Biology subjects

Monot, M.

Publications and source records attributed to Monot, M..

15 recordsLinked to original sources

bioRxiv2026

Host adaptation drives genome evolution and virulence diversification in a bacterial zoonotic pathogen

Understanding how zoonotic pathogens diversify across reservoir hosts remains a central question in evolutionary biology and infectious disease research. Here, we address this challenge using Leptospira interrogans, a globally distributed bacterial pathogen with an extremely wide range of animal reservoirs, as a model. After defining 13 distinct genogroups that largely align with serogroups, we selected the strongly host-adapted rodent-associated lineage, which is more frequently associated with fatal outcomes in patients, and the cattle-associated lineage for further analysis. A comprehensive approach integrating multi-omics analyses and host-specific infection assays showed that these two genogroups have followed distinct evolutionary trajectories associated with host specialization. Genomics demonstrated that specialized genogroups are genetically less diverse, characterized by divergence in membrane and signaling genes, and by the acquisition of host-adaptive functions. Changes in gene expression and protein production revealed distinct regulatory programs, predominantly affecting virulence pathways in rodent-borne lineage and stress responses in cattle-borne lineage. Consistently, rodent-borne lineage causes greater disruption of human epithelial barrier integrity and elicits an attenuated host-dependent macrophage inflammatory response relative to cattle-borne lineage. Collectively, these findings reveal distinct host-adaptive strategies and remarkable evolutionary plasticity in a major zoonotic bacterium, highlighting the central role of intraspecies heterogeneity in shaping host specialization.

microbiology↗

bioRxiv2026

High-quality proteins and RNAs extracted from exact same samples for proteomics and RNA-Seq analyses

Back to the 1990 the single step method developed by Chomczynski and Sacchi for RNA isolation was extended for sequential isolation of RNA, DNA and proteins from a same sample. Although the quality of the extracted RNA turned compatible with RNA-Seq analyses, the extraction of the protein fraction from the same sample was time-consuming and resulting in low yield and quality of proteins not compatible with LC-MS proteomic analyses. Here we report a novel procedure by isolating in parallel the protein fraction and the RNA fraction from the same exact minute mosquito samples. We provide evidence that each cognate fractions are compatible with LC-MS proteomic analysis on the one hand and RNA-Seq analysis on the other hand. This protocol is simple, time efficient and adequate for studies involving limited sample size and could be applied easily to a broad range of animal and human samples.

molecular biology↗

bioRxiv2025

Ecological constraints foster both extreme viral-host lineage stability and mobile element diversity in a marine community

Phages are typically viewed as very rapidly evolving biological entities. Little is known, however, about whether and how phages can establish long-term genetic stability. We addressed this eco-evolutionary question in an open marine animal associated system, through two longitudinal samplings years apart in an oyster farm, obtaining >1,000 virulent phages and >600 Vibrio crassostreae strains. Surprisingly, lineages of phage and bacteria were very persistent, with some phages remaining strictly identical after four years. The phage-vibrio infection network remained modular with multiple lineages co-habitating within individual oysters for long periods of time. Seasonal restriction of V. crassostreae, overwintering in wild oysters, and limited viral decay may explain phage stability. Oysters also act as hotspots of activity of diverse mobile genetic element (MGE), hosting plasmids, prophages, phage-plasmids and entirely new classes of satellite-plasmids. Our findings demonstrate how nested ecological constraints can stabilize viral lineages: oysters house vibrio populations that escape host immunity, vibrios restrict phages via receptors and defenses, and satellites parasitize their cognate helper phages. We suggest that ecological context can favor the long-term maintenance of viral lineages and stabilize MGE persistence in the ocean, even amid ongoing mutation and antagonistic co-evolution.

microbiology↗

bioRxiv2025

In Vivo Dual RNA-Seq uncovers key toxin-like effectors of epithelial barrier disruption and tissue colonization by an extracellular bacterial pathogen

