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Accueil > Départements > Microbiologie > Olga SOUTOURINA : ARNs régulateurs chez les Clostridies



  • A. Maikova, V. Kreis, A. Boutserin, K. Severinov, et O. Soutourina, « Using endogenous CRISPR-Cas system for genome editing in the human pathogen Clostridium difficile », Applied and Environmental Microbiology, août 2019.
    Résumé : Human enteropathogen Clostridium difficile constitutes a key public health issue in industrialized countries. Many aspects of C. difficile pathophysiology and adaptation inside the host remain poorly understood. We have recently reported that this bacterium possesses an active CRISPR-Cas system of subtype I-B for defence against phages and other mobile genetic elements that could contribute to its success during infection. In this paper, we demonstrate that redirecting this endogenous CRISPR-Cas system towards autoimmunity allows efficient genome editing in C. difficile We provide detailed description of this newly developed approach and show, as a proof of principle, its efficient application for deletion of a specific gene in reference 630Δerm and in epidemic R20291 C. difficile strains. The new method expands the arsenal of the currently limiting set of gene engineering tools available for investigation of C. difficile and may serve as the basis for new strategies to control C. difficile infections.ImportanceClostridium difficile represents today a real danger for human and animal health. It is the leading cause of diarrhoea associated with healthcare in adults in industrialized countries. The incidence of these infections continues to increase and this trend is accentuated by the general aging of the population. Many questions remain unanswered on the mechanisms contributing to C. difficile success inside the host. The set of genetic tools available for this pathogen is limited and new developments are badly needed. C. difficile has developed efficient defence systems that are directed against foreign DNA and could contribute to its survival in phage-rich gut communities. We show how one of such defence systems, named CRISPR-Cas, can be hijacked for C. difficile genome editing. Our results also show a great potential of CRISPR-Cas system for development of new therapeutic strategies against C. difficile infections.
    Mots-clés : ARNCLO, MICROBIO.

  • O. Soutourina, « Type I Toxin-Antitoxin Systems in Clostridia », Toxins, vol. 11, nᵒ 5, mai 2019.
    Résumé : Type I toxin-antitoxin (TA) modules are abundant in both bacterial plasmids and chromosomes and usually encode a small hydrophobic toxic protein and an antisense RNA acting as an antitoxin. The RNA antitoxin neutralizes toxin mRNA by inhibiting its translation and/or promoting its degradation. This review summarizes our current knowledge of the type I TA modules identified in Clostridia species focusing on the recent findings in the human pathogen Clostridium difficile. More than ten functional type I TA modules have been identified in the genome of this emerging enteropathogen that could potentially contribute to its fitness and success inside the host. Despite the absence of sequence homology, the comparison of these newly identified type I TA modules with previously studied systems in other Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, revealed some important common traits. These include the conservation of characteristic sequence features for small hydrophobic toxic proteins, the localization of several type I TA within prophage or prophage-like regions and strong connections with stress response. Potential functions in the stabilization of genome regions, adaptations to stress conditions and interactions with CRISPR-Cas defence system, as well as promising applications of TA for genome-editing and antimicrobial developments are discussed.
    Mots-clés : ARNCLO, Clostridium difficile, MICROBIO, prophages, RNA antitoxins, toxin-antitoxin systems, type I.


  • J. R. Garneau, O. Sekulovic, B. Dupuy, O. Soutourina, M. Monot, et L. - C. Fortier, « High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile », Applied and Environmental Microbiology, vol. 84, nᵒ 3, 2018.
    Résumé : Clostridioides difficile (formerly Clostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in C. difficile so far, with a genome of 131 kbp. It shares morphological and genomic similarity with other large siphophages, like phage 949, infecting Lactococcus lactis, and phage c-st, infecting Clostridium botulinum A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a cas3 gene. We screened 2,584 C. difficile genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in C. difficile strains corresponding to 21 different multilocus sequence type groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.IMPORTANCEClostridioides difficile is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could explain the apparent differences observed in the virulence of different C. difficile strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect C. difficile so far. Screening 2,584 C. difficile genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple-genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could influence bacterial biology and fitness. Our study further supports the relevance of studying phages in C. difficile to better understand the epidemiology of this clinically important human pathogen.
    Mots-clés : ARNCLO, bacteriophage, Clostridioides difficile, Clostridium difficile, DNA, Viral, Genetic Fitness, Genetic Variation, Genome, Bacterial, Genome, Viral, Genomics, Humans, MICROBIO, Multilocus Sequence Typing, phiCD211, phiCDIF1296T, Prevalence, prophage genomics, Prophages, Sequence Analysis, DNA, Virulence.

