Novel D-form of hybrid peptide (D-AP19) rapidly kills Acinetobacter baumannii while tolerating proteolytic enzymes

Abstract

Antimicrobial peptides (AMPs) are being developed as potent alternative treatments to conventional antibiotics which are unlikely to induce bacterial resistance. They can be designed and modified to possess several druggable properties. We report herein a novel hybrid peptide of modified aurein (A3) and cathelicidin (P7), or A3P7, by a flipping technique. It exhibited potent antibacterial activity against both Gram-negative and -positive pathogenic bacteria but had moderate hemolytic activity. To reduce the sequence length and toxicity, C-terminal truncation was serially performed and eight truncated derivatives (AP12-AP19) were obtained. They had significantly less hemolytic activity while preserving antibacterial activity. Secondary structures of the candidate peptides in environments simulating bacterial membranes (30 mM SDS and 50% TFE), determined by CD spectroscopy, showed α-helical structures consistent with predicted in silico 3D structural models. Among the peptides, AP19 demonstrated the best combination of broad-spectrum antibacterial activity (including toward Acinetobacter baumannii) and minimal hemolytic and cytotoxic activities. A D-form peptide (D-AP19), in which all L-enantiomers were substituted with the D-enantiomers, maintained antibacterial activity in the presence of pepsin, trypsin, proteinase K and human plasma. Both isomers exhibited potent antibacterial activity against multi-drug (MDR) and extensively-drug resistant (XDR) clinical isolates of A. baumannii comparable to the traditional antibiotic, meropenem. D-AP19 displayed rapid killing via membrane disruption and leakage of intracellular contents. Additionally, it showed a low tendency to induce bacterial resistance. Our work suggested that D-AP19 could be further optimized and developed as a novel compound potentially for fighting against MDR or XDR A. baumannii.

Figure 1

The hemolytic activity of parent peptide, A3P7, and its derivatives compared with that of melittin (a positive control). Three independent experiments were performed and the data are presented as mean ± SD. The statistical analyses utilized one-way ANOVA and Tukey’s test (GraphPad Prism7). Star indicates a significant difference from negative control (*p-value < 0.05).

Figure 2

The cytotoxicity of AP19 and D-AP19 against L929 mouse fibroblast cells compared with that of melittin (a positive control). Three independent experiments were performed and the data are presented as mean ± SD. The statistical analyses utilized one-way ANOVA and Tukey’s test at p-value < 0.05 (GraphPad Prism7). Star indicates a significant difference from negative control (p-value < 0.05).

Figure 3

The circular dichroism spectra of melittin, A3P7, AP19, AP18, AP17, AP16, AP13 and D-AP19 in PBS (A), 30 mM SDS micelles in PBS (B), and 50% (v/v) TFE in PBS (C). Peptides were dissolved in each solution, filled in a 0.1 cm path length rectangular quartz cell and measured at 25 °C by Jasco-815 spectropolarimeter. Three scans of CD spectra were analyzed in the wavelength range of 190 to 260 nm at scanning speed of 10 nm/min.

Figure 4

Time-kill kinetics of D-AP19 at 1 × MIC against A. baumannii ATCC 19,606 for 24 h. Control (blue line) indicates untreated bacteria grown in MHB-DI solution. Green line indicates A. baumannii ATCC 19,606 treated with D-AP19 at 1 × MIC.

Figure 5

Percentage of fluorescent positive cells stained with PI or BOX after treatment with D-AP19 for 2 h. A. baumannii ATCC 19,606 was treated with half MIC (A) or 1 × MIC (B), and stained with a fluorescent dye.

Figure 6

Scanning electron microscopic micrographs of A. baumannii ATCC 19,606 treated with D-AP19. Untreated bacterial cells (A), (B) and treated with half MIC of D-AP19 for 15 min (C)–(F).

Figure 7

Transmission electron microscopic micrographs of A. baumannii ATCC 19,606 treated with D-AP19. Untreated bacterial cells (A), (B) and cells treated with half MIC of D-AP19 for 15 min (C)–(F). Black, white and red arrows indicate inclusion bodies, intracellular leakage and ghost cells in bacterial cells, respectively.

Figure 8

Fold changes of MICs of D-AP19 and meropenem against A. baumannii ATCC 19,606. The serial passages of bacteria were grown for 20 days in the presence of test compounds at different concentrations. The MIC and MBC values were determined after each passage.