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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.
Pierre Boudry; Emma Piattelli; Emilie Drouineau; Johann Peltier; Anaïs Boutserin; Maxence Lejars; Eliane Hajnsdorf; Marc Monot; Bruno Dupuy; Isabelle Martin-Verstraete; Daniel Gautheret; Claire Toffano-Nioche; Olga Soutourina. Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile. RNA Biology 2021, 1 -22.
AMA StylePierre Boudry, Emma Piattelli, Emilie Drouineau, Johann Peltier, Anaïs Boutserin, Maxence Lejars, Eliane Hajnsdorf, Marc Monot, Bruno Dupuy, Isabelle Martin-Verstraete, Daniel Gautheret, Claire Toffano-Nioche, Olga Soutourina. Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile. RNA Biology. 2021; ():1-22.
Chicago/Turabian StylePierre Boudry; Emma Piattelli; Emilie Drouineau; Johann Peltier; Anaïs Boutserin; Maxence Lejars; Eliane Hajnsdorf; Marc Monot; Bruno Dupuy; Isabelle Martin-Verstraete; Daniel Gautheret; Claire Toffano-Nioche; Olga Soutourina. 2021. "Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile." RNA Biology , no. : 1-22.
Toxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosomes. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA. The first type I TA were recently identified in the human enteropathogen Clostridioides difficile. Here we report the characterization of five additional type I TA within phiCD630-1 (CD0977.1-RCd11, CD0904.1-RCd13 and CD0956.3-RCd14) and phiCD630-2 (CD2889-RCd12 and CD2907.2-RCd15) prophages of C. difficile strain 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest that is neutralized by antitoxin RNA co-expression. We show that type I TA located within the phiCD630-1 prophage contribute to its stability and heritability. We have made use of a type I TA toxin gene to generate an efficient mutagenesis tool for this bacterium that allowed investigation of the role of these widespread TA in prophage maintenance.
Johann Peltier; Audrey Hamiot; Julian R. Garneau; Pierre Boudry; Anna Maikova; Eliane Hajnsdorf; Louis-Charles Fortier; Bruno Dupuy; Olga Soutourina. Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile. Communications Biology 2020, 3, 1 -13.
AMA StyleJohann Peltier, Audrey Hamiot, Julian R. Garneau, Pierre Boudry, Anna Maikova, Eliane Hajnsdorf, Louis-Charles Fortier, Bruno Dupuy, Olga Soutourina. Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile. Communications Biology. 2020; 3 (1):1-13.
Chicago/Turabian StyleJohann Peltier; Audrey Hamiot; Julian R. Garneau; Pierre Boudry; Anna Maikova; Eliane Hajnsdorf; Louis-Charles Fortier; Bruno Dupuy; Olga Soutourina. 2020. "Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile." Communications Biology 3, no. 1: 1-13.
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.
Pierre Boudry; Emma Piattelli; Emilie Drouineau; Johann Peltier; Anaïs Boutserin; Maxence Lejars; Eliane Hajnsdorf; Marc Monot; Bruno Dupuy; Isabelle Martin-Verstraete; Daniel Gautheret; Claire Toffano-Nioche; Olga Soutourina. Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile. 2020, 1 .
AMA StylePierre Boudry, Emma Piattelli, Emilie Drouineau, Johann Peltier, Anaïs Boutserin, Maxence Lejars, Eliane Hajnsdorf, Marc Monot, Bruno Dupuy, Isabelle Martin-Verstraete, Daniel Gautheret, Claire Toffano-Nioche, Olga Soutourina. Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile. . 2020; ():1.
Chicago/Turabian StylePierre Boudry; Emma Piattelli; Emilie Drouineau; Johann Peltier; Anaïs Boutserin; Maxence Lejars; Eliane Hajnsdorf; Marc Monot; Bruno Dupuy; Isabelle Martin-Verstraete; Daniel Gautheret; Claire Toffano-Nioche; Olga Soutourina. 2020. "Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile." , no. : 1.
