Rational Design of a Potent Antimicrobial Peptide Based on the Active Region of a Gecko Cathelicidin

Abstract

The emergence of multidrug-resistant (MDR) bacteria presents a significant challenge to public health, increasing the risk of infections that are resistant to current antibiotic treatment. Antimicrobial peptides (AMPs) offer a promising alternative to conventional antibiotics in the prevention of MDR bacterial infections. In the present study, we identified a novel cathelicidin AMP from Gekko japonicus, which exhibited broadspectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging from 2.34 to 4.69 μg/mL. To improve its potential therapeutic application, a series of peptides was synthesized based on the active region of the gecko-derived cathelicidin. The lead peptide (RH-16) showed an antimicrobial activity comparable to that of the parent peptide. Structural characterization revealed that RH-16 adopted an amphipathic α-helical conformation. Furthermore, RH-16 demonstrated neither hemolytic nor cytotoxic activity but effectively killed a wide range of clinically isolated, drug-resistant bacteria. The antimicrobial activity of RH-16 was attributed to the nonspecific targeting of bacterial membranes, leading to rapid bacterial membrane permeabilization and rupture. RH-16 also retained its antibacterial activity in plasma and exhibited mild toxicity in vivo. Notably, RH-16 offered robust protection against skin infection in a murine model. Therefore, this newly identified cathelicidin AMP may be a strong candidate for future pharmacological development targeting multidrug resistance. The use of a rational design approach for isolating the minimal antimicrobial unit may accelerate the transition of natural AMPs to clinically applicable antibacterial agents.

TC-14, a cathelicidin-derived antimicrobial peptide with broad-spectrum antibacterial activity and high safety profile

“…The secondary structures of TC-14 were assessed by CD spectrometry (Chirascan V100, Applied Photophysics, UK) according to our previously described methods. 48 Briefly, TC-14 was first dissolved in either distilled water or sodium dodecyl sulfate (SDS) solution (0–64 mM) to a final concentration of 0.2 mg/mL. The CD spectra were recorded from 190 to 260 nm with a path length of 1 mm, step size of 1 nm, bandwidth of 1 nm, and time per point of 1 s at room temperature.…”

“…159077, Med Chem Express). 48 Human HaCaT keratinocytes (5 × 10 5 cells/well) were cultured in 96-well plates with Dulbecco’s modified Eagle’s medium (DMEM) for 24 h in a cell incubator (5% CO 2 , 37°C). Peptide solutions (final concentrations of 3.125–100 μg/mL) were then added to the wells and incubated at 37°C for 24 h. The CCK-8 kit was then used according to the manufacturer’s instructions.…”

“…To determine the effects of plasma on the antibacterial activity of TC-14, the peptide (400 μg/mL) was incubated in saline solution with an equal volume of diluted mouse plasma at 37°C for 0, 0.5, 1, 2, 4, 6, and 8 h. The antibacterial activity of the peptide against P. aeruginosa ATCC27853, A. baumannii ATCC19606, and S. aureus ATCC6538 was evaluated at each time point based on MIC values. 48 …”

“…The impact of TC-14 on bacterial membrane permeabilization was determined using fluorescence assay according to our previously reported method. 48 In brief, the four bacterial strains were cultured in LB broth to the logarithmic growth phase, with the density of the bacterial solution then adjusted to 2 × 10 8 CFU/mL. Subsequently, 100 μL of bacterial suspension was added to a 96-well plate, followed by the addition of 10 μL of PI (2.5 μg/mL).…”

“…Bacterial passaging was conducted every 24 h based on our previous research. 48 , 49 In brief, bacteria were cultured on LB medium containing final MICs of 25%, 50%, 75%, 100%, 150%, and 200% for generations 1–10, 11–20, 21–30, 31–40, 41–50, and 51–60, respectively. After 60 consecutive generations, the bacteria were removed from the culture at five-generation intervals, and the MICs of the antimicrobial peptides against the above bacteria were examined.…”