Discovery of psoralen as a quorum sensing inhibitor suppresses Pseudomonas aeruginosa virulence

Abstract

Pseudomonas aeruginosa is a common opportunistic pathogen with growing resistance and presents heightened treatment challenges. Quorum sensing (QS) is a cell-to-cell communication system that contributes to the production of a variety of virulence factors and is also related to biofilm formation of P. aeruginosa. Compared to traditional antibiotics which kill bacteria directly, the anti-virulence strategy by targeting QS is a promising strategy for combating pseudomonal infections. In this study, the QS inhibition potential of the compounds derived from the Traditional Chinese Medicines was evaluated by using in silico, in vitro, and in vivo analyses. The results showed that psoralen, a natural furocoumarin compound derived from Psoralea corylifolia L., was capable of simultaneously inhibiting the three main QS regulators, LasR, RhlR, and PqsR of P. aeruginosa. Psoralen had no bactericidal activity but could widely inhibit the production of extracellular proteases, pyocyanin, and biofilm, and the cell motilities of the model and clinical P. aeruginosa strains. RNA-sequencing and quantitative PCR analyses further demonstrated that a majority of QS-activated genes in P. aeruginosa were suppressed by psoralen. The supplementation of psoralen could protect Caenorhabditis elegans from P. aeruginosa challenge, especially for the hypervirulent strain PA14. Moreover, psoralen showed synergistic antibacterial effects with polymyxin B, levofloxacin, and kanamycin. In conclusions, this study identifies the anti-QS and antibiofilm effects of psoralen against P. aeruginosa strains and sheds light on the discovery of anti-pseudomonal drugs among Traditional Chinese Medicines. KEY POINTS: • Psoralen derived from Psoralea corylifolia L. inhibits the virulence-related phenotypes of P. aeruginosa. • Psoralen simultaneously targets the three core regulators of P. aeruginosa QS system and inhibits the expression of a large part of downstream genes. • Psoralen protects C. elegans from P. aeruginosa challenge and enhances the susceptibility of P. aeruginosa to antibiotics.

Keywords: Drug combination; Functional profiling; Pseudomonas aeruginosa; Psoralen; Quorum sensing inhibitor; Traditional Chinese Medicine.

Fig. 1

Structure of P. aeruginosa QS signal molecules and psoralen. Native signal molecules 3-oxo-C12-HSL (a), C4-HSL (b), PQS (c) of P. aeruginosa QS system. d Psoralen. e The growth of PAO1 in MHB containing different concentrations of psoralen (37 °C, 18 h). f The growth of PAO1 in LB broth containing different concentrations of psoralen (37 °C, 24 h). g The growth curves of PAO1 in M9-adenosine (0.1%, w/v) containing different concentrations of psoralen (37 °C, 36 h). The results were presented as mean ± SD and three independent experiments. The analysis method was used one-way ANOVA; ns, not significant

Fig. 2

Effects of psoralen on phenotypes of P. aeruginosa. a The growth of PAO1 in M9-casein (0.5%, w/v) medium containing different concentrations of psoralen. b The image of growth of PAO1 in M9-casein (0.5%, w/v) medium. c The pyocyanin production of PAO1 in LB broth containing different concentrations of psoralen. The effects of psoralen (200 μM) on motility of PAO1 (d–f). d Swarming motility. e Swimming motility. f Twitching motility. The effects of psoralen on biofilm formation of P. aeruginosa (g, h). g The effect on PAO1 biofilm formation. h The effects on clinical isolates biofilm formation. The results were presented as mean ± SD and three independent experiments. The analysis methods were used one-way ANOVA and t test; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Effects of psoralen (200 μM) supplementation with AIs on phenotypic experiments of P. aeruginosa. a The growth of PAO1 in M9-adenosine (0.1%, w/v) medium containing psoralen supplementation C12-HSL. b The growth of PAO1 in M9-casein (0.5%, w/v) medium containing psoralen supplementation with different AIs. c The pyocyanin production of PAO1 in LB broth containing psoralen supplementation with different AIs. The effects of psoralen supplementation with different AIs on motility of PAO1 (d, e). d Swimming motility. e Twitching motility. f The biofilm formation of PAO1 containing psoralen supplementation with different AIs. g The image of motility of PAO1 containing psoralen supplementation with different AIs. The results were presented as mean ± SD and three independent experiments. The analysis method was used one-way ANOVA; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

The mechanism of anti-QS and antibiofilm activity of psoralen. a Volcano plot of differentially expressed genes. b Psoralen significantly influenced KEGG pathways. c Venn diagram of differentially expressed genes. d Expression of important QS genes of psoralen-treated PAO1 was determined by qPCR. e The expression of lasB in PAO1-ΔlasI under different conditions. f The expression of rhlA in PAO1-ΔrhlI under different conditions. g The expression of phzA1 in PAO1-ΔpqsA under different conditions. The results were presented as mean ± SD and three independent experiments. C7 indicated psoralen-treated PAO1. The analysis methods were used one-way ANOVA and t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Effects of antibiotic-psoralen combinations on growth of PAO1 and clinical isolates. Effects on growth of PAO1 and clinical isolates combining antibiotics with psoralen (a, c, e). The growth curves of PAO1 and clinical isolates combining sub-MICs with different concentrations of psoralen (b, d, f). a PAO1. b PAO1, and polymyxin B (PB): 1 μg/mL. c Clinical isolate 3–100-1. d Clinical isolate 3–100-1, and levofloxacin (LVX): 1 μg/mL. e Clinical isolate 5-R5-3A. f Clinical isolate 5-R5-3A, and kanamycin (Kan): 6 μg/mL. The results were presented as mean ± SD and three independent experiments

Fig. 6

Protective effects of psoralen in C. elegans infection models. a PAO1-killing assay. b PA14-killing assay. c Clinical isolate P. aeruginosa 4–61-8-killing assay. The concentration of psoralen was 200 µM, and the concentration of kanamycin was 24 µg/mL. Log-rank (Mantel-Cox) test