  • A. Maikova, J. Peltier, P. Boudry, E. Hajnsdorf, N. Kint, M. Monot, I. Poquet, I. Martin-Verstraete, B. Dupuy, et O. Soutourina, « Discovery of new type I toxin-antitoxin systems adjacent to CRISPR arrays in Clostridium difficile », Nucleic Acids Research, vol. 46, nᵒ 9, p. 4733-4751, mai 2018.
    Résumé : Clostridium difficile, a major human enteropathogen, must cope with foreign DNA invaders and multiple stress factors inside the host. We have recently provided an experimental evidence of defensive function of the C. difficile CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system important for its survival within phage-rich gut communities. Here, we describe the identification of type I toxin-antitoxin (TA) systems with the first functional antisense RNAs in this pathogen. Through the analysis of deep-sequencing data, we demonstrate the general co-localization with CRISPR arrays for the majority of sequenced C. difficile strains. We provide a detailed characterization of the overlapping convergent transcripts for three selected TA pairs. The toxic nature of small membrane proteins is demonstrated by the growth arrest induced by their overexpression. The co-expression of antisense RNA acting as an antitoxin prevented this growth defect. Co-regulation of CRISPR-Cas and type I TA genes by the general stress response Sigma B and biofilm-related factors further suggests a possible link between these systems with a role in recurrent C. difficile infections. Our results provide the first description of genomic links between CRISPR and type I TA systems within defense islands in line with recently emerged concept of functional coupling of immunity and cell dormancy systems in prokaryotes.
    Mots-clés : ARNCLO, MICROBIO.

  • A. Maikova, K. Severinov, et O. Soutourina, « New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System », Frontiers in Microbiology, vol. 9, p. 1740, 2018.
    Résumé : Over the last decades the enteric bacterium Clostridium difficile (novel name Clostridioides difficile) - has emerged as an important human nosocomial pathogen. It is a leading cause of hospital-acquired diarrhea and represents a major challenge for healthcare providers. Many aspects of C. difficile pathogenesis and its evolution remain poorly understood. Efficient defense systems against phages and other genetic elements could have contributed to the success of this enteropathogen in the phage-rich gut communities. Recent studies demonstrated the presence of an active CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) subtype I-B system in C. difficile. In this mini-review, we will discuss the recent advances in characterization of original features of the C. difficile CRISPR-Cas system in laboratory and clinical strains, as well as interesting perspectives for our understanding of this defense system function and regulation in this important enteropathogen. This knowledge will pave the way for the development of promising biotechnological and therapeutic tools in the future. Possible applications for the C. difficile strain monitoring and genotyping, as well as for CRISPR-based genome editing and antimicrobials are also discussed.
    Mots-clés : antimicrobials, ARNCLO, C. difficile, CRISPR, CRISPR regulation, Genome editing, I-B subtype CRISPR-Cas system, MICROBIO.
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  • I. Poquet, L. Saujet, A. Canette, M. Monot, J. Mihajlovic, J. - M. Ghigo, O. Soutourina, R. Briandet, I. Martin-Verstraete, et B. Dupuy, « Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture », Frontiers in Microbiology, vol. 9, p. 2084, 2018.
    Résumé : Clostridium difficile is an opportunistic entero-pathogen causing post-antibiotic and nosocomial diarrhea upon microbiota dysbiosis. Although biofilms could contribute to colonization, little is known about their development and physiology. Strain 630Δerm is able to form, in continuous-flow micro-fermentors, macro-colonies and submersed biofilms loosely adhesive to glass. According to gene expression data, in biofilm/planktonic cells, central metabolism is active and fuels fatty acid biosynthesis rather than fermentations. Consistently, succinate is consumed and butyrate production is reduced. Toxin A expression, which is coordinated to metabolism, is down-regulated, while surface proteins, like adhesins and the primary Type IV pili subunits, are over-expressed. C-di-GMP level is probably tightly controlled through the expression of both diguanylate cyclase-encoding genes, like dccA, and phosphodiesterase-encoding genes. The coordinated expression of genes controlled by c-di-GMP and encoding the putative surface adhesin CD2831 and the major Type IV pilin PilA1, suggests that c-di-GMP could be high in biofilm cells. A Bacillus subtilis SinR-like regulator, CD2214, and/or CD2215, another regulator co-encoded in the same operon as CD2214, control many genes differentially expressed in biofilm, and in particular dccA, CD2831 and pilA1 in a positive way. After growth in micro-titer plates and disruption, the biofilm is composed of robust aggregated structures where cells are embedded into a polymorphic material. The intact biofilm observed in situ displays a sparse, heterogeneous and high 3D architecture made of rods and micro-aggregates. The biofilm is denser in a mutant of both CD2214 and CD2215 genes, but it is not affected by the inactivation of neither CD2831 nor pilA1 . dccA, when over-expressed, not only increases the biofilm but also triggers its architecture to become homogeneous and highly aggregated, in a way independent of CD2831 and barely dependent of pilA1 . Cell micro-aggregation is shown to play a major role in biofilm formation and architecture. This thorough analysis of gene expression reprogramming and architecture remodeling in biofilm lays the foundation for a deeper understanding of this lifestyle and could lead to novel strategies to limit C. difficile spread.
    Mots-clés : acid, aggregates, ARNCLO, bacillus-subtilis, bacteria, biofilm architecture, biofilm formation, Clostridium difficile, dependent regulation, gene expression profiling, gene-expression, infection, iv pili, MICROBIO, sporulation, staphylococcus-aureus, virulence.


