DOI: 10.25442/hku.28479020.v1
title: Supporting data for "<b>UNRAVELING</b><b> </b><b>A REWIRED QUORUM SENSING NETWORK AND REGULATION OF PYOCYANIN PRODUCTION IN THE</b><b> </b><b><i>LASR</i></b><b>-DEFICIENT</b><b> </b><b><i>PSEUDOMONAS AERUGINOSA</i></b><b><i> </i></b><b>GENOTYPE: INSIGHTS FROM THE</b> <b>CLINICAL ISOLATE PA154197</b><b>"</b>

description: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that represents a significant obstacle in global infection control owing to its capability to cause a broad range of infections. 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 is the master regulator. However, <i>lasR</i>-deficient QS cheaters are frequently isolated from clinical settings, which often display uncompromised virulence. This indicates that clinical isolates have evolved compensatory regulatory mechanisms that rewire the QS systems, thereby activating virulence factors independently of the master regulator, LasR. However, the precise molecular mechanisms underlying these compensatory pathways remain largely unknown.We demonstrated a multidrug-resistant, <i>lasR</i>-deficient clinical <i>P. aeruginosa </i>isolate PA154197, producing a high level of PYO and displaying uncompromised virulence. In this study, we found a mutation (Q98P) in the PA154197 LasR signal-binding domain compared to the reference strain PAO1. This mutation contributes to the low LasR-autoinducer binding affinity and to the delayed activation of the QS cascade However, this delay was compensated by the uncoupled Rhl QS and Pqs QS systems. The mutations in the promoter region of the <i>pqsABCDE</i> gene cluster prevent RhlR binding. RhlR can’t exert the negative regulation on <i>pqsABCDE</i>. Consequently, the Pqs QS system becomes hyperactive, leading to sustained high-level production of PYO.To uncover other genetic elements underlying the elevated PYO synthesis in PA154197, a genome-wide transposon insertion library screening was performed. We found transposon (Tn) insertions within rhlR, rhlI, pqsA, and pqsR in the Rhl and Pqs QS systems reduce PYO production. Interestingly, a Tn insertion in the <i>algZ</i> gene, which encodes the sensor kinase of the AlgZR two-component regulatory system, was found to completely abolish PYO production. Tn insertions at three distinct sites within the <i>rhlA</i> promoter, which controls rhamnosyltransferase in rhamnolipid biosynthesis under Rhl QS regulation, were shown to upregulate PYO production. Site-directed mutagenesis and RT-PCR identified a transcription unit within the promoter region of the <i>rhlA</i>. Under the regulation of Rhl QS, this promoter directs the extended transcript encompassing <i>rhlA</i>, <i>rhlB</i>, <i>rhlR</i>, and <i>rhlI</i>. Hence, this transcription unit establishes a positive feedback loop within the Rhl QS system, thereby regulating PYO production. Further analysis revealed that overexpression of <i>algR</i>, a gene downstream of <i>algZ</i>, resulted in reduced PYO production and downregulation of Las QS and Rhl QS genes by binding to the promoters of <i>rhlI</i> and <i>rhlA</i>, suggesting that AlgR exerts a negative regulatory effect on the Las QS and Rhl QS systems.Furthermore, PA154197 maintained higher algR expression levels than PAO1 during the log phase, correlating with the observed reduction in PYO production.Replacing the PA154197 LasR with an active LasR variant encoded in PAO1 or supplementing exogenous Las autoinducer suppressed the AlgR-mediated repression of PYO production in the PA154197 background. In PAO1, PYO production remains unchanged in the <i>algR</i> overexpression strain.However, inhibition of lasR expression in PAO1 using CRISPRi technology led to the complete suppression of PYO by AlgR. AlgR competes with LasR to repress the Rhl QS system and its downstream PYO production. In the presence of an active Las QS system, such as in PAO1, the repression effect of AlgR is negligible. In the <i>lasR</i>-deficient clinical strain PA154197, AlgR dominates the QS system and PYO downregulation during the log phase. PA154197 relies on the hyperactivated Pqs QS system to maintain high PYO levels in the stationary phase. This study revealed a rewired regulatory pathway in <i>lasR</i>-deficient clinical <i>P. aeruginosa</i> strains to maintain a high-level virulence production.