Quercetin: a promising virulence inhibitor of Pseudomonas aeruginosa LasB in vitro

Abstract

With the inappropriate use of antibiotics, antibiotic resistance has emerged as a major dilemma for patients infected with Pseudomonas aeruginosa. Elastase B (LasB), a crucial extracellular virulence factor secreted by P. aeruginosa, has been identified as a key target for antivirulence therapy. Quercetin, a natural flavonoid, exhibits promising potential as an antivirulence agent. We aim to evaluate the impact of quercetin on P. aeruginosa LasB and elucidate the underlying mechanism. Molecular docking and molecular dynamics simulation revealed a rather favorable intermolecular interaction between quercetin and LasB. At the sub-MICs of ≤256 μg/ml, quercetin was found to effectively inhibit the production and activity of LasB elastase, as well as downregulate the transcription level of the lasB gene in both PAO1 and clinical strains of P. aeruginosa. Through correlation analysis, significant positive correlations were shown between the virulence gene lasB and the QS system regulatory genes lasI, lasR, rhlI, and rhlR in clinical strains of P. aeruginosa. Then, we found the lasB gene expression and LasB activity were significantly deficient in PAO1 ΔlasI and ΔlasIΔrhlI mutants. In addition, quercetin significantly downregulated the expression levels of regulated genes lasI, lasR, rhlI, rhlR, pqsA, and pqsR as well as effectively attenuated the synthesis of signaling molecules 3-oxo-C12-HSL and C4-HSL in the QS system of PAO1. Quercetin was also able to compete with the natural ligands OdDHL, BHL, and PQS for binding to the receptor proteins LasR, RhlR, and PqsR, respectively, resulting in the formation of more stabilized complexes. Taken together, quercetin exhibits enormous potential in combating LasB production and activity by disrupting the QS system of P. aeruginosa in vitro, thereby offering an alternative approach for the antivirulence therapy of P. aeruginosa infections. KEY POINTS: • Quercetin diminished the content and activity of LasB elastase of P. aeruginosa. • Quercetin inhibited the QS system activity of P. aeruginosa. • Quercetin acted on LasB based on the QS system.

Keywords: Antivirulence therapy; LasB elastase; Pseudomonas aeruginosa; Quercetin; Quorum sensing.

Fig. 1

Molecular docking and molecular dynamics simulation of quercetin with LasB. a Overall 3D. b Local 3D. c Local 2D. d RMSD. e RMSF. f Rg. g H-bond number. H-bonds produced are in green dashed lines, while VDWs are in pink and brown dashed lines.

Fig. 2

Effects of quercetin on the growth and LasB elastase of P. aeruginosa PAO1. a MICs of quercetin and baicalin. b Bacterial colony counts of PAO1 treated with quercetin or baicalin at the interval of 2 h for a duration of 24 h. c OD600 nm of PAO1 treated with quercetin or baicalin after 24 h incubation. d Elastin agar assay. 1: control, 20 mm; 2: quercetin 256 μg/ml, 11 mm; 3: quercetin 128 μg/ml, 14 mm; 4: quercetin 64 μg /ml, 14.5 mm; 5: quercetin 32 μg/ml, 15.5mm; 6: quercetin 16 μg/ml, 16 mm; 7: quercetin 16 μg/ml, 16 mm; 8: baicalin 256 μg/ml, 13 mm; 9: LB, no hydrolysis area. mm represents the diameter size of the hydrolyzed area. e Elastin-Congo red assay. 1: control; 2: quercetin 16 μg/ml, 11 m; 3: quercetin 32 μg/ml; 4: quercetin 64 μg /ml; 5: quercetin 128 μg/ml; 6: quercetin 256 μg/ml; 7: baicalin 256 μg/ml. f OD495 nm of elastin-Congo red assay. g Fluorescent-labeled elastin assay. Ex/Em = 485 nm/530 nm. PC, positive control; NC, negative control; RFU, relative fluorescence unit. h Relative expression level of lasB by RT-qPCR. i LasB concentration by ELISA. The data are presented as M ± SD of three independent experiments. ***P<0.001, ****P<0.0001 vs. the control group.

Fig. 3

Effects of quercetin on LasB activity and lasB gene expression of P. aeruginosa clinical isolates. a Electrophoretic patterns of lasB of PA 1-12. M: DNA marker (100–2000 bp); lanes 1–12: lasB of PA 1–12, respectively. b MICs of quercetin against PA 1–12. c Elastin-Congo red assay. d OD495 nm of elastin-Congo red assay. e Fluorescent-labeled elastin assay. Ex/Em = 485 nm/530 nm. PC, positive control; NC, negative control; RFU, relative fluorescence unit. f Relative expression level of lasB by RT-qPCR. The data are presented as M ± SD of three independent experiments. **P<0.01, ***P<0.001, ****P<0.0001 vs. the control group.