The ability of pathogenic bacteria to stably infect the host depends on their capacity to respond and adapt to the host environment and on the efficiency of their defensive mechanisms. Bacterial envelope provides a physical barrier protecting against environmental threats. It also constitutes an important sensory interface where numerous sensing systems are located. Signal transduction systems include Two-Component Systems (TCSs) and alternative sigma factors. These systems are able to sense and respond to the ever-changing environment inside the host, altering the bacterial transcriptome to mitigate the impact of the stress. The regulatory networks associated with signal transduction systems comprise small regulatory RNAs (sRNAs) that can be directly involved in the expression of virulence factors. The aim of this review is to describe the importance of TCS- and alternative sigma factor-associated sRNAs in human pathogens during infection. The currently available genome-wide approaches for studies of TCS-regulated sRNAs will be discussed. The differences in the signal transduction mediated by TCSs between bacteria and higher eukaryotes and the specificity of regulatory RNAs for their targets make them appealing targets for discovery of new strategies to fight against multi-resistant bacteria.
Emma Piattelli; Johann Peltier; Olga Soutourina. Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection. Genes 2020, 11, 1209 .
AMA StyleEmma Piattelli, Johann Peltier, Olga Soutourina. Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection. Genes. 2020; 11 (10):1209.
Chicago/Turabian StyleEmma Piattelli; Johann Peltier; Olga Soutourina. 2020. "Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection." Genes 11, no. 10: 1209.
The emerging human enteropathogen Clostridioides difficile is the main cause of diarrhea associated with antibiotherapy. Regulatory pathways underlying the adaptive responses remain understudied and the global view of C. difficile promoter structure is still missing. In the genome of C. difficile 630, 22 genes encoding sigma factors are present suggesting a complex pattern of transcription in this bacterium. We present here the first transcriptional map of the C. difficile genome resulting from the identification of transcriptional start sites (TSS), promoter motifs and operon structures. By 5′-end RNA-seq approach, we mapped more than 1000 TSS upstream of genes. In addition to these primary TSS, this analysis revealed complex structure of transcriptional units such as alternative and internal promoters, potential RNA processing events and 5′ untranslated regions. By following an in silico iterative strategy that used as an input previously published consensus sequences and transcriptomic analysis, we identified candidate promoters upstream of most of protein-coding and non-coding RNAs genes. This strategy also led to refine consensus sequences of promoters recognized by major sigma factors of C. difficile. Detailed analysis focuses on the transcription in the pathogenicity locus and regulatory genes, as well as regulons of transition phase and sporulation sigma factors as important components of C. difficile regulatory network governing toxin gene expression and spore formation. Among the still uncharacterized regulons of the major sigma factors of C. difficile, we defined the SigL regulon by combining transcriptome and in silico analyses. We showed that the SigL regulon is largely involved in amino-acid degradation, a metabolism crucial for C. difficile gut colonization. Finally, we combined our TSS mapping, in silico identification of promoters and RNA-seq data to improve gene annotation and to suggest operon organization in C. difficile. These data will considerably improve our knowledge of global regulatory circuits controlling gene expression in C. difficile and will serve as a useful rich resource for scientific community both for the detailed analysis of specific genes and systems biology studies.
Olga Soutourina; Thomas Dubois; Marc Monot; Pavel V. Shelyakin; Laure Saujet; Pierre Boudry; Mikhail S. Gelfand; Bruno Dupuy; Isabelle Martin-Verstraete. Genome-Wide Transcription Start Site Mapping and Promoter Assignments to a Sigma Factor in the Human Enteropathogen Clostridioides difficile. Frontiers in Microbiology 2020, 11, 1939 .
AMA StyleOlga Soutourina, Thomas Dubois, Marc Monot, Pavel V. Shelyakin, Laure Saujet, Pierre Boudry, Mikhail S. Gelfand, Bruno Dupuy, Isabelle Martin-Verstraete. Genome-Wide Transcription Start Site Mapping and Promoter Assignments to a Sigma Factor in the Human Enteropathogen Clostridioides difficile. Frontiers in Microbiology. 2020; 11 ():1939.
