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Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile.

ABSTRACT: Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA-seq and differential 5'-end RNA-seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

SUBMITTER: Soutourina OA

PROVIDER: S-EPMC3649979 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundErwinia amylovora is a phytopathogenic bacterium and causal agent of fire blight disease in apples and pears. Although many virulence factors have been characterized, the coordination of expression of these virulence factors in E. amylovora is still not clear. Regulatory small RNAs (sRNAs) are important post-transcriptional regulatory components in bacteria. A large number of sRNAs require the RNA chaperone Hfq for both stability and functional activation. In E. amylovora, Hfq was identified as a major regulator of virulence and various virulence traits. However, information is still lacking about Hfq-dependent sRNAs on a genome scale, including the virulence regulatory functions of these sRNAs in E. amylovora.ResultsUsing both an RNA-seq analysis and a Rho-independent terminator search, 40 candidate Hfq-dependent sRNAs were identified in E. amylovora. The expression and sizes of 12 sRNAs and the sequence boundaries of seven sRNAs were confirmed by Northern blot and 5' RACE assay respectively. Sequence conservation analysis identified sRNAs conserved only in the Erwinia genus as well as E. amylovora species-specific sRNAs. In addition, a dynamic re-patterning of expression of Hfq-dependent sRNAs was observed at 6 and 12 hours after induction in Hrp-inducing minimal medium. Furthermore, sRNAs that control virulence traits were characterized, among which ArcZ positively controls the type III secretion system (T3SS), amylovoran exopolysaccahride production, biofilm formation, and motility, and negatively modulates attachment while RmaA (Hrs6) and OmrAB both negatively regulate amylovoran production and positively regulate motility.ConclusionsThis study has significantly enhanced our understanding of the Hfq-dependent sRNAs in E. amylovora at the genome level. The identification of multiple virulence-regulating sRNAs also suggests that post-transcriptional regulation by sRNAs may play a role in the deployment of virulence factors needed during varying stages of pathogenesis during host invasion by E. amylovora.

Project description:The 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 defense 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 toward autoimmunity allows efficient genome editing in C. difficile We provide a 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 strain 630Δerm and in epidemic C. difficile strain R20291. 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 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.

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Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile.

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