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

Abstract

The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to control its virulence. One of its major signal molecules, the Pseudomonas quinolone signal PQS, has high affinity to membranes and is known to be trafficked mainly via outer membrane vesicles (OMVs). We previously reported that several 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) catalyze the cleavage of PQS and thus act as quorum quenching enzymes. Further analysis showed that, in contrast to other HQDs, the activity of HQD from Streptomyces bingchenggensis (HQD_S._b.) was unexpectedly stabilized by culture supernatants of P. aeruginosa. Interestingly, the stabilizing effect was higher with supernatants from the strain PA14 than with supernatants from the strain PAO1. Heat treatment and lyophilization hardly affected the stabilizing effect; however, fractionation of the supernatant excluded small molecules as stabilizing agents. In a pull-down assay, HQD_S._b. appeared to interact with several P. aeruginosa proteins previously found in the OMV proteome. This prompted us to probe the physical interaction of HQD_S._b. with prepared extracellular membrane vesicles. Homo-FRET of fluorescently labeled HQD_S._b. indeed indicated a spatial clustering of the protein on the vesicles. Binding of a PQS-cleaving enzyme to the OMVs of P. aeruginosa may enhance PQS degradation and is highly reconcilable with its function as a quorum quenching enzyme.

Keywords: Pseudomonas aeruginosa; Pseudomonas quinolone signal; dioxygenase; homo-FRET; membrane-protein interaction; outer membrane vesicles; quorum quenching enzyme.

Figure 1

Residual enzymatic activity of HQD_S._b. upon incubation with (A) flow-through and retentate fractions of P. aeruginosa PA14 supernatant after ultrafiltration with 10 kDa, 30 kDa, 50 kDa cut-off membranes, (B) heat-treated (95 °C, 20 min), lyophilized, and untreated cell-free PA14 supernatant preparations and (C) supernatant fractions of P. aeruginosa PAO1, PAO1 ∆pslJ and PA14 strains, prepared in LB or M63 medium. In (B,C), the residual activity of HQD was determined after 120 min of incubation. All experiments were conducted at 37 °C. Sterile media were included as negative controls. Data represent the mean values ± SD of three replicates.

Figure 2

Stabilizing effect induced by the incubation of P. aeruginosa MVs with (A) HQD_S._b. and (B) AqdC. The enzymes were incubated at 37 °C with 0.2 mg/mL P. aeruginosa PA14 MVs (protein-based concentration), LPS (1 mg/mL in PBS) or PBS as a control. The residual enzymatic activity was determined at the specified time intervals. The data represent the mean ± SD of three replicates.

Figure 3

(A) Schematic illustration of the homo-FRET interaction assay with fluorescently labeled enzymes. The horizontal dark-blue arrow indicates the polarized excitation light. Interaction with or adsorption of the fluorescently labeled enzyme (red) to the vesicle surface brings the fluorophore molecules (blue) into spatial proximity, leading to FRET and hence to a decrease in polarization, indicated by the vertical light blue arrow (homo-FRET box, center). Without interaction with MVs, the emitted light (vertical blue arrow) remains highly polarized (left box). (B,C) Fluorescence polarization of (B) labeled HQD_S._b.-Dy488 and of (C) AqdC-Dy488 in the presence or absence (control) of 0.2 mg/mL of P. aeruginosa MVs (protein-based concentration), measured at 488 nm excitation and 519 nm emission wavelength over 150 min at the indicated time intervals. Three independent experiments were performed. The lines connecting the data points collected from each replicate are for illustrative purposes only.