Fig. 4

Correlation analysis of lasB gene and QS system regulatory genes in clinical isolates. a Electrophoretogram of lasI, lasR, rhlI, rhlR, and lasB gene of P. aeruginosa clinical strains. M: DNA marker (100–2000 bp); lanes 1–5, 6–10, and 11–15 represent lasI, lasR, rhlI, rhlR, and lasB of clinical strains 1, 11, and 22, respectively. b Correlation analysis of the relative expression levels of virulence gene lasB and QS system genes lasI, lasR, rhlI, and rhlR in clinical isolates. |r| ≥ 0.8, very highly correlated; 0.6 ≤ |r| < 0.8, highly correlated; 0.4 ≤ |r| < 0.6, moderately correlated; 0.2 ≤ |r| < 0.4, low correlated; |r| < 0.2, essentially uncorrelated.

Fig. 5

Effects of ΔlasI and ΔlasIΔrhlI mutant strains of PAO1 on lasB gene expression and LasB activity. a Electrophoretic patterns of PAO1, ΔlasI, and ΔlasIΔrhlI. M: DNA marker (100–2000 bp); lanes 1–4, 5–8, and 9–12 represent the expression of lasI, rhlI, lasR, and rhlR genes of PAO1, ΔlasI, and ΔlasIΔrhlI strains, respectively. b SDS-PAGE protein electrophoresis. M: protein marker (20–245 kDa); lanes 1, 3, and 5 represent the secretory protein of PAO1, ΔlasI, and ΔlasIΔrhlI culture supernatant, respectively; lanes 2, 4, and 6 represent the bacterial protein of PAO1, ΔlasI, and ΔlasIΔrhlI thallus, respectively. c Elastin-Congo red assay and d Fluorescent-labeled elastin assay were used to determine the LasB activity. c Relative expression levels of lasB by RT-qPCR. All the data are presented as the M ± SD of three independent experiments. *P<0.05, ****P<0.0001 vs. the control group.

Fig. 6

Relative expression levels of lasI, lasR, rhlI, rhlR, pqsA, and pqsR in the QS system of PAO1 treated with the sub-MICs of quercetin. a Relative expression level of lasI. b Relative expression level of lasR. c Relative expression level of rhlI. d Relative expression level of rhlR. d Relative expression level of pqsA. d Relative expression level of pqsR. All the data are presented as the M ± SD of three independent experiments. **P<0.01, ***P<0.001, ****P<0.0001 vs. the control group.

Fig. 7

The impacts of quercetin on the synthesis of AHLs extracted from cultural supernatant of PAO1 evaluated by LC-MS/MS. a Characteristic fragments originating from the 3-oxo-C12-HSL. b Effects of quercetin on the 3-oxo-C12-HSL synthesis of PAO1. c Characteristic fragments originating from the C4-HSL. d Effects of quercetin on C4-HSL synthesis of PAO1. Blue lines indicate peaks for characteristic fragments of the AHLs. All the data are presented as the M ± SD of three independent experiments. ****P<0.0001 vs. the control group.

Fig. 8

Molecular docking complexes and their interaction modes of small molecules with QS receptor proteins. a OdDHL with LasR. b Quercetin with LasR. c BHL with RhlR. d Quercetin with RhlR. e PQS with PqsR. f Quercetin with PqsR. I, II, and III represent overall 3D, local 3D, and local 2D structure, respectively. H-bonds produced are in green and light green dashed lines and VDWs are in pink, brown, and purple dashed lines.

Fig. 9

Molecular dynamics simulation of small molecule binding to QS receptor proteins. a RMSD of OdDHL/quercetin binding to LasR. b RMSF of OdDHL/quercetin binding to LasR. c Rg of OdDHL/quercetin binding to LasR. d H-bond number of OdDHL/quercetin binding to LasR. e RMSD of BHL/quercetin binding to RhlR. f RMSF of BHL/quercetin binding to RhlR. g Rg of BHL/quercetin binding to RhlR. h H-bond number of BHL/quercetin binding to RhlR. i RMSD of PQS/quercetin binding to PqsR. j RMSF of PQS/quercetin binding to PqsR. k Rg of PQS/quercetin binding to PqsR. l H-bond number of PQS/quercetin binding to PqsR.