Biology subjects

Toffano-Nioche, C.

Publications and source records attributed to Toffano-Nioche, C..

6 recordsLinked to original sources

bioRxiv2025

Deciphering the RNA-based regulation mechanism of the phage-encoded AbiF system in Clostridioides difficile

Clostridioides difficile is the major cause of nosocomial infections associated with antibiotic therapy. The severity of C. difficile infections increased worldwide with the emergence of hypervirulent strains, including 027 ribotype epidemic strains. Many aspects of C. difficiles adaptation strategies during pathogenesis remain poorly understood. This pathogen thrives in gut communities that are rich in microbes and phages. To regulate horizontal transfer of genetic material during its infection cycle, C. difficile relies on diverse mechanisms. More specifically, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas and Toxin-Antitoxin (TA) systems contribute to prophage maintenance, prevention of phage infection, and stress response. Abortive infection (Abi) systems can provide additional lines of anti-phage defense. RNAs have emerged as key components of these systems including CRISPR RNAs and antitoxin RNAs within type I and type III TA. We report here the identification of a new AbiF-like system within a prophage of the hypervirulent C. difficile strain R20291. It is associated with an Abi_2/AbiD/F protein family largely distributed in Bacillota and Pseudomonadota with structural links to ancestral Cas13 proteins at the origin of the RNA-targeting CRISPR-Cas13 systems. We demonstrated toxic activity of the AbiFCd protein in C. difficile and in Escherichia coli and negative regulation of the abiFCd expression by an associated non-coding RNA RCd22. RCd22 contains two conserved abiF motifs and is active both in cis and in trans to neutralize the toxin by direct RNA-protein interaction, similar to RNA antitoxin in type III TA. A mass spectrometry interactomics analysis of protein fractions from MS2-Affinity Purification coupled with RNA sequencing (MAPS) revealed the AbiFCd protein among the most enriched RCd22 partners in C. difficile. Structural modeling of the RNA-protein complex and mutagenesis analysis revealed key positions on both protein and RNA partners for this interaction and toxic activity. In summary, these findings provide valuable insights into the mechanisms of interaction between bacteria and phages, which are pertinent to the advancement of phage therapy, genome editing, epidemiological surveillance, and the formulation of novel therapeutic approaches.

microbiology↗

bioRxiv2024

A specialized bacterial group II intron is a highly efficient retrotransposon

Mobile group II introns are site-specific retrotransposons composed of a large self-splicing ribozyme and an intron-encoded reverse transcriptase that are widespread in bacterial and organellar genomes. Sequence and structural variations of the ribozyme and the associated reverse transcriptase define several lineages of bacterial group II introns. Interestingly, some of these intron families evolved different mobility strategies while others colonize particular genetic contexts. Here, we have investigated the mobility activity of an Escherichia coli group II intron that is inserted into the stop codon of the stress-response gene groEL. Using mobility assays based on over-expression from a donor plasmid, we demonstrate that this intron is a highly efficient and site-specific retrotransposon, capable of colonizing the groEL gene of an E. coli host strain according to the insertion pattern observed in natural genomes. Furthermore, we provide evidence that a chromosomal copy of the full-length retrotransposon can be expressed from its native genetic locus to yield mobile retroelement particles. This intron constitutes a novel model system that could help reveal original mobility strategies used by some group II intron retrotransposons to colonize bacterial genomes.

molecular biology↗

bioRxiv2024

Dual RNA-seq study of the dynamics of coding and non-coding RNAs expression during Clostridioides difficile infection in a mouse model

