A scorpion venom peptide derivative BmKn‒22 with potent antibiofilm activity against Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a leading cause of nosocomial and serious life-threatening infections and infections caused by this bacterium continue to pose a major medical challenge worldwide. The ability of P. aeruginosa to produce multiple virulence factors and in particular to form biofilms makes this bacterium resistant to all known antibiotics. As a consequence, standard antibiotic therapy are increasingly become ineffective to clear such infections associated with biofilms. In search for novel effective agents to combat P. aeruginosa biofilm infections, a series of the BmKn‒2 scorpion venom peptide and its truncated derivatives were synthesized and their antibiofilm activities assessed. Among the peptides tested, BmKn‒22 peptide, which was a modified peptide of the parental BmKn‒2 scorpion venom peptide, clearly demonstrated the most potential inhibitory activity against P. aeruginosa biofilms without affecting the bacterial growth. This peptide was not only capable of inhibiting the formation of P. aeruginosa biofilms, but also disrupting the established biofilms of P. aeruginosa. Additionally, BmKn‒22 peptide was able to inhibit the production of key virulence factor pyocyanin of P. aeruginosa. Our results also showed that BmKn‒22 peptide significantly reduced lasI and rhlR expression, and suggested that BmKn‒22 peptide-mediated inhibition of P. aeruginosa biofilms and virulence factors was achieved through the components of quorum-sensing systems. Combination of BmKn‒22 peptide with azithromycin resulted in a remarkable reduction P. aeruginosa biofilms. Since this peptide exhibited low toxicity to mammalian cells, all our results therefore indicate that the BmKn‒22 peptide is a promising antibiofilm agent against P. aeruginosa and warrant further development of this peptide as a novel therapeutic for treatment of P. aeruginosa‒associated biofilm infections.

Fig 1. Antibiofilm activities of BmKn‒2 peptide and its derivatives against P. aeruginosa.

Bacterial cells were incubated with BmKn‒2 peptide and derivatives at the final concentration of 800 μM. After incubation at 37°C for 24 h, biofilm biomass was assessed by crystal violet staining assay and OD measured at 550 nm. Data are expressed as mean ± SEM of three independent experiments. *, P < 0.05 and **, P < 0.01 compared with the untreated control. A dot black line represents an OD cut-off value.

Fig 2. Effect of BmKn‒2 peptide and its derivatives on P. aeruginosa growth.

Bacterial cells were treated with the test peptides and incubated at 37°C for 24 h. Bacterial growth was assessed by standard plate counts. Data are expressed as mean ± SEM of three independent experiments. **, P < 0.01 and ***, P < 0.001 compared with the untreated control.

Fig 3. Toxicity of BmKn‒2 peptide and its derivatives against mammalian cells.

Hemolytic activity of BmKn‒2 peptide and derivatives against sheep red blood cells (A), toxicity of BmKn‒2 peptide and derivatives against L929 cells determined by MTT assay (B). Values are expressed as mean ± SEM of three independent experiments. ***, P < 0.001 compared with the untreated control.

Fig 4. Inhibitory effects of BmKn‒22 and BmKn‒23 peptides on biofilm formation and preformed (24-h old) biofilms of P. aeruginosa.

Data are represented as mean ± SEM of three independent experiments.

Fig 5. Effect of BmKn‒22 peptide on pyocyanin production of P. aeruginosa.

P. aeruginosa were treated with BmKn‒22 peptide at concentrations of 200 ‒ 800 μM for 24 h, and pyocyanin was determined by chloroform extraction method. Data are expressed as mean ± SEM of three independent experiments. *, P < 0.05 and **, P < 0.01 compared with the untreated control.

Fig 6. Effect of BmKn‒22 peptide on mRNA expression of quorum sensing-related genes in P. aeruginosa.

Bacterial cells were treated with BmKn‒22 peptide for 10 h and the levels of mRNA expression determined by quantitative real-time PCR. Values are expressed as the mean ± SEM of two independent experiments done in quadruplicate. *, P < 0.05 compared with the untreated control.

Fig 7. Effect of BmKn‒22 peptide and azithromycin, tested alone or in combination, on P. aeruginosa biofilms.

Bacterial cells were treated with BmKn‒22 peptide alone or in combination with azithromycin for 24 h at 37°C, and biofilm biomass was assessed by crystal violet staining assay and OD measured at 550 nm. Values are expressed as the mean ± SEM of three independent experiments. **, P < 0.01 and ***, P < 0.001.