Chicago/Turabian StyleOlga Soutourina; Thomas Dubois; Marc Monot; Pavel V. Shelyakin; Laure Saujet; Pierre Boudry; Mikhail S. Gelfand; Bruno Dupuy; Isabelle Martin-Verstraete. 2020. "Genome-Wide Transcription Start Site Mapping and Promoter Assignments to a Sigma Factor in the Human Enteropathogen Clostridioides difficile." Frontiers in Microbiology 11, no. : 1939.
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.
Johann Peltier; Audrey Hamiot; Julian R. Garneau; Pierre Boudry; Anna Maikova; Louis-Charles Fortier; Bruno Dupuy; Olga Soutourina. 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. 2020, 1 .
AMA StyleJohann Peltier, Audrey Hamiot, Julian R. Garneau, Pierre Boudry, Anna Maikova, Louis-Charles Fortier, Bruno Dupuy, Olga Soutourina. 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. . 2020; ():1.
Chicago/Turabian StyleJohann Peltier; Audrey Hamiot; Julian R. Garneau; Pierre Boudry; Anna Maikova; Louis-Charles Fortier; Bruno Dupuy; Olga Soutourina. 2020. "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." , no. : 1.
Clostridium difficile represents today a real danger for human and animal health. It is the leading cause of diarrhea associated with health care 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 about the mechanisms contributing to C. difficile 's success inside the host remain unanswered. The set of genetic tools available for this pathogen is limited, and new developments are badly needed. C. difficile has developed efficient defense systems that are directed against foreign DNA and that could contribute to its survival in phage-rich gut communities. We show how one such defense system, named CRISPR-Cas, can be hijacked for C. difficile genome editing. Our results also show a great potential for the use of the CRISPR-Cas system for the development of new therapeutic strategies against C. difficile infections.
Anna Maikova; Victor Kreis; Anaïs Boutserin; Konstantin Severinov; Olga Soutourina. Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile. Applied and Environmental Microbiology 2019, 85, 1 .
AMA StyleAnna Maikova, Victor Kreis, Anaïs Boutserin, Konstantin Severinov, Olga Soutourina. Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile. Applied and Environmental Microbiology. 2019; 85 (20):1.
Chicago/Turabian StyleAnna Maikova; Victor Kreis; Anaïs Boutserin; Konstantin Severinov; Olga Soutourina. 2019. "Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile." Applied and Environmental Microbiology 85, no. 20: 1.
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.
Olga Soutourina. Type I Toxin-Antitoxin Systems in Clostridia. Toxins 2019, 11, 253 .
AMA StyleOlga Soutourina. Type I Toxin-Antitoxin Systems in Clostridia. Toxins. 2019; 11 (5):253.
Chicago/Turabian StyleOlga Soutourina. 2019. "Type I Toxin-Antitoxin Systems in Clostridia." Toxins 11, no. 5: 253.
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.
Isabelle Poquet; Laure Saujet; Alexis Canette; Marc Monot; Jovanna Mihajlovic; Jean-Marc Ghigo; Olga Soutourina; Romain Briandet; Isabelle Martin-Verstraete; Bruno Dupuy. Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture. Frontiers in Microbiology 2018, 9, 2084 .
AMA StyleIsabelle Poquet, Laure Saujet, Alexis Canette, Marc Monot, Jovanna Mihajlovic, Jean-Marc Ghigo, Olga Soutourina, Romain Briandet, Isabelle Martin-Verstraete, Bruno Dupuy. Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture. Frontiers in Microbiology. 2018; 9 ():2084.
Chicago/Turabian StyleIsabelle Poquet; Laure Saujet; Alexis Canette; Marc Monot; Jovanna Mihajlovic; Jean-Marc Ghigo; Olga Soutourina; Romain Briandet; Isabelle Martin-Verstraete; Bruno Dupuy. 2018. "Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture." Frontiers in Microbiology 9, no. : 2084.
