Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System

Abstract

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin's impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.

Keywords: PQS inhibition; Pseudomonas aeruginosa; Type III secretion system; baicalin; mouse lung infection model; virulence factors.

Figure 1

Inhibition of virulence genes expression and swarming motility by the extract of Scutellariae radix. (A) Inhibition on the expression of lasR, exoS, rhlI and rhlR by the ethanolic extract of Scutellariae radix. 1, 1/2, 1/4 and 1/8 represent the concentrations of exacts at 172.5 mg/mL, 86.25 mg/mL, 43.13 mg/mL, 21.56 mg/mL respectively; C, solvent as control. (B) Inhibition of swarming motility in P. aeruginosa PAO1 by the aqueous and ethanolic extracts. The concentration of extracts was 43.13 mg/mL.

Figure 2

Repression on the virulence genes expression and phenotypes in P. aeruginosa by baicalin (A) The chemical structure of baicalin. (B) Inhibition on the expression of T3SS genes exoS, exsD, and exoT by baicalin. Five microliters of baicalin solution was dropped on the plates. 1, 1/2 and 1/4 represent that the concentrations of baicalin are 25, 12.5 and 6.25 mg/mL respectively; C, solvent as control. (C) Repression of swarming motility bybaicalin. Baicalin was added at 250 µg/mL. Inhibition of rhamnolipid production (D) and elastase production (E) by baicalin. Baicalin was added at 250 µg/mL. Averages of triplicate experiments ± standard errors of the means are shown. The results shown are representative of three independent experiments with similar trends. * significant difference (p < 0.05).

Figure 3

Inhibition of cytotoxicity of P. aeruginosa PAO1 to eukaryotic cells by baicalin. 1/4 and 1/16 represent 250 μg/mL and 62.5 μg/mL of baicalin respectively; Averages of triplicate experiments ± standard errors of the means are shown. The results shown are representative of three independent experiments with similar trends. ** very significant difference (p < 0.01).

Figure 4

Inhibition of pathogenicity of PAO1 by baicalin in fly infection model. Baicalin was added at 250 µg/mL. Averages of three independent experiments ± standard errors of the means are shown.

Figure 5

Morphological pathology of rat lungs in different experimental groups. (A,B) Panels represent the lungs of rats in Model group. (C–E) represent the blank Control group, Cefepime-treated group and Baicalin-treated group.

Figure 6

Microscopic changes of the lung tissues of the different animal groups (H&E staining, magnification: ×200). Panels (A–D) show pathology of Model group, Blank uninfected control group, Baicalin-treated group and Cefepime-treated group respectively. (A) lung hyperemia, large amount of inflammatory cell infiltration, vascular and alveolar walls dilatation; (B) light lung hyperemia; (C) milder lung hyperemia and few inflammatory cell infiltrations; (D) alveolar walls widened, few inflammatory cell infiltration and milder lung hyperemia.

Figure 7

Levels of TNF-α in serum of rats in different groups. Averages of triplicate experiments ± standard errors of the means are shown. The results shown are representative of three independent experiments with similar trends. * significant difference (p < 0.05); ** very significant differences (p < 0.01); *** highly significant differences (p < 0.001).

Figure 8

Relative expression levels of exoS in PAO1, ΔpqsR and ΔpqsH under different conditions. The activity of exoS promoter in soft agar medium was measured as described in Materials and Methods. Baicalin was added at 250 µg/mL. PQS was added at 1 µg/mL. After overnight incubation at 37 °C, light production of the reporter strain was recorded by a LAS-3000 imaging system (Fuji Corp, Tokoyo, Japan). The relative promoter activity was evaluated by the software multi gauge version 3.0. AU strands for Arbitrary Unit, a unit to measure the amount of chemiluminescence recorded by LAS-3000. It signifies the relative density value accumulated as linear data by a CCD camera in the image surface. Averages of triplicate experiments ± standard errors of the means are shown. The results shown are representative of three independent experiments with similar trends. * significant difference (p < 0.05) ns, no significant difference.