Clostridioides difficile is the leading cause of healthcare associated diarrhoea in industrialized countries. Many questions remain to be answered about the mechanisms governing its interaction with the host during infection. Non-coding RNAs (ncRNAs) contribute to shaping virulence in many pathogens and modulate host responses, however, their role in C. difficile infection (CDI) has not been explored. To better understand the dynamics of ncRNAs expression contributing to C. difficile infectious cycle and host response, we used a dual RNA- seq approach in a conventional murine model. From the pathogen side, this transcriptomic analysis revealed the upregulation of virulence factors, metabolism and sporulation genes, as well as the identification of 61 ncRNAs differentially expressed during infection that correlated with the analysis of available raw RNA-seq datasets from two independent studies. From these data we identified 118 potential new transcripts in C. difficile including 106 new ncRNA genes. From the host side, we observed the induction of several pro-inflammatory pathways and, among the 185 differentially expressed ncRNAs, the overexpression of microRNAs (miRNAs) previously associated to inflammatory responses or unknown long ncRNAs and miRNAs. A particular host gene expression profile could be associated to the symptomatic infection. In accordance, the metatranscriptomic analysis revealed specific microbiota changes accompanying CDI and specific species associated with symptomatic infection in mice. This first adaptation of in vivo dual RNA-seq to C. difficile contributes to unravelling the regulatory networks involved in C. difficile infectious cycle and host response and provides valuable resources for further studies of RNA-based mechanisms during CDI. ImportanceClostridioides difficile is a major cause of nosocomial infections associated with antibiotic therapy classified as an urgent antibiotic resistance threat. This pathogen interacts with host and gut microbial communities during infection, but the mechanisms of these interactions remain largely to be uncovered. Noncoding RNAs contribute to bacterial virulence and host responses, but their expression has not been explored during C. difficile infection. We took advantage of the conventional mouse model of C. difficile infection to look simultaneously to the dynamics of gene expression in pathogen, its host and gut microbiota composition providing valuable resources for future studies. We identified a number of ncRNAs that could mediate the adaptation of C. difficile inside the host and the crosstalk with the host immune response. Promising inflammation markers and potential therapeutic targets emerged from this work open new directions for RNA-based and microbiota-modulatory strategies to improve the efficiency of C. difficile infection treatments.

microbiology↗

bioRxiv2024

Staphylococcal aconitase expression during iron deficiency is controlled by an sRNA-driven feedforward loop and moonlighting activity

Pathogenic bacteria employ complex systems to cope with metal ion shortage conditions and propagate in the host. IsrR is a regulatory RNA (sRNA) whose activity is decisive for optimum S. aureus fitness upon iron starvation and for full virulence. IsrR down-regulates several genes encoding iron-containing enzymes to spare iron for essential processes. Here we report that IsrR regulates the tricarboxylic acid (TCA) cycle by controlling aconitase (CitB), an iron-sulfur cluster-containing enzyme, and its transcriptional regulator, CcpE. This IsrR-dependent dual-regulatory mechanism provides an RNA-driven feedforward loop, underscoring the tight control required to prevent aconitase expression. Beyond its canonical enzymatic role, aconitase becomes an RNA-binding protein with regulatory activity in iron-deprived conditions, a feature that is conserved in S. aureus. Aconitase not only negatively regulates its own expression, but also impacts the enzymes involved in both its substrate supply and product utilization. This moonlighting activity concurrently upregulates pyruvate carboxylase expression, allowing it to compensate for the TCA cycle deficiency associated with iron scarcity. These results highlight the cascade of complex posttranscriptional regulations controlling S. aureus central metabolism in response to iron deficiency.

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↗

bioRxiv2020

Highly reproducible characterization of Escherichia coli tRNA epitranscriptome with a simple method of library preparation for deep sequencing

In conventional RNA high-throughput sequencing, modified bases prevent a large fraction of tRNA transcripts to be converted into cDNA libraries. Recent proposals aiming at resolving this issue take advantage of the interference of base modifications with RT enzymes to detect and identify them by establishing signals from aborted cDNA transcripts. Because some modifications, such as methyl groups, do almost not allow RT bypassing, demethylation and highly processive RT enzymes have been used to overcome these obstacles. Working with Escherichia coli as a model system, we show that with a conventional (albeit still engineered) RT enzyme and key optimizations in library preparation, all RT-impairing modifications can be highlighted along the entire tRNA length without a demethylation procedure. This is achieved by combining deep-sequencing samples, which allows to establish aborted transcription signal of higher accuracy and reproducibility, with the potential for differentiating tiny differences in the state of modification of all cellular tRNAs. In addition, our protocol provides estimates of the relative tRNA abundance.

genomics↗