Romo Catalán, Natalia Valentina
(2022)
New insights into the repressor role of PqsE over pqsA.
PhD thesis, University of Nottingham.
Abstract
Pseudomonas aeruginosa is an opportunistic pathogen able to thrive in a wide variety of environments. Its highly diverse and adaptable behaviour is an increasing threat in chronic and hospital acquired infections (HAI). The exceptional ecological success of P. aeruginosa can be attributed to its vast metabolic versatility and its sophisticated cell- to- cell communication system quorum sensing (QS). QS network enables P. aeruginosa control the expression of diverse virulence factors such as pyocyanin, elastase, exotoxins, rhamnolipids, and biofilm formation. QS in P. aeruginosa is closely regulated by at least three different but highly interconnected systems namely, the Las, Rhl, and quinolone-based QS system, PQS. The PQS system acts mainly via 2-heptyl-3-hydroxy-4-quinolone, named the Pseudomonas quinolone signal (PQS), which connects the LysR-type transcriptional regulator PqsR to stimulate their own synthesis along with the expression of several virulence factors. The synthesis of PQS is driven by the pqsABCDE operon, phnAB and pqsH, located further in the P. aeruginosa genome. PqsE, encoded by the last gene of the pqs operon, functions as a pathway-specific thioesterase involved in the synthesis of HHQ and PQS, however its role in this pathway can be replaced by the broad-specificity thioesterase TesB, which reveals why pqsE deletion mutants maintains the synthesis of PQS. PqsE also adds independent roles onto the control of bacterial virulence through an unidentified mechanism. Among others, it balances the levels of QS signal molecules and secondary metabolites deriving from the PQS pathway by repressing the pqsA promoter, however, since PqsE does not possess DNA binding domain, its molecular mechanism remains elusive. Previously at the University of Nottingham, two DNA promoter pull down analysis were performed in a PAO1 pqsE Ind strain. In this setup, the overexpression of pqsE at the early and late stage of growth of P. aeruginosa showed a diverse protein profile binding the pqsA promoter, suggesting that they could potentially act as intermediate of the action of this effector in this regulation.
Chapter 3 shows PA2705 bound the pqsA promoter in high abundance and only when pqsE was overexpressed, hence becoming a potential candidate to mediate the action of this effector in early growth. Due to significant changes in the pqsA expression using two pMiniCTX-lux reporters in a PA2705 mutant, in depth analysis of the promoter regions included in these constructs revealed that the -311 rhlR-box was interrupted in one of reporters and that has a pivotal role in regulation of the PpqsA. This analysis contributed to elucidate that (i) PA2705 is an inductor of the pqsA expression and that (ii) PqsE is a repressor of pqsA at the post-transcriptional level. Furthermore, it was evidenced that PA2705 is under the regulation of PqsE, but it is not essential for the PqsE-mediated repression of pqsA.
At the late stage of growth of P. aeruginosa, fewer proteins were found binding the pqsA promoter. In Chapter 4, mutation of the main candidates genes showed that the denitrification regulatory protein nirQ caused the major disruption towards the expression of pqsA, becoming the main candidate to analyse in further studies. qRT-PCR in P. aeruginosa grown anaerobically indicated that nirQ is a repressor of pqsA. Moreover, gene expression analysis unveiled that nirQ is under the regulation of PqsE, but like PA2705, it is dispensable for the PqsE-mediated repression of pqsA.
The analysis of PA2705 and nirQ in the PqsE-mediated repression of pqsA evidenced that this regulation is especially complex and seems to involve other regulatory elements. In the search for the mediator of PqsE and to better understand the regulation of pqsA at the post-transcriptional level, further work in this direction was addressed in Chapter 5. The mutation of the main QS regulators lasR, rhlR and pqsR within the wild-type and the pqsE Ind genetic background revealed that the PqsE-mediated repression of pqsA is dependent on PqsR. In addition, it is suggested that a ncRNA expressed at the -339 transcriptional start site of the pqsA and that is under the control of RhlR participates in the repression of pqsA. Nevertheless, this finding differs to that indicated in another study and conflicts with the mechanism proposed by the authors. Last, a broader view of the pqsABCDE operon led to investigate whether pqsE is independently regulated. A CRP like box found upstream the pqsE coding sequence elucidated the regulatory role of Vfr towards pqsE.
In summary, various regulatory venues were explored in the P. aeruginosa pqsA regulation that reveals new perspectives to understand the cell- to- cell communication in this bacterium and exhibits the dynamics and yet obscure events occurring within this regulation, knowledge of which could pave the path towards new studies in this pathogen.
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