Biology subjects

Soutourina, O.

Publications and source records attributed to Soutourina, O..

6 recordsLinked to original sources

bioRxiv2026

Structural analysis reveals three families of bacterial tRNA mimics

Comparative genomics has identified over 1,000 candidate structured RNA motifs, yet most remain functionally uncharacterized. Here, we combine crystallography, comparative analysis, and bacterial genetics to identify three families of bacterial tRNA-like RNAs. The skipping-rope RNA adopts an L-shaped fold resembling tRNA despite distinct secondary-structure connectivity and tertiary interactions. In Clostridioides difficile, the skipping-rope RNA RCd1 is abundantly expressed and processed into a highly stable RNA species. RCd1 overexpression suppresses sporulation, whereas its deletion enhances early spore formation, supporting a role in sporulation control. Guided by the skipping-rope structure, we identify a conserved architectural core and use it to recognize additional candidate tRNA mimics. Crystal structures of DUF2693-FD and salivarius-1 RNAs confirm a shared tRNA-like scaffold assembled through distinct interaction networks. Together, these findings expand the repertoire of bacterial tRNA mimics and establish a structural framework for recognizing shared architectures among uncharacterized ncRNAs, linking one such family to bacterial developmental regulation.

biochemistry↗

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

The L,D-transpeptidation pathway is inhibited by antibiotics of the β-lactam class in Clostridioides difficile

The resistance of Clostridioides difficile to the {beta}-lactam antibiotics cephalosporins, which target the peptidoglycan (PG) assembly, is a leading contributor to the development of C. difficile infections. C. difficile has an original PG structure with a predominance of 3[->]3 cross-links generated by L,D-transpeptidases (LDTs). C. difficile forms spores and we show that the spore cortex PG contains exclusively 3[->]3 cross-links. PG and spore cortex of C. difficile cells were largely unaffected by the deletion of the three predicted LDTs, revealing the implication of a new family of LDTS. The D,D-carboxypeptidases producing the essential LDT substrate were inactivated by cephalosporins, resulting in the inhibition of the L,D-transpeptidation pathway. In contrast, the participation of penicillin-binding proteins (PBPs) to PG cross-linking increased in the presence of the antibiotics. Our findings highlight that cephalosporin resistance is not primarily mediated by LDTs and illustrate the plasticity of the PG biosynthesis machinery in C. difficile.

microbiology↗

bioRxiv2022

The Clostridioides difficile S-Layer Protein A (SlpA) serves as a general phage receptor

Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, but the lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but clear and unambiguous experimental evidence is lacking. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by phiCD38-2, phiCDIII and phiCD146, which normally infect the parental strain. Complementation assays with 12 different Slayer Cassette Types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by phiCD111, and SLCT-11 enabled infection by phiCD38-2 and phiCD146. Of note, expression of SLCTs 1, 6, 8, 9, 10 or 12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain, namely phiMMP02, phiMMP03, phiMMP04, phiCD506 and phiCD508. Adsorption assays showed that >50% adsorption was required for productive phage infection. Altogether, our data suggest that many phages use SlpA as their receptor and most importantly, morphologically distinct phages of the Siphoviridae and Myoviridae families target SlpA despite major differences in their tail structures. Our study therefore represents an important breakthrough in our understanding of the molecular interaction between C. difficile and its phages. IMPORTANCEPhage therapy represents an interesting alternative to treat Clostridioides difficile infections because contrary to antibiotics, most phages are highly species-specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage RBP binds to a specific SLCT, identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.

microbiology↗

bioRxiv2020

Predictive regulatory and metabolic network models for systems analysis of Clostridioides difficile

Though Clostridioides difficile is among the most studied anaerobes, the interplay of metabolism and regulation that underlies its ability to colonize the human gut is unknown. We have compiled public resources into three models and a portal to support comprehensive systems analysis of C. difficile. First, by leveraging 151 transcriptomes from 11 studies we generated a regulatory model (EGRIN) that organizes 90% of C. difficile genes into 297 high quality conditional co-regulation modules. EGRIN predictions, validated with independent datasets, recapitulated and extended regulons of key transcription factors, implicating new genes for sporulation, carbohydrate transport and metabolism. Second, by advancing a metabolic model, we discovered that 15 amino acids, diverse carbohydrates, and 10 metabolic genes are essential for C. difficile growth within an intestinal environment. Finally, by integrating EGRIN with the metabolic model, we developed a PRIME model that revealed unprecedented insights into combinatorial control of essential processes for in vivo colonization of C. difficile and its interactions with commensals. We have developed an interactive web portal (http://networks.systemsbiology.net/cdiff-portal/) to disseminate all data, algorithms, and models to support collaborative systems analyses of C. difficile.

systems biology↗

bioRxiv2020

Type I toxin-antitoxin systems contribute to mobile genetic elements maintenance in Clostridioides difficile and can be used as a counter-selectable marker for chromosomal manipulation

Toxin-antitoxin (TA) systems are widespread on mobile genetic elements as well as in bacterial chromosomes. According to the nature of the antitoxin and its mode of action for toxin inhibition, TA systems are subdivided into different types. The first type I TA modules were recently identified in the human enteropathogen Clostridioides (formerly Clostridium) difficile. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA during normal growth. Here, we report the characterization of five additional type I TA systems present within phiCD630-1 and phiCD630-2 prophage regions of C. difficile 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest. Growth is restored when the antitoxin RNAs, transcribed from the opposite strand, are co-expressed together with the toxin genes. In addition, we show that type I TA modules located within the phiCD630-1 prophage contribute to its stability and mediate phiCD630-1 heritability. Type I TA systems were found to be widespread in genomes of C. difficile phages, further suggesting their functional importance. We have made use of a toxin gene from one of type I TA modules of C. difficile as a counter-selectable marker to generate an efficient mutagenesis tool for this bacterium. This tool enabled us to delete all identified toxin genes within the phiCD630-1 prophage, thus allowing investigation of the role of TA in prophage maintenance. Furthermore, we were able to delete the large 49 kb phiCD630-2 prophage region using this improved procedure.

microbiology↗