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Tractable Mammalian Cell Infections with Protozoan-primed Bacteria
Published on: April 2, 2013
The Legionella pneumophila genome evolved to accommodate multiple regulatory mechanisms controlled by the CsrA-system
Tobias Sahr1,2, Christophe Rusniok1,2, Francis Impens3,4
1Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris France.
Abstract:
The carbon storage regulator protein CsrA regulates cellular processes post-transcriptionally by binding to target-RNAs altering translation efficiency and/or their stability. Here we identified and analyzed the direct targets of CsrA in the human pathogen Legionella pneumophila. Genome wide transcriptome, proteome and RNA co-immunoprecipitation followed by deep sequencing of a wild type and a csrA mutant strain identified 479 RNAs with potential CsrA interaction sites located in the untranslated and/or coding regions of mRNAs or of known non-coding sRNAs. Further analyses revealed that CsrA exhibits a dual regulatory role in virulence as it affects the expression of the regulators FleQ, LqsR, LetE and RpoS but it also directly regulates the timely expression of over 40 Dot/Icm substrates. CsrA controls its own expression and the stringent response through a regulatory feedback loop as evidenced by its binding to RelA-mRNA and links it to quorum sensing and motility. CsrA is a central player in the carbon, amino acid, fatty acid metabolism and energy transfer and directly affects the biosynthesis of cofactors, vitamins and secondary metabolites. We describe the first L. pneumophila riboswitch, a thiamine pyrophosphate riboswitch whose regulatory impact is fine-tuned by CsrA, and identified a unique regulatory mode of CsrA, the active stabilization of RNA anti-terminator conformations inside a coding sequence preventing Rho-dependent termination of the gap operon through transcriptional polarity effects. This allows L. pneumophila to regulate the pentose phosphate pathway and the glycolysis combined or individually although they share genes in a single operon. Thus the L. pneumophila genome has evolved to acclimate at least five different modes of regulation by CsrA giving it a truly unique position in its life cycle.
Insights
The carbon storage regulator (CsrA) protein in Legionella pneumophila controls gene expression post-transcriptionally, impacting virulence, metabolism, and RNA stability. This study reveals CsrA
Area of Science:
- Microbiology
- Molecular Biology
- Genomics
Background:
- The carbon storage regulator (CsrA) protein post-transcriptionally modulates cellular processes by binding to target RNAs, affecting translation and stability.
- CsrA is a crucial regulator in various bacteria, influencing diverse physiological functions.
Purpose of the Study:
- To identify and analyze the direct RNA targets of CsrA in the human pathogen Legionella pneumophila.
- To elucidate the multifaceted regulatory roles of CsrA in L. pneumophila virulence, metabolism, and RNA processing.
Main Methods:
- Genome-wide transcriptome and proteome analyses of wild-type and csrA mutant L. pneumophila strains.
- RNA immunoprecipitation followed by deep sequencing (RIP-Seq) to identify CsrA-RNA interactions.
- Bioinformatic analysis to map CsrA binding sites and predict regulatory effects.
Main Results:
- Identified 479 potential CsrA-target RNAs, including mRNAs and small RNAs, with binding sites in untranslated and coding regions.
- Demonstrated CsrA's dual role in virulence, regulating key transcriptional regulators (FleQ, LqsR, LetE, RpoS) and over 40 Dot/Icm secretion system substrates.
- Uncovered CsrA's involvement in a feedback loop controlling its own expression and the stringent response via RelA-mRNA binding, linking it to quorum sensing and motility.
- Revealed CsrA's central role in carbon, amino acid, and fatty acid metabolism, cofactor biosynthesis, and energy transfer.
- Described the first L. pneumophila riboswitch (thiamine pyrophosphate riboswitch) fine-tuned by CsrA and a novel CsrA regulatory mode involving stabilization of anti-terminator RNA structures to control Rho-dependent termination.
Conclusions:
- CsrA is a master regulator in L. pneumophila, orchestrating complex cellular processes through diverse regulatory mechanisms.
- The identified CsrA targets and regulatory modes highlight its critical importance in L. pneumophila pathogenesis and adaptation.
- L. pneumophila employs at least five distinct regulatory strategies involving CsrA, underscoring its unique role in the bacterium's life cycle.
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