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.
Anna Maikova; Konstantin Severinov; Olga Soutourina. New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System. Frontiers in Microbiology 2018, 9, 1740 .
AMA StyleAnna Maikova, Konstantin Severinov, Olga Soutourina. New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System. Frontiers in Microbiology. 2018; 9 ():1740.
Chicago/Turabian StyleAnna Maikova; Konstantin Severinov; Olga Soutourina. 2018. "New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System." Frontiers in Microbiology 9, no. : 1740.
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.
Anna Maikova; Johann Peltier; Pierre Boudry; Eliane Hajnsdorf; Nicolas Kint; Marc Monot; Isabelle Poquet; Isabelle Martin-Verstraete; Bruno Dupuy; Olga Soutourina. Discovery of new type I toxin–antitoxin systems adjacent to CRISPR arrays in Clostridium difficile. Nucleic Acids Research 2018, 46, 4733 -4751.
AMA StyleAnna Maikova, Johann Peltier, Pierre Boudry, Eliane Hajnsdorf, Nicolas Kint, Marc Monot, Isabelle Poquet, Isabelle Martin-Verstraete, Bruno Dupuy, Olga Soutourina. Discovery of new type I toxin–antitoxin systems adjacent to CRISPR arrays in Clostridium difficile. Nucleic Acids Research. 2018; 46 (9):4733-4751.
Chicago/Turabian StyleAnna Maikova; Johann Peltier; Pierre Boudry; Eliane Hajnsdorf; Nicolas Kint; Marc Monot; Isabelle Poquet; Isabelle Martin-Verstraete; Bruno Dupuy; Olga Soutourina. 2018. "Discovery of new type I toxin–antitoxin systems adjacent to CRISPR arrays in Clostridium difficile." Nucleic Acids Research 46, no. 9: 4733-4751.
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. IMPORTANCE Clostridioides 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.
Julian R. Garneau; Ognjen Sekulovic; Bruno Dupuy; Olga Soutourina; Marc Monot; Louis-Charles Fortier. High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile. Applied and Environmental Microbiology 2018, 84, 1 .
AMA StyleJulian R. Garneau, Ognjen Sekulovic, Bruno Dupuy, Olga Soutourina, Marc Monot, Louis-Charles Fortier. High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile. Applied and Environmental Microbiology. 2018; 84 (3):1.
Chicago/Turabian StyleJulian R. Garneau; Ognjen Sekulovic; Bruno Dupuy; Olga Soutourina; Marc Monot; Louis-Charles Fortier. 2018. "High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile." Applied and Environmental Microbiology 84, no. 3: 1.
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.
Johann Peltier; Olga Soutourina. Identification of c-di-GMP-Responsive Riboswitches. Advanced Structural Safety Studies 2017, 1657, 377 -402.
AMA StyleJohann Peltier, Olga Soutourina. Identification of c-di-GMP-Responsive Riboswitches. Advanced Structural Safety Studies. 2017; 1657 ():377-402.
Chicago/Turabian StyleJohann Peltier; Olga Soutourina. 2017. "Identification of c-di-GMP-Responsive Riboswitches." Advanced Structural Safety Studies 1657, no. : 377-402.
Olga Soutourina. RNA-based control mechanisms of Clostridium difficile. Current Opinion in Microbiology 2017, 36, 62 -68.
AMA StyleOlga Soutourina. RNA-based control mechanisms of Clostridium difficile. Current Opinion in Microbiology. 2017; 36 ():62-68.
Chicago/Turabian StyleOlga Soutourina. 2017. "RNA-based control mechanisms of Clostridium difficile." Current Opinion in Microbiology 36, no. : 62-68.
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 σB. In this work, we studied the role of σB 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 σB controls the expression of ∼25% of genes including genes involved in sporulation, metabolism, cell surface biogenesis and the management of stresses. By contrast, σB 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 σB 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.
