The Trp-rich Antimicrobial Amphiphiles With Intramolecular Aromatic Interactions for the Treatment of Bacterial Infection

Abstract

Antibiotic resistance is emerging as a hot issue with the abuse and overuse of antibiotics, and the shortage of effective antimicrobial agents against multidrug resistant bacteria creates a huge problem to treat the threatening nosocomial skin and soft tissue infection. Antimicrobial peptides (AMPs) exhibite enormous potential as one of the most promising candidates of antibiotic to fight against pathogenic infections because of its unique membrane penetration mechanism to kill pathogens, whereas the clinical application of AMPs still faces the challenges of production cost, stability, safety, and design strategy. Herein, a series of Trp-rich peptides was designed following the principle of paired Trp plated at the ith and ith+4 position on the backbone of peptides, based on the template (VKKX)₄, where X represents W, A, or L, to study the effect of intramolecular aromatic interactions on the bioactivity of AMPs. Through comparing the antimicrobial performance, hemolysis, cytotoxicity, and stability, VW5 which is equipped with the characters of direct antimicrobial efficacy (GM=1.68μM) and physical destruction of bacterial membrane (SEM and electron microscopy) stood out from the engineering peptides. VW5 also performed well in mice models, which could significantly decrease the bacterial colony (VW5 vs infection group, 12.72±2.26 vs 5.52±2.01×10⁹CFU/abscess), the area of dermo-necrosis (VW5 vs infection group, 0.74±0.29 vs 1.86±0.98mm²) and the inflammation cytokine levels at the abscess site without causing toxicity to the skin. Overall, this study provides a strategy and template to diminish the randomness in the exploration and design of novel peptides.

Keywords: antimicrobial activity; antimicrobial peptide; bacterial infection; intramolecular aromatic interaction; subcutaneous abscess.

Figure 1

(A) Schematic structure of peptides based on the template (VKKX)₄, where X represents W, A, or L. (B) The backbone structure and potential surface of peptides. The green structure represents Ala residues, the red structure is Trp residues, and the blue structure represents other amino acid residues involved in the sequence. (C) The helical wheel projections of peptides. The charged amino acid residues are colored as dark blue and the hydrophobic amino acid residues are painted as dark yellow. (D) The circular dichroism (CD) spectrum of peptides.

Figure 2

(A) The hemolysis activity of peptides at different concentration (0.25μM, 0.5μM, 1μM, 2μM, 4μM, 8μM, 16μM, 32μM, 64μM, and 128μM). (B) Cytotoxicity of engineering peptide to HEK293T (B1), IPEC-J2 (B2), and RAW 264.7 cells (B3) followed by the MTT methods at different concentrations (2μM, 4μM, 8μM, 16μM, 32μM, 64μM, and 128μM). The data represented as means ± standard deviations of three independent experiments. (C) The killing curve of peptides against bacteria. The eruginosa ATCC 27853 and S. aureus ATCC 29213 were diluted into1×10⁵CFU/ml, and treated with VW5 (C1,C3) and melittin (C3, C4) at their 1×MIC, 2×MIC and 4×MIC after 0, 5, 10, 30, 60, and 120min treatment. The colony counting was performed to determine the survival rate of bacterial cells by plating on MHA plates.

Figure 3

(A) Schematic of the model of action of antimicrobial peptide. (B) LPS binding affinity of VW5 at different concentrations. The BODIPY-TR cadaverine fluorescent dye displacement assay was employed to evaluate the LPS binding activity of peptides at an excitation wavelength of 580nm and an emission wavelength of 620nm. (C) The outer membrane permeabilization of P. aeruginosa ATCC 27853 cells. The fluorescence change of 1-N-phenylnaphthylamine (NPN) dye was used to study the outer membrane permeabilization after the treatment of VW5 and melittin at a series of concentrations (1μM, 2μM, 4μM, 8μM, 16μM, 32μM, and 64μM). (D) Cytoplasmic membrane depolarization of P. aeruginosa ATCC 27853 after incubation of VW5 and melittin at 2 MIC, MIC, and 1/2 MIC concentration. (E) Scanning electron microscopic micrographs of P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 after the treatment of VW5 at MIC (1μM for P. aeruginosa ATCC 27853 and 2μM for S. aureus ATCC 29213). (F) Transmission electron microscope of bacterial cells treated with VW5. The bacterial specimen of control received no treatment.

Figure 4

(A) Illustration of experimental protocol. (B) The therapeutic effect of peptides on bacterial abscess. The colony counting of abscess tissue (B1) and the area of lesion after treatment at 3 d. The saline group is the infected mice treated with equivalent saline and the control group refers to the uninfected mice. (C) The histological examination of abscess site by HE staining. The black arrows indicate the condition of the epidermis. The epidermis of the saline treatment was infiltrated with inflammatory cells, whereas the epidermis of VW5 treatment and ciprofloxacin treatment maintained integrity. Large-area necrosis tissue overlayed the epidermis (red arrow) of saline treatment at the abscess site. The cell debris and large amount of inflammatory cell infiltration could be observed in the dermis layer (green arrow). (D) The inflammatory cytokines level after the treatment of saline, VW5, and ciprofloxacin. (E) The skin toxicity of VW5. The TUNEL assay was performed to detect the apoptotic cells (green) in mice skin, and the nuclei of the cells were counterstained with DAPI (blue). DMSO (dimethyl sulfoxide) injection serves as a positive control.