The Legionella pneumophila genome evolved to accommodate multiple regulatory mechanisms controlled by the CsrA-system

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.

Global Regulation by CsrA and Its RNA Antagonists

“…Recent findings from transcriptomics studies have led to the discovery that CsrA binding occurs at the 3′ UTR of a few transcripts, but occurs predominantly deep within the coding regions of mRNAs (29, 31, 34, 35). Furthermore, CsrA binding within mRNA coding regions is significantly increased in frequency near ribosome pause sites (35).…”

The pleiotropic Legionella transcription factor LvbR links the Lqs and c‐di‐GMP regulatory networks to control biofilm architecture and virulence

“…In the post-exponential growth phase L. pneumophila induces (de-represses) transmissive traits such as virulence, motility and stress resistance. The phase switch is controlled by a complex regulatory network, including the twocomponent system LetAS (Segal, 2013), the RNA-binding global regulator CsrA (carbon storage regulator A) (Sahr et al, 2017) and the Lqs system ( Fig. 1A) (Hochstrasser and Hilbi, 2017).…”

The single‐domain response regulator LerC functions as a connector protein in the Legionella pneumophila effectors regulatory network

“…Numerous L. pneumophila EEGs have been shown before to be directly regulated by the PmrAB TCS (Zusman et al, ; Al‐Khodor et al, ) and by the LetAS‐RsmYZ‐CsrA regulatory cascade (Rasis and Segal, ; Nevo et al, ; Sahr et al, ). However, no EEGs (or any other genes) which are regulated by both of these regulatory systems were previously described.…”

“…These four TCSs are responsible for the regulation of more than 100 L. pneumophila effector-encoding genes (EEGs) (Segal, 2013). These TCSs consist of (i) the PmrAB TCS, which includes the PmrA response regulator (RR) and the PmrB sensor histidine kinase (SHK), that was shown to directly activate the expression of 42 EEGs (Zusman et al, 2007;Al-Khodor et al, 2009;Speiser et al, 2017); (ii) the CpxRA TCS, which includes the CpxR RR and the CpxA SHK, that was shown to directly activate or repress the expression of 27 EEGs and four Icm/Dot components (Gal-Mor and Segal, 2003a;Feldman and Segal, 2007;Altman and Segal, 2008;Feldheim et al, 2016;Tanner et al, 2016); (iii) the LetAS TCS, which includes the LetA RR and the LetS SHK (Hammer et al, 2002;Gal-Mor and Segal, 2003b), that was shown to activate the transcription of two small regulatory RNAs (RsmY and RsmZ) during stationary phase (Hovel-Miner et al, 2009;Rasis and Segal, 2009;Sahr et al, 2009), which act in a redundant fashion to jointly antagonize CsrA, an RNA-binding protein that negatively regulates the expression of about 40 EEGs at the translational level (Nevo et al, 2014;Sahr et al, 2017); (vi) the LqsRS TCS and its signaling molecule LAI-1, which were also shown to affect the expression of EEGs, but since LqsR lacks a DNA binding domain, the way by which this regulation is mediated is currently not known (Tiaden et al, 2007;Schell et al, 2014;2016).…”

“…the LetAS-RsmYZ-CsrA regulatory cascade (Rasis and Segal, 2009;Nevo et al, 2014;Sahr et al, 2017). However, no EEGs (or any other genes) which are regulated by both of these regulatory systems were previously described.…”