An Enhanced Variant Designed From DLP4 Cationic Peptide Against Staphylococcus aureus CVCC 546.

Bing Li,Ruoyu Mao,Ya Hao,Zhanzhan Li,Jianhua Wang,Da Teng,Na Yang,Xiumin Wang,Zhenlong Wang

doi:10.3389/fmicb.2020.01057

Abstract

Insect defensins are promising candidates for the development of potent antimicrobials against antibiotic-resistant Staphylococcus aureus (S. aureus). An insect defensin, DLP4, isolated from the hemolymph of Hermetia illucens larvae, showed low antimicrobial activity against Gram-positive (G+) pathogens and high cytotoxicity, which limited its effective therapeutic application. To obtain more potent and low cytotoxicity molecules, a series of peptides was designed based on the DLP4 template by changing the conservative site, secondary structure, charge, or hydrophobicity. Among them, a variant designated as ID13 exhibited strong antibacterial activity at low MIC values of 4–8 μg/mL to G+ pathogens (S. aureus: 4 μg/mL; Staphylococcus epidermidis: 8 μg/mL; Streptococcus pneumoniae: 4 μg/mL; Streptococcus suis: 4 μg/mL), which were lower than those of DLP4 (S. aureus: 16 μg/mL; S. epidermidis: 64 μg/mL; S. pneumoniae: 32 μg/mL; S. suis: 16 μg/mL), and cytotoxicity of ID13 (71.4% viability) was less than that of DLP4 (63.8% viability). ID13 could penetrate and destroy the cell membrane of S. aureus CVCC 546, resulting in an increase in potassium ion leakage; it bound to genomic DNA (gDNA) and led to the change of gDNA conformation. After treatment with ID13, perforated, wrinkled, and collapsed S. aureus CVCC 546 cells were observed in electron microscopy. Additionally, ID13 killed over 99.99% of S. aureus within 1 h, 2 × MIC of ID13 induced a post-antibiotic effect (PAE) of 12.78 ± 0.28 h, and 10 mg/kg ID13 caused a 1.8 log10 (CFU/g) (CFU: colony-forming units) reduction of S. aureus in infected mouse thigh muscles and a downregulation of TNF-α, IL-6, and IL-10 levels, which were superior to those of DLP4 or vancomycin. These findings indicate that ID13 may be a promising peptide antimicrobial agent for therapeutic application.

Highlights

Antibiotics play pivotal roles in disease prevention, growth promotion, and production improvement in animal husbandry (Ferber, 2002; Cheng et al, 2014; Van Boeckel et al, 2015)
The helix always ends with the R23 residue, followed by a G residue which is involved in a hydrogen bond with the carbonyl group of C3 (Landon et al, 2008)
Hydrophobic side chains distributed over the surface present a number of hydrophobic spots, some of which correspond to conservative amino acid (AA): A1, T2, L5, L6, A15, and A17; others are more specific: P8, A14, and W29 for DLP4, I9, L18, L21, and F22 for Sapecin C; and I11 for Phormicin A

Summary

Introduction

Antibiotics play pivotal roles in disease prevention, growth promotion, and production improvement in animal husbandry (Ferber, 2002; Cheng et al, 2014; Van Boeckel et al, 2015). The long-term use of antibiotics in animal breeding can accelerate the emergence of antibiotic-resistant bacteria (ARB), including Staphylococcus aureus, which can cause trauma infection, abscess, cellulitis, mastitis, endometritis, arthritis, septicemia, and sepsis in animals. The interactions among animals, humans, and the environment have strengthened the spreading of ARB (Cheng et al, 2013; Laxminarayan et al, 2013; Maertens et al, 2019). These factors affect the effectiveness of prevention and treatment of animal diseases and may endanger public safety (Coyne et al, 2016, 2019). Alternatives to antibiotics have become the spotlights of research in recent years (Lammie and Hughes, 2016; Moravej et al, 2018a)

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