Les mécanismes de résistance aux antibiotiques chez Staphylococcus Aureus

RENNES1-BU Santé (352382103) / SudocSudocFranceF

The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems.

CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool

RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes

Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.Originally published in manuscript form.</p

Profiling the intragenic toxicity determinants of toxin–antitoxin systems: revisiting hok / Sok regulation

International audienceType I toxin–antitoxin systems (T1TAs) are extremely potent bacterial killing systems difficult to characterize using classical approaches. To assess the killing capability of type I toxins and to identify mutations suppressing the toxin expression or activity, we previously developed the FASTBAC-Seq (Functional AnalysiS of Toxin–Antitoxin Systems in BACteria by Deep Sequencing) method in Helicobacter pylori. This method combines a life and death selection with deep sequencing. Here, we adapted and improved our method to investigate T1TAs in the model organism Escherichia coli. As a proof of concept, we revisited the regulation of the plasmidic hok/Sok T1TA system. We revealed the death-inducing phenotype of the Hok toxin when it is expressed from the chromosome in the absence of the antitoxin and recovered previously described intragenic toxicity determinants of this system. We identified nucleotides that are essential for the transcription, translation or activity of Hok. We also discovered single-nucleotide substitutions leading to structural changes affecting either the translation or the stability of the hok mRNA. Overall, we provide the community with an easy-to-use approach to widely characterize TA systems from diverse types and bacteria

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5′ untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5′ UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5′ UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5′ UTR and on the role of RNase Y in speB regulation

In vivo 3′-to-5′ exoribonuclease targetomes of Streptococcus pyogenes

mRNA decay plays an essential role in the control of gene expression in bacteria. Exoribonucleases (exoRNases), which trim transcripts starting from the 5′ or 3′ end, are particularly important to fully degrade unwanted transcripts and renew the pool of nucleotides available in the cell. While recent techniques have allowed genome-wide identification of ribonuclease (RNase) targets in bacteria in vivo, none of the 3′-to-5′ exoRNase targetomes (i.e., global processing sites) have been studied so far. Here, we report the targetomes of YhaM, polynucleotide phosphorylase (PNPase), and RNase R of the human pathogen Streptococcus pyogenes. We determined that YhaM is an unspecific enzyme that trims a few nucleotides and targets the majority of transcript ends, generated either by transcription termination or by endonucleolytic activity. The molecular determinants for YhaM-limited processivity are yet to be deciphered. We showed that PNPase clears the cell from mRNA decay fragments produced by endoribonucleases (endoRNases) and is the major 3′-to-5′ exoRNase for RNA turnover in S. pyogenes. In particular, PNPase is responsible for the degradation of regulatory elements from 5′ untranslated regions. However, we observed little RNase R activity in standard culture conditions. Overall, our study sheds light on the very distinct features of S. pyogenes 3′-to-5′ exoRNases

RNA sequencing uncovers antisense RNAs and novel small RNAs in <i>Streptococcus pyogenes</i>

<p><i>Streptococcus pyogenes</i> is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in <i>S. pyogenes.</i> In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in <i>S. pyogenes</i>. By comparing our list of predicted sRNAs with previous <i>S. pyogenes</i> sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.</p