Disruption of host cell barriers is a fundamental strategy enabling pathogens to establish a paracellular infection. Using dual RNA-Seq, we determined the in vivo host-pathogen transcriptomic landscape upon infection by the extracellular pathogen Leptospira interrogans and uncovered a novel mechanism of cell-cell junction disruption. We demonstrated that, upon infection, an increase in intracellular calcium triggered tight junction destabilization, by activating the calmodulin and myosin light chain kinase signalization. We identified two novel bacterial effectors of the Virulence-Modifying (VM) proteins family, structurally related to toxin-like proteins, that promoted modulation of calcium homeostasis and disruption of cell-cell junctions, thereby allowing Leptospira translocation across epithelium barriers, tissue colonization and pathogenicity. Furthermore, we demonstrated that at least one of these VM proteins was internalized inside host cells. Altogether, these findings reveal a unique strategy by which an extracellular pathogen secretes toxin-like proteins to exploit host calcium signaling for breaching epithelial barriers.

microbiology↗

bioRxiv2024

Transcriptional landscape of the archaeal cell cycle is broadly conserved in eukaryotes

The cell cycle is a series of events that occur from the moment of cell birth to cell division. In eukaryotes, cell growth, genome replication, genome segregation, and cytokinesis are strictly coordinated, defining discrete cell cycle phases. In contrast, these key processes may occur concurrently in bacteria. Thermoacidophilic archaea in the genus Saccharolobus follow a defined cell cycle program, with the first pre-replicative growth (G1) phase, followed by the chromosome replication (S) phase, the second growth (G2) phase, and rapid genome segregation (M) and cytokinesis (D) phases. However, whether other processes, such as metabolism, catabolism, protein translation, and antiviral defense also occur at specific cell cycle phases, as in eukaryotes, or are active throughout the cell cycle, as in bacteria, remains unclear. To address this question, we synchronized cultures of S. islandicus and performed an in-depth transcriptomic analysis of samples enriched in cells undergoing the M-G1, S, and G2 phases. Differential gene expression and consensus gene co-expression network analyses provided a holistic view of the S. islandicus cell cycle. In addition to the core transcriptome network, which is expressed throughout the cell cycle, we show that diverse metabolic pathways, protein synthesis, cell motility and even antiviral defense systems, are expressed in a cell cycle dependent fashion. Our data also refines understanding of the processes previously known to be linked to the cell cycle, such as DNA replication. We show that most DNA replication genes are expressed prior to the S phase, during the M-G1, whereas expression of the major chromatin genes, and accordingly, chromatinization are concomitant with replication. A statistical model was used to define sets of signature genes characteristic of each of the analyzed cell cycle phases, emphasizing transcriptional stratification of the phases. Signature genes are more conserved across Thermoproteota than non-signature genes and their peak expression, especially for the M-G1 and G2 specific genes, matches that of homologs in yeast. Collectively, our data elucidate the complexity of the S. islandicus cell cycle and suggest that it more closely resembles the cell cycle of eukaryotes than previously appreciated.

microbiology↗

bioRxiv2024

Revisiting oxygen toxicity: evolution and adaptation to superoxide in a SOD-deficient bacterial pathogen

Defenses against oxidants are crucial for the virulence of pathogens, with superoxide scavenging enzymes (SOSEs) playing a vital role for most aerobes. However, our knowledge of superoxide adaptation primarily stems from the study of SOSE-encoding bacteria. Here, we investigated the evolution of a naturally SOSE-deficient pathogen (Leptospira spp.), along with the alternative mechanisms it recruits to combat superoxide stress. We demonstrate that emergence of pathogenic Leptospira correlated with SOD loss, but that a long-lasting adaptation to superoxide remains possible. We reveal that cysteine and leucine biosynthesis are the most induced pathways in response to superoxide and demonstrate the importance of sulfur metabolism in superoxide adaptation in this SOSE-deficient model. We also propose cysteine oxidation as a key mediator of superoxide toxicity in the absence of SOSEs. This study challenges our conventional understanding of the oxygen toxicity theory and proposes a new model of superoxide adaptation through metabolic rewiring in bacteria.

microbiology↗

bioRxiv2023

TALEN-induced contraction of CTG trinucleotide repeats in myotonic dystrophy type 1 cells

