Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile

Soutourina, Olga; Monot, Marc; Boudry, Pierre; Saujet, Laure; Pichon, Christophe; Sismeiro, Odile; Semenova, Ekaterina; Severinov, Konstantin; Bouguénec, Chantal Le; Coppée, Jean‐Yves; Dupuy, Bruno; Martin‐Verstraete, Isabelle

doi:10.1371/journal.pgen.1003493

Cited by 210 publications

(380 citation statements)

References 88 publications

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“…Our data are consistent with findings from other groups that have mapped the ends of sRNAs in Yersinia and in other bacterial species, insofar as many individual sRNAs seem to have multiple start and end sites within the cell at any given time (33,67). The prevailing theory is that, unlike their eukaryotic counterparts, microRNAs, bacterial sRNAs are not generally processed (21).…”

Section: Discussionsupporting

confidence: 91%

Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System

et al. 2014

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bSmall noncoding RNA (sRNA) molecules are integral components of the regulatory machinery for many bacterial species and are known to posttranscriptionally regulate metabolic and stress-response pathways, quorum sensing, virulence factors, and more. The Yop-Ysc type III secretion system (T3SS) is a critical virulence component for the pathogenic Yersinia species, and the regulation of this system is tightly controlled at each step from transcription to translocation of effectors into host cells. The contribution of sRNAs to the regulation of the T3SS in Yersinia has been largely unstudied, however. Previously, our lab identified a role for the sRNA chaperone protein Hfq in the regulation of components of the T3SS in the gastrointestinal pathogen Yersinia pseudotuberculosis. Here we present data demonstrating a similar requirement for Hfq in the closely related species Yersinia pestis. Through deep sequencing analysis of the Y. pestis sRNA-ome, we found 63 previously unidentified putative sRNAs in this species. We identified a Yersinia-specific sRNA, Ysr141, carried by the T3SS plasmid pCD1 that is required for the production of multiple T3SS proteins. In addition, we show that Ysr141 targets an untranslated region upstream of yopJ to posttranscriptionally activate the synthesis of the YopJ protein. Furthermore, Ysr141 may be an unstable and/or processed sRNA, which could contribute to its function in the regulation of the T3SS. The discovery of an sRNA that influences the synthesis of the T3SS adds an additional layer of regulation to this tightly controlled virulence determinant of Y. pestis.

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Section: Discussionsupporting

confidence: 91%

Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System

et al. 2014

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“…In C. difficile, pilus gene expression promotes autoaggregation, a process that has been proposed to contribute to biofilm formation in 630/ 630⌬erm (21,24,25). Consistent with this, C. difficile biofilm formation is also stimulated by elevated c-di-GMP (25).…”

mentioning

confidence: 62%

“…In other bacterial species, TFP mediate surface motility, bacterial adhesion to surfaces and other bacteria, biofilm formation, and/or adherence to host cells (27)(28)(29)(30)(31). In C. difficile, pilus gene expression promotes autoaggregation, a process that has been proposed to contribute to biofilm formation in 630/ 630⌬erm (21,24,25). Consistent with this, C. difficile biofilm formation is also stimulated by elevated c-di-GMP (25).…”

mentioning

confidence: 73%

“…Regulation occurs via a c-di-GMP-responsive riboswitch that, in the presence of its ligand, promotes transcription elongation of pilA1, which encodes the major pilin subunit (24)(25)(26). In other bacterial species, TFP mediate surface motility, bacterial adhesion to surfaces and other bacteria, biofilm formation, and/or adherence to host cells (27)(28)(29)(30)(31).…”

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confidence: 99%

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Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

et al. 2016

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The intestinal pathogen Clostridium difficile is an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States. C. difficile rates of recurrence and mortality have increased in recent years due to the emergence of so-called "hypervirulent" epidemic strains. A great deal of the basic biology of C. difficile has not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger for C. difficile gene regulation. However, the function(s) of TFP in C. difficile remains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains of C. difficile. We demonstrate that TFP contribute to C. difficile biofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described in C. difficile. The expression and regulation of the pilA1 pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences in pilA1 expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemic C. difficile strains may contribute to the different infection outcomes presented by these strains. IMPORTANCEHow Clostridium difficile establishes and maintains colonization of the host bowel is poorly understood. Surface behaviors of C. difficile are likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation by C. difficile. TFP are also required for surface motility, which has not previously been demonstrated for C. difficile. Furthermore, an epidemic-associated C. difficile strain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains.

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“…Cas6 homologues, Sulfolobus islandicus (Held et al, 2013) I-B Cas8b Cascade Clostridium difficile (Soutourina et al, 2013) I-C Cas8c, repeat group 3 Cas5d Xanthomonas oryzae (Semenova et al, 2009)…”

Section: Structure Of the Crispr-cas Systemmentioning

confidence: 99%

CRISPR-Cas Systems in Prokaryotes

Burmistrz

Pyrć

2015

Pol J Microbiol

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A b s t r a c tProkaryotic organisms possess numerous strategies that enable survival in hostile conditions. Among others, these conditions include the invasion of foreign nucleic acids such as bacteriophages and plasmids. The clustered regularly interspaced palindromic repeats-CRISPRassociated proteins (CRISPR-Cas) system provides the majority of bacteria and archaea with adaptive and hereditary immunity against this threat. This mechanism of immunity is based on short fragments of foreign DNA incorporated within the hosts genome. After transcription, these fragments guide protein complexes that target foreign nucleic acids and promote their degradation. The aim of this review is to summarize the current status of CRISPR-Cas research, including the mechanisms of action, the classification of different types and subtypes of these systems, and the development of new CRISPR-Cas-based molecular biology tools. K e y w o r d s: CRISPR-Cas, prokaryotesBurmistrz M. and Pyrć K. 3 194 spacer sequences can be of either foreign or self-origin (Stern et al., 2010;Vercoe et al., 2013). The proteomic component is responsible for the incorporation of new template sequences, processing them into a form that enables base pairing with target nucleic acids, as well as for scanning and cleavage of target DNA or RNA. Of note, not all CRISPR-Cas systems discovered to date are active (Haft et al., 2005;van der Ploeg, 2009). Structure of the CRISPR-Cas systemThe genomic component of the CRISPR-Cas system is formed by a series of variable spacers, which in some cases share sequence similarity with viruses, plasmids, or bacteria. These regions are interspaced with repeat sequences that are identical or almost identical within a single CRISPR cassette. The length of the repeat sequences varies between 25 and 40 nt, whereas the length of the spacer sequences varies between 21 and 72 nt. As mentioned above, some spacers show high homology with foreign nucleic acids, but the origin of a significant percentage of spacers remains unknown. The sequence homology between the spacer and the target is the major determinant of nucleic acid degradation of the target. Some bacterial species contain more than one CRISPR locus within their genome (Louwen et al., 2014). Depending on the specific bacterial species or strain, a CRISPR locus may contain from a few to several hundred repeat spacer units; however, most commonly, a single CRISPR locus contains approximately 50 units. The CRISPR repeat sequences play an important role during both the acquisition of new spacers and the transcription and maturation of CRISPR RNA (crRNA). Based on the sequence similarity, the CRISPR repeats are assigned into groups (Kunin et al., 2007). These groups were taken into account in the current classification of CRISPR-Cas systems (Makarova et al., 2011). Although most CRISPR arrays are located on chromosomal DNA, there are examples of CRISPRs located on plasmids (Godde and Bickerton, 2006). In general, CRISPR loci are flanked by A/T rich leader sequences containing...

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Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile

Cited by 210 publications

References 88 publications

Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System

Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System

Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

CRISPR-Cas Systems in Prokaryotes

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