Anti-Biofilm and Anti-Inflammatory Properties of the Truncated Analogs of the Scorpion Venom-Derived Peptide IsCT against Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen in humans and a frequent cause of severe nosocomial infections and fatal infections in immunocompromised individuals. Its ability to form biofilms has been the main driving force behind its resistance to almost all conventional antibiotics, thereby limiting treatment efficacy. In an effort to discover novel therapeutic agents to fight P. aeruginosa-associated biofilm infections, the truncated analogs of scorpion venom-derived peptide IsCT were synthesized and their anti-biofilm properties were examined. Among the investigated peptides, the IsCT-Δ6-8 peptide evidently showed the most potential anti-P. aeruginosa biofilm activity and the effect was not due to bacterial growth inhibition. The IsCT-Δ6-8 peptide also exhibited inhibitory activity against the production of pyocyanin, an important virulence factor of P. aeruginosa. Furthermore, the IsCT-Δ6-8 peptide significantly suppressed the production of inflammatory mediators nitric oxide and interleukin-6 in P. aeruginosa LPS-induced macrophages. Due to its low cytotoxicity to mammalian cells, the IsCT-Δ6-8 peptide emerges as a promising candidate with significant anti-biofilm and anti-inflammatory properties. These findings highlight its potential application in treating P. aeruginosa-related biofilm infections.

Keywords: P. aeruginosa; anti-biofilm; anti-inflammation; pyocyanin; scorpion peptide.

Figure 1

The effects of the truncated IsCT analogs against P. aeruginosa biofilms. P. aeruginosa culture cells were incubated in the absence or presence of the truncated IsCT analogs (final concentration, 150 μM) at 37 °C for 24 h. Biofilm biomass was determined by crystal violet staining and the OD was measured at 550 nm. Values are expressed as the mean ± SEM of two independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01 compared with the untreated control.

Figure 2

The effects of the IsCT-Δ6-8 peptide on the growth of P. aeruginosa. The culture of P. aeruginosa was treated with the IsCT-Δ6-8 peptide at a final concentration of 150 μM. The bacterial culture alone served as an untreated control. The cultures were incubated at 37 °C and bacterial growth was assessed by measuring OD600 at the indicated time points. Values are expressed as the mean ± SEM of two independent experiments performed in duplicate. #, not statistically different to the untreated control (p > 0.5).

Figure 3

Effects of the IsCT-Δ6-8 peptide on pyocyanin production in P. aeruginosa. A culture of P. aeruginosa was treated with varying concentrations of the IsCT-Δ6-8 peptide (37.5, 75 and 150 μM) for 24 h, and pyocyanin levels were quantified using the chloroform extraction method. Values are expressed as mean ± SEM of two independent experiments performed in duplicate. *, p < 0.05 compared with the untreated control.

Figure 4

Effects of the IsCT-Δ6-8 peptide on the viability of RAW 264.7 macrophages. RAW 264.7 macrophage cells were treated with various concentrations of the IsCT-Δ6-8 peptide (37.5, 75 and 150 μM) for 48 h. Cell viability was assessed by the MTT assay.

Figure 5

Effects of the IsCT-Δ6-8 peptide on NO production in P. aeruginosa LPS-stimulated RAW 264.7 macrophages. RAW 264.7 macrophages were incubated with P. aeruginosa LPS in the presence or absence of the IsCT-Δ6-8 peptide (37.5–150 μM) for 48 h at 37 °C under 5% CO₂. Nitrite levels in the culture supernatant were determined by Griess reagents. ###, p < 0.001 compared with unstimulated RAW 264.7 macrophages; *, p < 0.05 compared with the P. aeruginosa LPS-stimulated RAW 264.7 macrophages.

Figure 6

Effects of the IsCT-Δ6-8 peptide on IL-6 production in P. aeruginosa LPS-stimulated RAW 264.7 macrophages. RAW 264.7 macrophages were incubated with P. aeruginosa LPS in the presence or absence of IsCT-Δ6-8 peptide (37.5–150 μM) for 48 h at 37 °C under 5% CO₂. The level of IL-6 in culture supernatant was measured by sandwich ELISA. ###, p < 0.001 compared with the unstimulated RAW 264.7 macrophages; *, p < 0.05 compared with the P. aeruginosa LPS-stimulated RAW 264.7 macrophages.