RT Journal Article
SR Electronic
T1 Evaluation of Host Defense Peptide (CaD23)-Antibiotic Interaction and Mechanism of Action: Insights from Experimental and Molecular Dynamics Simulations Studies
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.06.26.450050
DO 10.1101/2021.06.26.450050
A1 Darren Shu Jeng Ting
A1 Jianguo Li
A1 Chandra S. Verma
A1 Eunice T. L. Goh
A1 Mario Nubile
A1 Leonardo Mastropasqua
A1 Dalia G. Said
A1 Roger W. Beuerman
A1 Rajamani Lakshminarayanan
A1 Imran Mohammed
A1 Harminder S. Dua
YR 2021
UL http://biorxiv.org/content/early/2021/06/27/2021.06.26.450050.abstract
AB Background/aim Host 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.Methods Antimicrobial 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.Results CaD23-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.Conclusions CaD23 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.Competing Interest StatementThe authors have declared no competing interest.