ABSTRACT
Purpose Bacterial keratitis (BK) represents the leading cause of corneal blindness worldwide. This study aimed to generate potent hybridized human-derived host defense peptides (HDPs) as novel topical antimicrobial therapy for bacterial keratitis.
Methods Hybrid peptides were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to −3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells (HCE-2) and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively.
Results A second-generation hybrid peptide (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, demonstrated good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC=12.5-25.0μg/ml (5.2-10.4μM)] and S. epidermidis [MIC=3.1-12.5μg/ml (1.3-5.2μM)], and moderate efficacy against P. aeruginosa [MIC=50μg/ml (20.9μM)]. CaD23 (at 25μg/ml or 2x MIC) killed all the bacteria within 30 mins, which was 8 times faster than amikacin (25μg/ml or 20x MIC). At 200μg/ml (16x MIC), CaD23 was shown to be relatively safe against HCE-2 (<30% toxicity) and erythrocytes (<10% toxicity). Pre-clinical murine studies showed that CaD23 0.05% (500μg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal healing.
Conclusions Rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Funding / support: D.S.J.T. is supported by the Medical Research Council / Fight for Sight (FFS) Clinical Research Fellowship (MR/T001674/1), the FFS / John Lee, Royal College of Ophthalmologists Primer Fellowship (24CO4), and the University of Nottingham International Research Collaboration Fund (A2RRG1). I.M. acknowledges funding support from the Medical Research Council – Confidence in Concept Scheme (MRC-CIC_2019-028).
Conflict of interest: None