Trinucleotide repeat expansions are the cause of two dozen neurodegenerative and developmental disorders. One of these, myotonic dystrophy type 1 (Steinert disease, or DM1) is due to the expansion of a CTG triplet in the 3 UTR of the DMPK gene. We used highly specific DNA endonucleases to induce a double-strand break in the repeat tract to contract it below pathological length. Expression of a TALE Nuclease (TALEN) in human DM1 cells induced moderate CTG repeat contractions in 27% of the clones analyzed. These clones exhibited large internal deletions within the TALEN, occurring by homologous recombination between internal TALE repeats, inactivating the nuclease, and explaining its reduced efficacy. Taking advantage of the degeneracy of the genetic code, we recoded the TALEN sequence, to decrease internal redundancy and optimize codon usage. The new recoded TALEN showed increased efficacy in DM1 cells, with 68% of clones exhibiting a moderate to large contraction of the CTG repeat tract. In contrast, Staphylococcus aureus Cas9 (SaCas9) was unable to contract the CTG repeat tract. In parallel, we completely sequenced to very high coverage the DM1 genome using the PacBio technology. Several clones in which the TALEN was induced were also totally sequenced. In some of them, length changes of other long CTG repeats were detected, possibly corresponding to off-target effects, all of them in introns or intergenic regions. Repeat contractions were never associated with recombination of flanking markers, suggesting that contractions most probably occur by an intra-allelic mechanism such as single-strand annealing. TALENs should now be considered as a promising gene therapy approach, not only for DM1 but also for many other microsatellite expansion disorders.

genomics↗

bioRxiv2023

Spatially-resolved metabolomic identifies colibactin-specific principles of reprogrammed lipid metabolism to promote cancer progression.

Intratumoral bacteria locally contribute to cellular and molecular tumor heterogeneity that support cancer stemness through poorly understood mechanisms. This study aims to explore how Colibactin-producing Escherichia coli (CoPEC) flexibly alters the tumor microenvironment in right-sided colorectal cancer (CRC). Metabolomic and transcriptomic spatial profiling uncovered that CoPEC colonization establishes a high-glycerophospholipid microenvironment within the tumor that is conducive to exhaustion of infiltrated CD8+ T cell and has a lowered prognostic value in right-sided CRC. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress and supply with sufficient energy for sustaining cell survival and lowering tumor immunogenicity. Specifically, a heightened phosphatidylcholine remodeling of CoPEC-infected cancer cells by the enzyme of the Lands cycle coincided with a lowered accumulation of proapoptotic ceramide and lysophosphatidylcholine. Consequently, a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-{gamma} was found where invading bacteria have been geolocated. By contrast, such an immunosuppressive dysmetabolic process was not observed when human colon cancer cells were infected with the mutant strain that did not produce colibactin (11G5{delta}ClbQ). This work revealed an unexpected property of CoPEC on lipid overload within tumors that could locally provide an inflammatory environment leading to immunosuppressive mechanisms and tumor expansion. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.

cancer biology↗

bioRxiv2022

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

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 hypothesised 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.

microbiology↗

bioRxiv2022

Divergence in bacterial ecology is reflected by difference in population genetic structure, phage-predator load and host range

Phages depend on their bacterial host to replicate, but how habitat, density and diversity of the host population drive phage ecology is not well understood. Here, we addressed this question by comparing two populations of marine bacteria and their phages collected during a time series sampling in an oyster farm. Vibrio crassostreae reproduces more specifically in oysters. This population is genetically structured into clades of near clonal strains favoring infection by closely related phages and leading to a modular structure of the phage-bacterial infection network. Vibrio chagasii, on the other hand, blooms in the water column from where it can colonize oysters via filter-feeding. We found higher phage predation pressure on V. chagasii that did not result from a broader host range of the phages but rather from a greater burst size generating more infectious particles in the environment. We showed that contrasting patterns of genetic diversity for host and phage lead to different infection network architectures. We also provided evidence that a bloom of phages generates epigenetic and genetic variability that can be selected to counteract host defense systems.

microbiology↗

bioRxiv2022

Chromosome folding and prophage activation reveal gut-specific genome dynamics of bacteria in the OMM12 consortium

Bacteria and their viruses, bacteriophages, are the most abundant entities of the gut microbiota, a complex community of microorganisms associated with human health and disease. In this ecosystem the interactions between these two key components are still largely unknown. In particular, the impact of the gut environment on bacteria and their associated prophages is yet to be deciphered. To gain insight into the activity of lysogenic phages within the context of their host genomes, we performed Hi-C on the 12 strains of the OMM12 synthetic bacterial community stably associated within mice gut (gnotobiotic mouse line OMM12) in both in vitro and in vivo conditions. High-resolution contact maps of the chromosome 3D organization of the bacterial genomes revealed a wide diversity of architectures, differences between environments and an overall stability over time in the gut of mice. The DNA contacts also pointed at 3D signatures of prophages leading to predict 16 of them as functional. We identified circularization signals and observed different 3D patterns depending on the condition. Concurrent virome analysis showed that 11 of these prophages produced viral particles in vivo and/or in vitro, and that OMM12 mice do not carry other intestinal viruses. By predicting functional prophages, the Hi-C approach unlocks the study of phage-bacteria interaction dynamics.

