Unraveling a rewired quorum sensing network and regulation of pyocyanin production in the LasR-deficient Pseudomonas aeruginosa genotype : insights from a clinical isolate PA154197

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that represents a significant obstacle in global infection control owing to its capability of causing a broad range of infections and ready development of antibiotic resistance. The production of virulence factors in the species, such as pyocyanin (PYO), is primarily controlled by three hierarchically organized quorum sensing (QS) systems, Las, Rhl, and Pqs, in which Las serves as the master regulator. However, lasR-deficient strains are frequently isolated from clinical settings, which often display uncompromised virulence. This indicates that clinical isolates may have evolved compensatory regulatory mechanisms that rewire the QS systems, thereby activating virulence factors independent of the master regulator LasR. However, the molecular mechanisms underlying these compensatory strategies remain largely unknown. Previously, our laboratory demonstrated that a multidrug-resistant, lasR-deficient clinical P. aeruginosa isolate PA154197 produces a high level of PYO and displays uncompromised virulence compared to the reference strain PAO1. In my thesis, I set out to investigate the potentially rewired QS network in this representative lasR-deficient genotype. First, targeted deletion of lasR and lasI were constructed and were found to have no effect on the QS dynamics and responses in PA154197. Sequence alignment identified a Q98P mutation in the LasR ligand-binding domain which was predicted to cause a decrease in binding affinity to the 3-oxo-C12-HSL autoinducer by molecular docking. Next, I constructed ΔrhlR and ΔrhlI, and ΔpqsR and ΔpqsA responsible for the Rhl and Pqs QS systems. It was found that deletion of rhlR and rhlI abolished Las- and Rhl- controlled genes but had no effect on the expression of pqsR and pqsA, whereas ΔpqsR and ΔpqsA led to a significant reduction of Las-, Rhl- and Pqs- controlled genes, indicating a dominant role of the Pqs system in the lasR-deficient genotype. Further analysis revealed that PA154197 compensated for the lasR-deficiency by the introduction of T-380C mutation in the pqsABCDE promoter to enable a hyperactivity of the Pqs system and hyperproduction of PqsE which activates the Rhl system and followingly Las system through the PqsE-RhlR complex. To uncover how the QS-controlled virulence factor PYO is regulated in the QS-rewired PA154197 genotype, a transposon (Tn) insertion library screening was performed. As expected, Tn insertions within rhlR, rhlI, pqsA, and pqsR loci abolished PYO production. Interestingly, a Tn insertion in the algZ locus, which encodes the sensor kinase of the two-component AlgZR regulatory system, was found to abolish PYO production. Overexpression of algR, a gene downstream of algZ, instead of ΔalgZ, resulted in reduced PYO production through downregulating the Rhl QS and Las QS systems. Further analysis revealed that overexpressed AlgR exerts a negative regulatory effect on the Rhl QS systems by binding to the promoters of rhlI and rhlA. In the reference strain PAO1 in which LasR serves as the master regulator of the QS network, overexpression of AlgR was found to result in a minimal impact on PYO production. This observation suggested competition between an active transcription activator LasR and a repressor AlgR in the rhlI and rhlA promoters. As expected, replacing the PA154197 LasR with an active LasR variant as encoded in PAO1, or supplementing exogenous Las autoinducer 3-oxo-C12-HSL in the PA154197 culture, suppressed the AlgR-mediated repression of PYO biosynthesis in the cell. Furthermore, gradually reducing the expression of lasR in PAO1 through CRISPRi enabled AlgR-mediated repression of PYO biosynthesis in the PAO1 background. Collectively, these results indicated that in the presence of an active Las QS system, such as in PAO1, the transcription activator LasR outcompetes the repressor AlgR. In the lasR-deficient genotype PA154197, AlgR would compete to bind to rhlI and rhlA promoters and downregulate the Rhl and Las QS systems. In summary, these studies revealed a compensatory QS rewire strategy in clinical lasR-deficient genotypes and provided insight into targeted therapy for these variant pathogens.