Abstract
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 the secondary structure and underlying mechanism of action of CaD23. Methods: Peptide-antibiotic interaction was evaluated against S. aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa using established checkerboard and time-kill assays. Fractional inhibitory concentration index (FICI) was calculated and interpreted as synergistic (FIC<0.5), additive (FIC between 0.5–1.0), indifferent (FIC between >1.0 and ≤4), or antagonistic (FIC>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. Circular dichroism (CD) spectroscopy was also performed to examine the secondary structures of CaD23. Results: CaD23-amikacin and CaD23-levofloxacin combination treatment exhibited a strong additive effect against S. aureus SH1000 (FICI = 0.60–0.69) and MRSA43300 (FICI = 0.56–0.60) but an indifferent effect against P. aeruginosa (FIC = 1.03–1.15). CaD23 (at 25 μg/ml; 2xMIC) completely killed S. aureus within 30 min. When used at sub-MIC concentration (3.1 μg/ml; 0.25xMIC), 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. The secondary structures of CaD23 observed in MD simulations were validated by CD spectroscopy. Conclusion: CaD23 is a novel alpha-helical, membrane-active synthetic HDP that can enhance and expedite the antimicrobial action of antibiotics against Gram-positive bacteria when used in combination. MD simulations serves as a powerful tool in revealing the peptide secondary structure, dissecting the mechanism of action, and guiding the design and optimisation of HDPs.
Highlights
Our group had demonstrated that CaD23, a hybrid derivative of human cathelicidin (LL-37) and human betadefensin (HBD)-2, exhibited a more rapid in vitro antimicrobial action than conventional antibiotics such as amikacin, with no risk of Antimicrobial resistance (AMR) observed among the CaD23treated bacteria. (Ting et al, 2021d) the mechanism of action has not been fully elucidated
In view of the hydrophobicity, CaD23 was first fully dissolved in 50 μL of dimethyl sulfoxide (DMSO) followed by dilution in sterile, deionised water to achieve a final concentration of 1 mg/ml peptide in 99.5% 0.5% v/v DMSO
Standard deviation is not presented as the same results are consistently observed in all three independent experiments
Summary
Antimicrobial resistance (AMR) is currently one of the major global health threats. (Prestinaci et al, 2015; Ventola, 2015) By 2050, it is estimated to cause 10 million deaths and cost the global economy up to 100 trillion USD if the issue remains untackled. (O’Neill, 2016) In addition, non-systemic infections, including ocular and skin infections, are being increasingly affected by drug-resistant pathogens, which usually result in poor prognosis (Ventola, 2015; Pulido-Cejudo et al, 2017; Ting et al, 2021a). (Hernandez-Camarena et al, 2015; Lalitha et al, 2017; Lin et al, 2019; Asbell et al, 2020) In addition, adjuvant procedures/surgeries such as therapeutic corneal cross-linking, (Ting et al, 2019c) amniotic membrane transplantation, (Ting et al, 2021c) and therapeutic/tectonic keratoplasty (Hossain et al, 2018; Ting et al, 2020a) are often required to manage uncontrolled infection and its complications, including corneal melting and perforation All these issues highlight the need for new treatment for IK. While CaD23 exhibited reasonable in vivo efficacy at a concentration of 0.05% (500 μg/ml), the use of a higher concentration of CaD23 to achieve stronger antimicrobial effect was prohibited by the toxicity, as observed in the wound healing study To overcome this limitation, we aimed to examine the potential synergism/interaction between CaD23 and commonly used antibiotics for IK, including levofloxacin and amikacin. To overcome this limitation, we aimed to examine the potential synergism/interaction between CaD23 and commonly used antibiotics for IK, including levofloxacin and amikacin. (Ting et al, 2021e) In addition, we aimed to determine the secondary structures and mechanism of action of CaD23 using a combination of experimental and molecular dynamics (MD) simulations studies
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