  • N. Kint, C. Janoir, M. Monot, S. Hoys, O. Soutourina, B. Dupuy, et I. Martin-Verstraete, « The alternative sigma factor σ(B) plays a crucial role in adaptive strategies of Clostridium difficile during gut infection », Environmental Microbiology, févr. 2017.
    Résumé : Clostridium difficile is a major cause of diarrhoea associated with antibiotherapy. Exposed to stresses in the gut, C. difficile can survive by inducing protection, detoxification and repair systems. In several firmicutes, most of these systems are controlled by the general stress response involving σ(B) . In this work, we studied the role of σ(B) in the physiopathology of C. difficile. We showed that the survival of the sigB mutant during the stationary phase was reduced. Using a transcriptome analysis, we showed that σ(B) controls the expression of ∼25% of genes including genes involved in sporulation, metabolism, cell surface biogenesis and the management of stresses. By contrast, σ(B) does not control toxin gene expression. In agreement with the up-regulation of sporulation genes, the sporulation efficiency is higher in the sigB mutant than in the wild-type strain. sigB inactivation also led to increased sensitivity to acidification, cationic antimicrobial peptides, nitric oxide and ROS. In addition, we showed for the first time that σ(B) also plays a crucial role in oxygen tolerance in this strict anaerobe. Finally, we demonstrated that the fitness of colonisation by the sigB mutant is greatly affected in a dixenic mouse model of colonisation when compared to the wild-type strain.
    Mots-clés : ARNCLO, MICROBIO.

  • J. Peltier et O. Soutourina, « Identification of c-di-GMP-Responsive Riboswitches », Methods in Molecular Biology (Clifton, N.J.), vol. 1657, p. 377-402, 2017.
    Résumé : Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important signaling molecule for community behavior control, cell morphogenesis, and virulence in bacteria. In addition to protein effectors, this second messenger binds RNA molecules that act as riboswitches to control target gene expression. In this chapter, we describe a method for experimental validation of the functionality of c-di-GMP-responsive riboswitches and the analysis of c-di-GMP control of target gene expression by qRT-PCR and Northern blot. This procedure can be used for the studies of in silico-predicted riboswitch candidates, as well as a targeted experimental approach for exploring the data from next-generation sequencing. The examples on the analysis of type I and type II c-di-GMP-responsive riboswitches in Clostridium difficile are provided to illustrate the application of the method.
    Mots-clés : ARNCLO, c-di-GMP, MICROBIO, Northern blot, Premature termination of transcription, qRT-PCR, Riboswitches, Target gene.

