Evaluation of Host Defense Peptide (CaD23)-Antibiotic Interaction and Mechanism of Action: Insights from Experimental and Molecular Dynamics Simulations Studies

Abstract

ABSTRACTBackground/aimHost defense peptides (HDPs) have the potential to provide a novel solution to antimicrobial resistance (AMR) in view of their unique and broad-spectrum antimicrobial activities. We had recently developed a novel hybrid HDP based on LL-37 and human beta-defensin-2, named CaD23, which was shown to exhibit good in vivo antimicrobial efficacy against Staphylococcus aureus in a bacterial keratitis murine model. This study aimed to examine the potential CaD23-antibiotic synergism and to evaluate the underlying mechanism of action of CaD23.MethodsAntimicrobial efficacy was determined using minimum inhibitory concentration (MIC) assay with broth microdilution method. Peptide-antibiotic interaction was evaluated against S. aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa using established checkerboard assay and time-kill kinetics assay. Fractional inhibitory concentration index (FICI) was calculated and interpreted as synergistic (FICI<0.5), additive (FICI between 0.5-1.0), indifferent (FICI between >1.0 and ≤4), or antagonistic (FICI>4). SYTOX green uptake assay was performed to determine the membrane-permeabilising action of CaD23. Molecular dynamics (MD) simulations were performed to evaluate the interaction of CaD23 with bacterial and mammalian mimetic membranes.ResultsCaD23-amikacin and CaD23-levofloxacin combination treatment exhibited a strong additive effect against S. aureus SH1000 (FICI=0.56) and MRSA43300 (FICI=0.56) but a borderline additive-to-indifferent effect against P. aeruginosa (FIC=1.0-2.0). CaD23 (at 25 μg/ml; 2x MIC) was able to achieve complete killing of S. aureus within 30 mins. When used at sub-MIC concentration (3.1 μg/ml; 0.25x MIC), it was able to expedite the antimicrobial action of amikacin against S. aureus by 50%. The rapid antimicrobial action of CaD23 was attributed to the underlying membrane-permeabilising mechanism of action, evidenced by the SYTOX green uptake assay and MD simulations studies. MD simulations revealed that cationicity, alpha-helicity, amphiphilicity and hydrophobicity (related to the Trp residue at C-terminal) play important roles in the antimicrobial action of CaD23.ConclusionsCaD23 is a novel membrane-active synthetic HDP that can enhance and expedite the antimicrobial action of antibiotics against Gram-positive bacteria when used in combination. MD simulation serves as a useful tool in dissecting the mechanism of action and guiding the design and optimisation of HDPs.