Identification of FDA-approved antivirulence drugs targeting the Pseudomonas aeruginosa quorum sensing effector protein PqsE

Abstract

The ability of the bacterial pathogen Pseudomonas aeruginosa to cause both chronic and acute infections mainly relies on its capacity to finely modulate the expression of virulence factors through a complex network of regulatory circuits, including the pqs quorum sensing (QS) system. While in most QS systems the signal molecule/receptor complexes act as global regulators that modulate the expression of QS-controlled genes, the main effector protein of the pqs system is PqsE. This protein is involved in the synthesis of the QS signal molecules 2-alkyl-4(1H)-quinolones (AQs), but it also modulates the expression of genes involved in virulence factors production and biofilm formation via AQ-independent pathway(s). P. aeruginosa pqsE mutants disclose attenuated virulence in plant and animal infection models, hence PqsE is considered a good target for the development of antivirulence drugs against P. aeruginosa. In this study, the negative regulation exerted by PqsE on its own transcription has been exploited to develop a screening system for the identification of PqsE inhibitors in a library of FDA-approved drugs. This led to the identification of nitrofurazone and erythromycin estolate, two antibiotic compounds that reduce the expression of PqsE-dependent virulence traits and biofilm formation in the model strain P. aeruginosa PAO1 at concentrations far below those affecting the bacterial growth rate. Notably, both drugs reduce the production of the PqsE-controlled virulence factor pyocyanin also in P. aeruginosa strains isolated from cystic fibrosis patients, and do not antagonize the activity of antibiotics commonly used to treat P. aeruginosa infection.

Keywords: Pseudomonas aeruginosa; PqsE; antivirulence strategy; erythromycin estolate; nitrofurazone; quorum sensing inhibition; screening.

Figure 1.

Screening system developed for the identification of PqsE inhibitors. (a) Schematic representation of the PqsE-Rep-based reporter system. The PqsE-Rep strain contains the PpqsA::lux transcriptional fusion and a genetic cassette for IPTG-inducible expression of the pqsE gene. Since in P. aeruginosa PqsE represses PpqsA promoter activity, the PqsE-Rep biosensor emits light at basal level when grown in LB supplemented with IPTG; as a consequence, molecules affecting PqsE functionality are expected to increase light emission by PpqsA derepression. (b) Activity of the PpqsA promoter in the PqsE-Rep strain grown in LB supplemented with the indicated concentrations of IPTG. PqsE-Rep was inoculated at an OD₆₀₀ of 0.08 in 0.2 mL of LB in 96-well microtiter plates and light emission was measured after 5 h of incubation at 37°C in shaking conditions. The average of three independent experiments is reported with SD.

Figure 2.

Selected hits increase PpqsA activity and reduce pyocyanin production. Effect of nitrofurazone (white bars), erythromycin estolate (light-gray bars) and diminazene aceturate (dark-gray bars) on PqsE-Rep bioluminescent emission (a) and pyocyanin production (b). Solvent vehicle control samples were considered as 100%. The average of at least three independent experiments is reported with SD. Statistical significance relative to the untreated control is indicated with asterisks: *, p < 0.05; **, p < 0.005; ***, p < 0.001 (ANOVA).

Figure 3.

Nitrofurazone and erythromycin estolate increase PpqsA activity only in a pqsE-proficient background. Effect of 100 µM nitrofurazone (a) or 50 µM erythromycin estolate (b) on PpqsA promoter activity in the indicated strains. Promoter activity is reported as percentage with respect to the corresponding solvent vehicle control sample, considered as 100%. The average of three independent experiments is reported with SD. Statistical significance relative to the untreated control is indicated with asterisks: ***, p < 0.001 (ANOVA).

Figure 4.

Nitrofurazone and erythromycin estolate inhibit the expression of PqsE-controlled virulence traits. Effect of 100 µM nitrofurazone or 50 µM erythromycin estolate on pyocyanin (a) and rhamnolipids (b) production, on swarming motility (c) and on biofilm formation (d) in P. aeruginosa PAO1. The isogenic ∆pqsE mutant was used as a control. For pyocyanin and rhamnolipids production, the average of three independent experiments is reported with SD; representative supernatants are shown in the inset picture in (A). Statistical significance relative to the untreated control is indicated with asterisks: ***, p < 0.001 (ANOVA). For swarming motility and biofilm formation, one representative picture of three independent experiments is shown.

Figure 5.

Nitrofurazone and erythromycin estolate are active against P. aeruginosa CF isolates. (a) Residual pyocyanin production in CF isolates grown in the presence of 100 μM nitrofurazone (white diamonds) or 50 µM erythromycin estolate (black diamonds) relative to solvent vehicle control samples, considered as 100%. Black lines represent the median values. The average of three independent experiments is reported. (b) Empirical cumulative distribution plots based on the data in (A). Differences between the distribution plots of nitrofurazone (white diamonds) and erythromycin estolate (black diamonds) are statistically significant (p < 0.001; ANOVA). Dashed lines indicate the residual pyocyanin production in 11 strains out of 21 (cumulative strains fraction = 0.524): ≤ 13.2% for erythromycin estolate and ≤ 61.2% for nitrofurazone. (c) Data from (A) clustered on the basis of the stage of infection: diamonds, CF strains isolated for the first time from patients; squares, CF strains isolated from patients with chronic infection from 2 to 3 years; triangles, CF strains isolated from patients with chronic infection for more than 5 years. **, p < 0.005; ***, p < 0.001 (ANOVA).