Nicolas Kint; Claire Janoir; Marc Monot; Sandra Hoys; Olga Soutourina; Bruno Dupuy; Isabelle Martin-Verstraete. The alternative sigma factor σBplays a crucial role in adaptive strategies ofClostridium difficileduring gut infection. Environmental Microbiology 2017, 19, 1933 -1958.
AMA StyleNicolas Kint, Claire Janoir, Marc Monot, Sandra Hoys, Olga Soutourina, Bruno Dupuy, Isabelle Martin-Verstraete. The alternative sigma factor σBplays a crucial role in adaptive strategies ofClostridium difficileduring gut infection. Environmental Microbiology. 2017; 19 (5):1933-1958.
Chicago/Turabian StyleNicolas Kint; Claire Janoir; Marc Monot; Sandra Hoys; Olga Soutourina; Bruno Dupuy; Isabelle Martin-Verstraete. 2017. "The alternative sigma factor σBplays a crucial role in adaptive strategies ofClostridium difficileduring gut infection." Environmental Microbiology 19, no. 5: 1933-1958.
The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H 2 S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601.
Thomas Dubois; Marie Dancer-Thibonnier; Marc Monot; Audrey Hamiot; Laurent Bouillaut; Olga Soutourina; Isabelle Martin-Verstraete; Bruno Dupuy. Control of Clostridium difficile Physiopathology in Response to Cysteine Availability. Infection and Immunity 2016, 84, 2389 -2405.
AMA StyleThomas Dubois, Marie Dancer-Thibonnier, Marc Monot, Audrey Hamiot, Laurent Bouillaut, Olga Soutourina, Isabelle Martin-Verstraete, Bruno Dupuy. Control of Clostridium difficile Physiopathology in Response to Cysteine Availability. Infection and Immunity. 2016; 84 (8):2389-2405.
Chicago/Turabian StyleThomas Dubois; Marie Dancer-Thibonnier; Marc Monot; Audrey Hamiot; Laurent Bouillaut; Olga Soutourina; Isabelle Martin-Verstraete; Bruno Dupuy. 2016. "Control of Clostridium difficile Physiopathology in Response to Cysteine Availability." Infection and Immunity 84, no. 8: 2389-2405.
Pierre Boudry; Ekaterina Semenova; Marc Monot; Kirill A. Datsenko; Anna Lopatina; Ognjen Sekulovic; Maicol Ospina-Bedoya; Louis-Charles Fortier; Konstantin Severinov; Bruno Dupuy; Olga Soutourina. Erratum for Boudry et al., Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile. mBio 2015, 6, e01508-15 -15.
AMA StylePierre Boudry, Ekaterina Semenova, Marc Monot, Kirill A. Datsenko, Anna Lopatina, Ognjen Sekulovic, Maicol Ospina-Bedoya, Louis-Charles Fortier, Konstantin Severinov, Bruno Dupuy, Olga Soutourina. Erratum for Boudry et al., Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile. mBio. 2015; 6 (5):e01508-15-15.
Chicago/Turabian StylePierre Boudry; Ekaterina Semenova; Marc Monot; Kirill A. Datsenko; Anna Lopatina; Ognjen Sekulovic; Maicol Ospina-Bedoya; Louis-Charles Fortier; Konstantin Severinov; Bruno Dupuy; Olga Soutourina. 2015. "Erratum for Boudry et al., Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile." mBio 6, no. 5: e01508-15-15.
Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli . These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. IMPORTANCE Clostridium difficile is the major cause of nosocomial infections associated with antibiotic therapy worldwide. To survive in bacteriophage-rich gut communities, enteropathogens must develop efficient systems for defense against foreign DNA elements. CRISPR-Cas systems have recently taken center stage among various anti-invader bacterial defense systems. We provide experimental evidence for the function of the C. difficile CRISPR system against plasmid DNA and bacteriophages. These data demonstrate the original features of active C. difficile CRISPR system and bring important insights into the interactions of this major enteropathogen with foreign DNA invaders during its infection cycle.
