Dissertation
Post-transcriptional control of Pseudomonas aeruginosa virulence genes by global regulatory networks
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2022
DOI: 10.25820/etd.006468
Abstract
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.
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.
Details
- Title: Subtitle
- Post-transcriptional control of Pseudomonas aeruginosa virulence genes by global regulatory networks
- Creators
- Jodi Marie Corley
- Contributors
- Timothy L Yahr (Advisor)Craig D Ellermeier (Committee Member)Dominique Limoli (Committee Member)David A Stoltz (Committee Member)David S Weiss (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Spring 2022
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.006468
- Number of pages
- xvii, 223 pages
- Copyright
- Copyright 2022 Jodi Marie Corley
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references.
- Public Abstract (ETD)
Pseudomonas aeruginosa is a bacterium that can cause destructive infections in humans. Infections can be especially detrimental to the lungs of people with cystic fibrosis. To cause infection, P. aeruginosa uses several weapons known as virulence factors. Production and utilization of virulence factors depends upon the environment and are highly controlled at genetic levels. Small non-coding RNAs (sRNAs) 0161 and 179, and the RNA binding proteins Hfq and RsmA are some regulators of virulence factor expression. P. aeruginosa produces a syringe and needle-like structure called the type III secretion system (T3SS) that injects toxins into host cells during some infections. Production of this machine is aided by RsmA but blocked by sRNAs 0161 and 179 along with Hfq. We sought to determine how these sRNAs and Hfq prevent T3SS gene expression. Furthermore, RsmA not only contributes to T3SS biogenesis, it also prevents expression of additional virulence factors like biofilms that contribute to other types of infection. Biofilms make infections more difficult to treat with antibiotics. We also aimed to unravel mechanisms important for the balance of RsmA within P. aeruginosa, which would coincide with an infection type. We discovered a mechanism that explains how RsmA reduces its own levels within the cell. Together, these contribute to new information on how virulence genes are regulated in P. aeruginosa.
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984270956302771
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