Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS): A Comparison between Naturally Occurring and Engineered PQS-Cleaving Dioxygenases

Abstract

The opportunistic pathogen Pseudomonas aeruginosa employs quorum sensing to govern the production of many virulence factors. Interference with quorum sensing signaling has therefore been put forward as an attractive approach to disarm this pathogen. Here, we analyzed the quorum quenching properties of natural and engineered (2-alkyl-)3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) that inactivate the P. aeruginosa signal molecule PQS (Pseudomonas quinolone signal; 2-heptyl-3-hydroxy-4(1H)-quinolone). When added exogenously to P. aeruginosa cultures, all HQDs tested significantly reduced the levels of PQS and other alkylquinolone-type secondary metabolites deriving from the biosynthetic pathway, such as the respiratory inhibitor 2-heptyl-4-hydroxyquinoline N-oxide. HQDs from Nocardia farcinica and Streptomyces bingchenggensis, which combine low KM values for PQS with thermal stability and resilience in the presence of P. aeruginosa exoproducts, respectively, attenuated production of the virulence factors pyocyanin and pyoverdine. A delay in mortality was observed when Galleria mellonella larvae were infected with P. aeruginosa suspensions treated with the S. bingchenggensis HQD or with inhibitors of alkylquinolone biosynthesis. Our data indicate that quenching of PQS signaling has potential as an anti-virulence strategy; however, an efficient anti-virulence therapy against P. aeruginosa likely requires a combination of agents addressing multiple targets.

Atomic-scale interactions between quorum sensing autoinducer molecules and the mucoid P. aeruginosa exopolysaccharide matrix

“…With significantly reduced EPS mobility, the PQS molecule is identified as a potential target for quorum quenching (QQ). In fact, enzymatic PQS deactivation, for example, through exogenous supplementation 2,4-dioxygenase, has proved to be a viable strategy for eliminating PQS and PQS related virulence from P. aeruginosa biofilms 61 , 62 .…”

A PQS-Cleaving Quorum Quenching Enzyme Targets Extracellular Membrane Vesicles of Pseudomonas aeruginosa

“…b . are quite instable at 37 °C, as reflected by a catalytic half-life (in PBS) of 0.49 h and 0.22 h, respectively [ 19 ]. When incubated in a 1:1 mixture of a 20 mM Tris-HCl buffer, pH 8, and sterile LB at 37 °C, AqdC and HQD S .…”

“…b . lost 52% and 79% of their activity, respectively, within 30 min [ 19 ]. Interestingly, culture supernatant of P. aeruginosa strain PA14 stabilized HQD S .…”

“…b . , as suggested by the observation that the enzyme retained 86% of activity when incubated for 30 min at 37 °C in a 1:1 mixture of buffer and supernatant of a stationary-phase PA14 culture grown in LB [ 19 ], raising the question of the causative agent(s). Remarkably, the stabilizing effect of the culture supernatant was not observed with any of the other PQS dioxygenases studied [ 19 ].…”

“…b . to P. aeruginosa cultures significantly quenched AQ levels and attenuated the production of pyocyanin and pyoverdine; moreover, the pre-incubation of P. aeruginosa PAO1 with this enzyme increased the survival rate of Galleria mellonella larvae when infected with this strain [ 19 ]. During our studies, and much to our surprise, we observed a pronounced stabilization of the HQD S .…”