Pierre Boudry; Ekaterina Semenova; Marc Monot; Kirill A. Datsenko; Anna Lopatina; Ognjen Sekulovic; Maicol Ospina-Bedoya; Louis-Charles Fortier; Konstantin Severinov; Bruno Dupuy; Olga Soutourina. Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile. mBio 2015, 6, e01112-15 -15.
AMA StylePierre Boudry, Ekaterina Semenova, Marc Monot, Kirill A. Datsenko, Anna Lopatina, Ognjen Sekulovic, Maicol Ospina-Bedoya, Louis-Charles Fortier, Konstantin Severinov, Bruno Dupuy, Olga Soutourina. Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile. mBio. 2015; 6 (5):e01112-15-15.
Chicago/Turabian StylePierre Boudry; Ekaterina Semenova; Marc Monot; Kirill A. Datsenko; Anna Lopatina; Ognjen Sekulovic; Maicol Ospina-Bedoya; Louis-Charles Fortier; Konstantin Severinov; Bruno Dupuy; Olga Soutourina. 2015. "Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile." mBio 6, no. 5: e01112-15-15.
Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile . In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile . Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile , including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria.
P. Boudry; C. Gracia; M. Monot; J. Caillet; L. Saujet; E. Hajnsdorf; B. Dupuy; I. Martin-Verstraete; O. Soutourina. Pleiotropic Role of the RNA Chaperone Protein Hfq in the Human Pathogen Clostridium difficile. Journal of Bacteriology 2014, 196, 3234 -3248.
AMA StyleP. Boudry, C. Gracia, M. Monot, J. Caillet, L. Saujet, E. Hajnsdorf, B. Dupuy, I. Martin-Verstraete, O. Soutourina. Pleiotropic Role of the RNA Chaperone Protein Hfq in the Human Pathogen Clostridium difficile. Journal of Bacteriology. 2014; 196 (18):3234-3248.
Chicago/Turabian StyleP. Boudry; C. Gracia; M. Monot; J. Caillet; L. Saujet; E. Hajnsdorf; B. Dupuy; I. Martin-Verstraete; O. Soutourina. 2014. "Pleiotropic Role of the RNA Chaperone Protein Hfq in the Human Pathogen Clostridium difficile." Journal of Bacteriology 196, no. 18: 3234-3248.
Deep-sequencing technologies applied to RNA have tremendous potential to identify novel transcripts with single-nucleotide resolution. By combining whole-transcript cDNA sequencing (RNA-seq) and genome-wide identification of transcription start sites (dRNA-seq), it is possible to characterize long 5'-untranslated regions potentially endowed with regulatory capacities and to detect premature termination of transcription. This can be used to identify new potential riboswitches. In this chapter, we provide a detailed protocol of the dRNA-seq method based on differential pretreatment of RNAs with tobacco acid pyrophosphatase to differentiate between 5'-ends of primary and processed RNAs. We also give a briefer protocol of the preparation of RNA-seq libraries and of how to go through data bioinformatics analysis and data visualization using genome browsers. This approach is powerful to identify novel riboswitches and to demonstrate the functionality of riboswitches predicted in silico.
Isabelle Rosinski-Chupin; Olga Soutourina; Isabelle Martin-Verstraete. Riboswitch Discovery by Combining RNA-Seq and Genome-Wide Identification of Transcriptional Start Sites. Methods in Enzymology 2014, 549, 3 -27.
AMA StyleIsabelle Rosinski-Chupin, Olga Soutourina, Isabelle Martin-Verstraete. Riboswitch Discovery by Combining RNA-Seq and Genome-Wide Identification of Transcriptional Start Sites. Methods in Enzymology. 2014; 549 ():3-27.
Chicago/Turabian StyleIsabelle Rosinski-Chupin; Olga Soutourina; Isabelle Martin-Verstraete. 2014. "Riboswitch Discovery by Combining RNA-Seq and Genome-Wide Identification of Transcriptional Start Sites." Methods in Enzymology 549, no. : 3-27.