Determining antimicrobial susceptibility and hemolytic activity of indolicidin derivatives

Antibiotic-resistant microorganisms have posed significant challenges to the development of novel antimicrobial agents. Antibiotic-resistant bacteria are increasingly being detected in humans and animals treated with antibiotics, making commercially available antibiotics less effective than they once were. Microbial compounds from various species are being studied extensively for potentially better and more effective antibacterial properties that may be used as an alternative. Antimicrobial peptides (AMPs) appear to be potential antibacterial medication candidates; however, the development of AMPs is impeded by their cytotoxicity. In the current study, indolicidin, a known antimicrobial originally isolated from bovine neutrophils, was modified by changing its tryptophan content. We predicted that replacing tryptophan with a less hydrophobic amino acid, alanine, would minimize hemolytic activity and maximize bioactivity. To address this question, we analyzed four indolicidin derivatives in which two of its tryptophan residues were replaced with alanine at different positions. We assessed their antibacterial and antifungal activity against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Candida albicans, Staphylococcus aureus, and Methicillin-Resistant Staphylococcus aureus (MRSA). Antimicrobial testing identified two active derivatives with minimum inhibitory concentrations in the 250-500 μg mL-1 range against all microbes, except Pseudomonas aeruginosa. Notably, the substitution of tryptophan residues at position 4 significantly affected antimicrobial activity. All derivatives exhibited a substantial decrease or complete removal of hemolytic activity at concentrations above their minimum inhibitory concentration (MIC). Additionally, derivatives containing the Proline-Tryptophan-Tryptophan-Proline (PWWP) domain showed DNA-binding capability, while those lacking this domain did not. These findings highlight the potential of modified indolicidin derivatives as effective and less cytotoxic antimicrobial agents, offering a potential solution for combating antibiotic resistance.