Quorum sensing antiactivators constrain Pseudomonas aeruginosa RhlR activity

Pseudomonas aeruginosa, an opportunistic pathogen, uses a cell-cell communication system called quorum sensing (QS) to regulate gene expression in response to population density. P. aeruginosa QS involves, in part, two transcription factors, LasR and RhlR, that respond to N-acyl homoserine lactone (AHL) signals. Two proteins known as "antiactivators," QteE and QslA, attenuate QS by inhibiting LasR, RhlR, or both. While initial characterization of antiactivation has revealed the considerable influence these factors may have on dampening QS, details regarding the individual impacts of these antiactivators on P. aeruginosa QS activity remain scant. Additionally, the effects of antiactivators on RhlR QS activity and in QS systems in isolates or strain lacking LasR have yet to be explored. To investigate how QteE and QslA each modulate LasR or RhlR independently, we combined gene deletion and over-expression analysis of each antiactivator in wild-type P. aeruginosa (PAO1) and two strains with rhl-dominated QS: clinical isolate E90 and PAO {Delta}lasR {Delta}mexT. As measured with a transcriptional reporter, over-expression of qteE or qslA notably reduced RhlR activity in PAO1 and PAO {Delta}lasR {Delta}mexT, but only expression of qteE had a marked effect on RhlR activity in E90. Expression analysis indicates LasR and RhlR repress QteE transcription, but not QslA. By over-expressing qslA in the absence of QteE and vice versa, we demonstrate that QslA activity and corresponding effects on QS phenotypes can be QteE-dependent in some scenarios. Our results reveal a nuanced role for individual antiactivator proteins in affecting the layered P. aeruginosa QS circuitry. ImportanceQuorum sensing (QS) is a cell signaling mechanism that enables populations of Pseudomonas aeruginosa to coordinate group behaviors such as biofilm formation, virulence factor production, and antibiotic tolerance once a critical cell-density threshold is reached. P. aeruginosa employs two "antiactivator" proteins that attenuate QS at low cell densities, dampening QS activation. The specific effects of individual antiactivators on the complex and hierarchically-arranged P. aeruginosa QS systems remain undefined. Here, we use two strains with rewired QS circuits to independently assess the effects of QS antiactivators on each QS circuit. We find that while one antiactivator selectively targets one QS circuit, the other can broadly target both with strong effects on QS activity. This work reveals an additional layer of complexity to counter-regulation of QS signalling and further defines antiactivation as a mechanism P. aeruginosa uses to finely tune QS responses.