Panagiotopoulou, Dimitra
(2024)
Post-transcriptional regulation of the Pqs Quorum Sensing System in Pseudomonas aeruginosa.
PhD thesis, University of Nottingham.
Abstract
Pseudomonas aeruginosa is a leading cause of morbidity in patients with respiratory diseases and is a very frequent pathogen in medical device-associated infections. The adaptability and potent virulence of this pathogen are mainly attributed to virulence coordination at the population level by the three Quorum Sensing (QS) systems Las, Rhl, and Pqs, in conjunction with small, regulatory non-coding RNAs (sRNA). Regulation by sRNAs provides quick responses to environmental cues by altering gene expression at the post-transcriptional level, aiding to swift adaptation to new environments.
In the present study, the expression of the sRNA PqsX in P. aeruginosa PAO1-L was explored. In the first results chapter, the construction of an aptamer-based reporter and a bioluminescent reporter fusion to the pqsX promoter were attempted. Although the aptamer reporter was marred by technical difficulties, the bioluminescence reporter showed that pqsX was under the control of the Las and the Rhl QS systems, as well as underscored PqsE as a significant inducer of this gene in both static and aerobic conditions. Northern Blot analysis revealed the presence of at least two forms of PqsX of less than 300 nt each, and these two forms could be detected from late exponential growth phase. The lack of longer PqsX transcripts that would run into pqsA was not due to steric inhibition of transcription by PqsR binding downstream of pqsX. In silico analysis of PqsX pointed towards two major stem loops and two probable RNase E recognition sites which might be important for PqsX maturation and function.
The second results chapter was focused on the potential roles of PqsX. PqsX could not be confirmed to control any of the QS systems under aerobic conditions, in contrast to a previously reported positive regulation of lasR in surface-attached cells. To reveal candidate RNA targets of PqsX, GRIL-Seq was employed. As PqsX was not known to bind RNAs or proteins, PhrS was chosen as positive control of the process. Both sRNAs were ligated to multiple different mRNA targets, including lasB, rsaL, mRNAs of aromatic amino acid catabolic enzymes, phzS, arcD, and bfrB, spanning a broad range of metabolic pathways and virulence determinants. They were also bound to other sRNAs, with PqsX binding to PhrS, CrcZ, and ErsA, while PhrS was found to bind to ErsA, expanding their regulatory roles.
A selection of target genes were explored in vitro and phenotypically in chapter 3. PqsX interacted with RNAs of rsaL, clpX, bfrB, mvaT, CrcZ, and PhrS in vitro, and PhrS with ErsA, CrcZ, and phzS. Although no interaction was seen between PqsX and phzS mRNA, deletion of pqsX resulted in reduced pyocyanin production compared with the wild type. The PqsX-CrcZ interaction prompted investigation on the connection of PqsX in the presence of non-preferred carbon sources. PqsX was more abundant and expressed in stationary phase of growth in presence of mannitol than in glucose or succinate where it was found in exponential phase. This upregulation was dependent on the presence of rhlR.
Considering the extensive regulome of PqsX, the final step was to identify the effect of this sRNA on the whole transcriptome by means of RNA-Seq. Overexpression of pqsX in microaerophilic conditions did not have any significant impact on the PAO1-L transcriptome. However, in aerobic conditions, pqsX overexpression led to downregulation of the pyochelin biosynthesis genes but increase in pyoverdine biosynthetic genes as well as genes involved in iron uptake and signalling in low iron environments. Employing the bioluminescent reporter fusion of pqsX, the activity of the promoter was inversely related to the iron concentration in the media, peaking at 0.15 μΜ FeCl3. A pqsX mutant produced less pyoverdine in iron-rich media than the wild type, and the opposite effect was observed in iron scarcity. Comparison of RNA-Seq and Gril-Seq revealed one common target, bfrB, leading to hypothesise that the effect on iron scavenging seen in the RNA-Seq is an indirect result of bfrB translational repression by PqsX.
This study uncovered the broad functions of the sRNA PqsX in connection with the virulence and response to low iron of the multi-drug resistant P. aeruginosa. Furthermore, it showed that sRNAs may interact with each other adding a new layer in the regulation of gene expression. These results will help understand how this pathogen adapts in the host environment and design novel antimicrobial therapies against P. aeruginosa.
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