Effects of human β-defensin 3 fused with carbohydrate-binding domain on the function of type III secretion system in Pseudomonas aeruginosa PA14

Abstract

Antimicrobial peptides are considered to be one of the candidate antimicrobial agents for antibiotic-resistant bacterial infection in the future. The effects of antimicrobial peptide hBD3-CBD on Pseudomonas aeruginosa PA14 and PA14 ΔexsA were analyzed by the bactericidal effects, hemolysis assays, pyocyanin pigment productions, and virulence factor expressions (exoU, exoS, hcnA, and lasB). Pyocyanin production and virulence factor expressions are important features of the type III secretion system in Pseudomonas aeruginosa. HBD3-CBD killed PA14 and PA14 ΔexsA with similar efficiency; it lowered the hemolysis levels of PA14 and PA14 ΔexsA and reduced the pyocyanin production, biofilm formation, and exoU, exoS, and lasB expressions in PA14. Compared with PA14, PA14 ΔexsA showed a lower hemolysis effect, pyocyanin production, exoU, and lasB expressions. The effects of hBD3-CBD on the PA14 toxin secretion were similar to the changes in the type III secretion system mutant isolate PA14 ΔexsA. Our results demonstrated that the type III secretion system was involved in the biological functions on PA 14 from hBD3-CBD.

Keywords: Antimicrobial peptide; Pseudomonas aeruginosa; T3SS; hBD3.

Fig. 1

Bactericidal activity in vitro of the peptide against P. aeruginosa PA14 and PA14 ΔexsA. Killing curves at different peptide concentrations (0, 4, 8, 16, 32, and 64 μg/mL) were performed using PA14 and PA14 ΔexsA. Data are presented as the mean ± standard deviation of three independent experiments. Statistical analysis comparing groups was performed by Student–Newman–Keuls t test. Significance was accepted when the P value was < 0.05. There was no difference between the bactericidal activities on these two bacteria

Fig. 2

Peptide hBD3-CBD’s effects on the hemolysis effects of P. aeruginosa PA14 and PA14 ΔexsA. Sheep erythrocytes were used in the hemolysis analysis, and the hemoglobin release was assessed by the absorbances at 540 nm. Peptide hBD3-CBD significantly reduced the hemolysis activities of PA14 and PA14 ΔexsA. Data are presented as the mean ± standard deviation of three independent experiments. Statistical analysis comparing groups was performed by Student–Newman–Keuls t test. Significance was accepted when the P value was < 0.05. * means a significant difference to PA14; # means a significant difference to PA14 ΔexsA

Fig. 3

Peptide hBD3-CBD’s effects on the pyocyanin productions (a) and biofilm formation (b) of P. aeruginosa PA14 and PA14 ΔexsA. Pyocyanin productions and biofilm formation in type III inducing conditions were detected by the absorbances at 520 nm and 570 nm, respectively. Peptide hBD3-CBD decreased the pyocyanin productions and the biofilm formation in PA14 and PA14 ΔexsA. Data are presented as the mean ± standard deviation of three independent experiments. Statistical analysis comparing groups was performed by Student–Newman–Keuls t test. Significance was accepted when the P value was < 0.05. * means a significant difference to PA14; # means a significant difference to PA14 ΔexsA

Fig. 4

Gene expressions of peptide-treated P. aeruginosa PA 14 and PA14 ΔexsA. T3SS-related gene exoU (a), exoS (b), hcnA (c), and lasB (d) were detected by quantitative real-time PCR. Gene expression levels were showed as the ratio to the expression in PA14. Peptide hBD3-CBD treatment can decrease the expressions of exoU, exoS, and lasB in the PA14 group and all four genes in the PA14 ΔexsA group. Gene lasB was high-regulated in the PA14 ΔexsA group than the PA14 group. Data are presented as the mean ± standard deviation of three independent experiments. Statistical analysis comparing groups was performed by Student–Newman–Keuls t test. Significance was accepted when the P value was < 0.05. * means a significant difference to PA14; # means a significant difference to PA14 ΔexsA