Pseudomonas aeruginosa is a Gram-negative bacterium capable of causing infections in immunocompromised individuals. The CsrA family of RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the post-transcriptional level. P. aeruginosa has a canonical CsrA family member (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for both RsmA and RsmF. The initial target aptamer was a 57 nt RNA transcript containing a central core randomized at 15 sequential positions. Most of the selected aptamers were the expected size and shared a common consensus sequence (CAnGGAyG). Longer aptamers (80-140 nts) containing two consensus-binding sites were also identified. Representative short (single consensus site) and long (two consensus sites) aptamers were tested for RsmA and RsmF binding. Whereas RsmA bound the short aptamers with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long aptamers with high affinity. Mutation of either consensus GGA site in the long aptamers reduced or eliminated RsmF binding, suggesting a requirement for two binding sites. Based on our observations that high affinity binding by RsmF appears to require two binding sites, we used an in-silico approach to search for candidate RsmF targets in the P. aeruginosa genome. We queried a library of 5’ UTRs (untranslated regions) for potential targets of RsmF based on the number and positions of GGA motifs, and secondary structure. Experimental validation of potential targets yielded few direct targets for both RsmA and RsmF indicating that additional factors contribute to differential binding in vivo.
P. aeruginosa has distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA activity is controlled by two small, non-coding regulatory RNAs (RsmY and RsmZ). In addition, we recently identified a sRNA (RsmV) that also contributes to RsmA and RsmF activity. Bioinformatic analyses suggest that these sRNAs each have 3-4 putative RsmA/RsmF bindings sites. Each site contains a GGA motif presented in the loop portion of a predicted stem-loop structure. These sRNAs regulate RsmA activity, and possibly RsmF, by sequestering RsmA and/or RsmF from target mRNAs. We characterize the contribution of each GGA site in RsmV, RsmY, and RsmZ using functional assays. We provided evidence that RsmF has more restrictive binding preferences compared to RsmA.
The type III secretion system (T3SS) is an important virulence factor that contributes to P. aeruginosa pathogenesis. Production of the T3SS is activated by host-associated signals and is tightly controlled at several levels. Global regulators including cAMP-Vfr signaling and Hfq contribute to tight regulation of the T3SS. Vfr (virulence factor regulator) is a transcription factor that responds to increased intracellular levels of cAMP. Vfr directly activates exsA transcription. ExsA activates transcription of the entire T3SS regulon. Hfq is an RNA chaperone that stabilizes sRNA and/or facilitates their binding to mRNA targets. Hfq is found in many bacteria and regulates stress responses, metabolism, and virulence. P. aeruginosa Hfq regulates about 5% of the genome and has a role in post-transcriptional control of T3SS in many Gram-negative bacteria. The mechanism of Hfq control of P. aeruginosa T3SS remains to be described. To better understand how Hfq regulates the T3SS we sought to identify mRNA targets of Hfq. Utilizing several reporters to genes involved in T3SS gene expression, we found that exsA transcription is decreased by Hfq activity. ExsA translation is also decreased by Hfq in conjunction with sRNA 179. Our findings show that Hfq may indirectly and directly regulate exsA translation.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-7433 |
| Date | 01 January 2017 |
| Creators | Janssen, Kayley Hope |
| Contributors | Yahr, Timothy L. |
| Publisher | University of Iowa |
| Source Sets | University of Iowa |
| Language | English |
| Detected Language | English |
| Type | dissertation |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | Copyright © 2017 Kayley Hope Janssen |
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