microbiology↗

bioRxiv2022

Transcriptomic landscapes of SARS-CoV-2-infected and bystander lung cells reveal a selective upregulation of NF-κB-dependent coding and non-coding proviral transcripts

Investigations of cellular responses to viral infection are commonly performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, leading to inaccurate and low-resolution gene expression interpretations. Here, we performed deep polyA+ transcriptome analyses and novel RNA profiling of SARS-CoV-2 infected lung epithelial cells, sorted based on the expression of the viral spike (S) protein. Infection caused a massive reduction in mRNAs and lncRNAs, including transcripts coding for antiviral factors, such as interferons (IFN). This absence of IFN signaling probably explained the poor transcriptomic response of bystander cells co-cultured with S+ ones. NF-{kappa}B pathway and the inflammatory response escaped the global shutoff in S+ cells. Functional investigations revealed the proviral function of the NF-{kappa}B pathway and the antiviral activity of CYLD, a negative regulator of the pathway. Thus, our transcriptomic analysis on sorted cells revealed additional genes that modulate SARS-CoV-2 replication in lung cells.

microbiology↗

bioRxiv2021

Metabolic adaption to extracellular pyruvate triggers biofilm formation in Clostridioides difficile

Clostridioides difficile infections are associated with gut microbiome dysbiosis and are the leading cause of hospital acquired diarrhoea. The infectious process is strongly influenced by the microbiota and successful infection relies on the absence of specific microbiota-produced metabolites. Deoxycholic acid (DOC) and short chain fatty acids are microbiota-produced metabolites that limit the growth of C. difficile and protect the host against this infection. In a previous study, we showed that DOC causes C. difficile to form strongly adherent biofilms after 48 h. Here, our objectives were to identify and characterize key molecules and events required for biofilm formation in the presence of DOC. We applied time-course transcriptomics and genetics to identify sigma factors, metabolic processes and type IV pili that drive biofilm formation. These analyses revealed that extracellular pyruvate induces biofilm formation in the presence of DOC. In the absence of DOC, pyruvate supplementation was sufficient to induce biofilm formation in a process that was dependent on pyruvate uptake by the membrane protein CstA. In the context of the human gut, microbiota-generated pyruvate is a metabolite that limits pathogen colonization. Taken together our results suggest that pyruvate-induced biofilm formation might act as a key process driving C. difficile persistence in the gut.

microbiology↗

bioRxiv2020

Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.

microbiology↗

bioRxiv2017

PhageTerm: a Fast and User-friendly Software to Determine Bacteriophage Termini and Packaging Mode using randomly fragmented NGS data

Bacteriophages are the most abundant viruses on earth and display an impressive genetic as well as morphologic diversity. Among those, the most common order of phages is the Caudovirales, whose viral particles packages linear double stranded DNA (dsDNA). In this study we investigated how the information gathered by high throughput sequencing technologies can be used to determine the DNA termini and packaging mechanisms of dsDNA phages. The wet-lab procedures traditionally used for this purpose rely on the identification and cloning of restriction fragment which can be delicate and cumbersome. Here, we developed a theoretical and statistical framework to analyze DNA termini and phage packaging mechanisms using next-generation sequencing data. Our methods, implemented in the PhageTerm software, work with sequencing reads in fastq format and the corresponding assembled phage genome.\n\nPhageTerm was validated on a set of phages with well-established packaging mechanisms representative of the termini diversity: 5cos (lambda), 3cos (HK97), pac (P1), headful without a pac site (T4), DTR (T7) and host fragment (Mu). In addition, we determined the termini of 9 Clostridium difficile phages and 6 phages whose sequences where retrieved from the sequence read archive (SRA).\n\nA direct graphical interface is available as a Galaxy wrapper version at https://galaxy.pasteur.fr and a standalone version is accessible at https://sourceforge.net/projects/phageterm/.

microbiology↗