Symmetrical Modification of Minimized Dermaseptins to Extend the Spectrum of Antimicrobials with Endotoxin Neutralization Potency

Abstract

Antimicrobial peptides (AMPs) have emerged as a promising class of antimicrobial agents that could potentially address the global antibiotic resistance. Generating mirror-like peptides by minimizing dermaseptin family sequences is an effective strategy for designing AMPs. However, the previous research still had some limitations such as lower effectiveness and a narrow spectrum of antibacterial activity. To further expand and hone this strategy, we designed a series of AMPs consisting of the WXMXW-NH₂ motif (X represents V, I, F, and W; M represents KAAAKAAAK). The peptides formed α-helices and displayed broad-spectrum antimicrobial activities against eleven types of clinical bacteria including both Gram-negative and Gram-positive bacteria. The optimized peptide WW exhibited high physical rupture by inducing membrane shrinkage, disruption, and lysis. Moreover, WW effectively neutralized endotoxins and inhibited the inflammatory response while having the highest therapeutic index. In conclusion, these results indicated that the peptide WW has potential as a broad-spectrum antimicrobial agent or preservative for overcoming the risk of multidrug resistance in localized or external therapeutic applications.

Keywords: antibacterial mechanism; antimicrobial peptide; endotoxin neutralization; minimized dermaseptins.

Figure 1

The structure of α-helix forming peptides. (A) The 3D structure of the peptides predicted by I-TASSER. The peptides are displayed by a solid ribbon style which was colored online according to secondary structures. red square: α-helix. (B,C) The CD spectra of the peptides in 10 mM PBS (pH 7.4) (B) and 30 mM SDS (C). An average of three scans was recorded for each peptide.

Figure 2

Cytotoxicity of the peptides against human red blood cells (hRBCs) (A), RAW 264.7 (B), and HEK293T (C). Results were given as the mean ± standard deviation (SD) of three independent trials. Differences between groups exposed to the same concentration were determined by one-way ANOVA followed by Turkey’s post-hoc analysis (n = 3). The values with different superscripts (a, b, c, d) indicate a significant difference (p < 0.05).

Figure 3

The influence of WW and melittin on the outer membrane permeability (A), cytoplasmic membrane depolarization (B), and membrane integrity (C). Results in (A) are given as the mean ± standard deviation (SD) of three independent experiments and the difference (* p < 0.05) between two groups exposed to the same peptide concentration was analyzed by the Student’s t test. The results in (B) were expressed as an average of three independent trials. The data in (C) are representative of three independent experiments.

Figure 4

Scanning electron micrographs of E. coli 25922 and S. aureus 29213 treated with WW at 1 × MIC. All data are representative of three independent experiments.

Figure 5

Transmission electron micrographs of E. coli 25922 and S. aureus 29213 treated with WW at 1 × MIC. All data are representative of three independent experiments.

Figure 6

(A) Peptide binding affinity to LPS from E. coli 0111:B4. Results in Figure 6A are given as mean ± standard deviation (SD) of three independent experiments and the difference (n.s., not significant at p < 0.05) between two groups exposed to the same peptide concentration was analyzed by the Student’s t test. (B,C) WW inhibited the LPS-induced gene expression of pro-inflammatory cytokine effects in the murine macrophage cell line RAW264.7. Results are given as the mean ± SD. Differences between groups were determined by one-way ANOVA followed by Turkey’s post-hoc analysis (n = 3). The values with different superscripts (a, b, c) indicated a significant difference (p < 0.05).