but little is known about recurrence due to reinfection.Methodology/principal findings: We describe two patients with Buruli ulcer recurrence due to reinfection with M. ulcerans, as demonstrated by comparisons of DNA from the strains isolated at the time of the first diagnosis and at recurrence. Based on the spatial distribution of M. ulcerans genotypes in this region and a detailed study of the behavior of these two patients with respect to sources of water as well as water bodies and streams, we formulated hypotheses concerning the sites at which they may have been contaminated.Conclusions/significance: Second episodes of Buruli ulcer may occur through reinfection, relapse or a paradoxical reaction. We formally demonstrated that the recurrence in these two patients was due to reinfection. Based on the sites at which the patients reported engaging in activities relating to water, we were able to identify possible sites of contamination. Our findings indicate that the non-random distribution of M. ulcerans genotypes in this region may provide useful information about activities at risk