Post-transcriptional control of Pseudomonas aeruginosa virulence genes by global regulatory networks
Creators: name: Corley, Jodi Marie
Publisher: name: University of Iowa
Year: 2022
Subjects: public abstract

description: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen causing skin and soft tissue, respiratory, and bloodstream infections. The type III secretion system (T3SS) is one important virulence factor. Production of the T3SS is controlled by ExsA, a transcription factor that activates expression of the entire T3SS regulon. Global regulators including Vfr, RsmA, and Hfq also contribute to regulation of the T3SS. Vfr is a cAMP-responsive transcription factor that activates exsA transcription. RsmA, an RNA-binding protein, inversely controls expression of the T3SS and the type VI secretion system (T6SS). Hfq is an RNA chaperone that functions by stabilizing sRNAs and/or facilitating base pairing between small non-coding RNAs (sRNA) and mRNA targets. A previous study identified sRNA 0161, which directly targets the exsA mRNA and likely inhibits ExsA synthesis. We screened an sRNA expression library and identified sRNA 179 as an Hfq-dependent inhibitor of T3SS gene expression by inhibiting the synthesis of both ExsA and Vfr. The previous finding that RsmA stimulates ExsA and Vfr synthesis suggested that sRNA 179 impacts the Gac/Rsm system. Consistent with that idea, the inhibitory activity of sRNA 179 is suppressed in a mutant lacking rsmY and rsmZ, and sRNA 179 expression stimulates rsmY transcription. RsmY and RsmZ are small non-coding RNAs that sequester RsmA from target mRNAs. More recent work demonstrates that sRNA 179 inhibits T3SS in a mutant lacking gacA. This suggests sRNA 179 could be inhibiting or activating targets that activate RsmY expression independently of GacA, such as RpoS. However, expressing sRNA 179 in a rpoS deletion mutant still led to decreased T3SS gene expression. RNA-seq analysis showed that expressing sRNA 179 increases and decreases transcript levels of many genes important for the T3SS and cAMP-Vfr regulon, and others with no known associated connection. Our combined findings show that Hfq and sRNA 179 indirectly regulate ExsA and Vfr synthesis by reducing the available pool of RsmA leading to reduced expression of the T3SS and cAMP-Vfr regulons but the exact mechanism has yet to be determined.
description: The Gac/Rsm system is a global regulator of Pseudomonas aeruginosa gene expression. The primary effectors are RsmA and RsmF. Both are RNA-binding proteins that interact with target mRNAs to modulate protein synthesis. RsmA/RsmF recognize GGA sequences presented in the loop portion of stem-loop structures. For repressed targets, the GGA sites usually overlap the ribosome binding site (RBS) and RsmA/RsmF binding inhibits translation initiation. RsmA/RsmF activity is controlled by several small non-coding RNAs (sRNA) that sequester RsmA/RsmF from target mRNAs. The most important sequestering sRNAs are RsmY and RsmZ. Transcription of rsmY/rsmZ is directly controlled by the GacSA two-component regulatory system. GacSA activity is antagonized by RetS, a hybrid sensor kinase. In the absence of retS, rsmY/rsmZ transcription is derepressed and RsmA/RsmF are sequestered by RsmY/RsmZ. Gac/Rsm system homeostasis is tightly controlled by at least two mechanisms. First, direct binding of RsmA to the rsmA and rsmF mRNAs inhibits further synthesis of both proteins. Second, RsmA stimulates rsmY/rsmZ transcription through an undefined mechanism. In this study we demonstrate that RsmA stimulates rsmY/rsmZ transcription by directly inhibiting RetS synthesis. RetS protein levels are elevated 2.5-fold in an rsmA mutant. Epistasis experiments demonstrate that the rsmA requirement for rsmY/rsmZ transcription is entirely suppressed in an rsmA, retS double mutant. RsmA directly interacts with the retS mRNA and requires two distinct GGA sites, one of which overlaps the RBS. We propose a model wherein RsmA inhibits RetS synthesis to promote rsmY/rsmZ transcription and that this acts as a checkpoint to limit RsmA/RsmF availability.