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

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 log 10 (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.

Design and Pharmacodynamics of Recombinant Fungus Defensin NZL with Improved Activity against Staphylococcus hyicus In Vitro and In Vivo

“…As shown in Figure 2 A, the circular dichroism (CD) spectra showed that NZ2114 and NZL had a positive peak at 196 nm and two negative peaks at approximately 208 and 228 nm, respectively, which demonstrated that the peptides were characterized by α-helix and β-sheet or random coil in various environments [ 26 ]. However, the CD peak of NZL showed a significant increase in α-helix in 40 mM sodium dodecyl sulfate (SDS) mimicking the bacterial membrane, indicating that NZL tended to form α-helix structure and enhance membrane interactions [ 27 ]. Additionally, the three-dimensional structure modeling result predicted that similar to NZ2114, NZL possessed a typical cystine-stabilized alpha-beta structure (CSαβ) conformation, including an α-helix (residues 12–20), an antiparallel β-sheet (residues 17–31 and 36–40), and three disulfide bonds (Cys4–Cys30, Cys15–Cys37, and Cys19–Cys39) ( Figure 2 B).…”

“…The presence of resistant S. hyicus and high morbidity rates (up to 90%) during exudative epidermitis outbreaks often make it a laborious and economically impractical task [ 6 , 7 , 29 , 30 ]. AMPs, as new therapeutic agents in the future, are currently under evaluation, whereas some challenges such as low antimicrobial activity and relatively high cytotoxicity limit their clinical applications [ 27 , 31 ]. It has been argued that the most difficult problem in the development of AMPs is that new candidates without optimization have been put into preclinical and clinical trials too quickly, thereby leading to failure, which highlights the importance of optimization in the early stages of laboratory development [ 32 , 33 ].…”

“…The MIC value obtained is a crucial factor in the initial selection of candidate peptides [ 32 , 40 ]. Besides, the toxicity and stability of AMPs are the developmental obstacles for their clinical applications [ 27 ]. In this work, compared with NZ2114, NZL had lower MIC values (0.23~0.92 μM) ( Table 2 ), hemolytic activity (1.11%) ( Figure 3 C) and cytotoxicity (60.28% viability) ( Figure 3 D), which may be related to the increase of hydrophobicity and α-helix content in 40 mM SDS mimicking the bacterial membrane ( Table 1 and Figure 2 A) and thereby enhancing the affinity of peptide to cell membrane [ 22 , 24 ].…”

Unveiling the Impact of Antibiotics and Alternative Methods for Animal Husbandry: A Review

“…Another interesting study by Li, et al [ 134 ] showed that insect defensins are promising candidates against antibiotic-resistant Staphylococcus aureus . A variant designed from insect defensin DLP 4 , ID 13 exhibited strong antibacterial activity at low MIC values of 4–8 μg/mL to Gram-positive pathogens ( S. aureus, S. epidermidis, S. pneumoniae , S. suis ), which were lower than those of DLP 4 , and cytotoxicity of ID 13 (71.4% viability) was less than that of DLP 4 (63.8% viability).…”

“…A variant designed from insect defensin DLP 4 , ID 13 exhibited strong antibacterial activity at low MIC values of 4–8 μg/mL to Gram-positive pathogens ( S. aureus, S. epidermidis, S. pneumoniae , S. suis ), which were lower than those of DLP 4 , and cytotoxicity of ID 13 (71.4% viability) was less than that of DLP 4 (63.8% viability). The authors concluded that ID 13 could be a promising peptide antibiotic agent for therapeutic applications in farms [ 134 ].…”