  • O. Soutourina, « RNA-based control mechanisms of Clostridium difficile », Current Opinion in Microbiology, vol. 36, p. 62-68, févr. 2017.
    Résumé : Clostridium difficile (CD)-associated diarrhoea is currently the most prevalent nosocomial diarrhoea worldwide. Many characteristics of CD pathogenicity remain poorly understood. Recent data strongly indicate the importance of an RNA network for the control of gene expression in CD. More than 200 regulatory RNAs have been identified by deep sequencing and targeted approaches, including Hfq-dependent trans riboregulators, cis-antisense RNAs, CRISPR RNAs, and c-di-GMP-responsive riboswitches. These regulatory RNAs are involved in the control of major processes in the CD infection cycle, for example motility, biofilm formation, adhesion, sporulation, stress response, and defence against bacteriophages. We will discuss recent advances in elucidation of the original features of RNA-based mechanisms in this important enteropathogen. This knowledge may pave the way for further discoveries in this emergent field.
    Mots-clés : ARNCLO, MICROBIO.
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Selection of publications before 2017

- Dubois T, Dancer-Thibonnier M, Monot M, Hamiot A, Bouillaut L, Soutourina O, Martin-Verstraete I, and Dupuy B. Control of Clostridium difficile physiopathology in response to cysteine availability. (2016). Infection and immunity 84(8) : 2389-2405.

- Boudry P, Semenova E, Monot M, Datsenko K, Lopatina A, Sekulovic O, Ospina-Bedoya M, Fortier L-C, Severinov K, Dupuy B, Soutourina O.* Function of the CRISPR-Cas system of the human pathogen Clostridium difficile. (2015). mBio 6(5) : e01112-15.

- Rosinski-Chupin I.§, Soutourina O§, Martin-Verstraete I. Riboswitch discovery by combining RNA-seq and Genome-Wide identification of transcriptional start sites. (2014). Methods in Enzymology. Vol. 549 : 3-27. §Equal contribution.

- Boudry P, Gracia C, Monot M, Caillet J, Saujet L, Hajnsdorf E, Dupuy B, Martin-Verstraete I, Soutourina O.* Pleiotropic Role of the RNA Chaperone Protein Hfq in the Human Pathogen Clostridium difficile. (2014). J Bacteriol. 196(18):3234-48.

- El Meouche I., Peltier J., Monot M., Soutourina O., Pestel-Caron M., Dupuy B., Pons J-L. Characterization of the SigD regulon of C. difficile and its positive control of toxin production through the regulation of tcdR. (2013). PLoS One. 8(12) : e83748.

- Saujet L., Pereira F.C., Soutourina O., Shelyakin P., Monot M., Gelfand M., Dupuy B., Henriques A. O. and Martin-Verstraete I. Genome-wide Analysis of Cell type-specific Gene Transcription during Spore Formation in Clostridium difficile. (2013). PLoS Genetics. 9(10) : e1003756.

- Soutourina O.*, Monot, M., Boudry, P., Saujet, L., Pichon, C., Sismeiro, O., Semenova, E., Severinov, K., Le Bouguenec, C., Coppée, J.-Y., Dupuy, B. and Martin-Verstraete, I. Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile. (2013). PLoS Genetics. 9(5) :e1003493.

- Shepard W, Soutourina O, Courtois E, England P, Haouz A, Martin-Verstraete I. Insights into the Rrf2 Repressor Family : The Structure of CymR, the Global Cysteine Regulator of Bacillus subtilis. (2011). FEBS J. 278(15):2689-701.

- Saujet L, Monot M, Dupuy B, Soutourina O, Martin-Verstraete I. The key sigma factor of transition phase, SigH, controls sporulation, metabolism and virulence factor expression in Clostridium difficile. (2011). Journal of Bacteriology. 193(13) : 3186-3196.

- Soutourina, O.*, Dubrac, S., Poupel, O., Msadek, T. and Martin-Verstraete, I. The Pleiotropic CymR Regulator of Staphylococcus aureus Plays an Important Role in Virulence and Stress Response. (2010). PLoS Pathogens. 6(5) :e1000894.

- Soutourina, O.*, Poupel, O., Coppée, JY., Danchin, A., Msadek, T. and Martin-Verstraete I. CymR, the master regulator of cysteine metabolism in Staphylococcus aureus, controls human sulphur sources utilization and plays a role in biofilm formation (2009). Mol Microbiol. 73(2) :194-211.

- André, G., Even, S., Putzer, H., Burguière, P., Danchin, A., Martin-Verstraete, I. and Soutourina, O*. S-box and T-box riboswitches and antisense RNA control a sulfur metabolic operon of Clostridium acetobutylicum. (2008). Nucleic Acids Res. 36(18):5955-69. 69 citations.

* corresponding author

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