Evaluation of Phenylalanine Substitution on the Antimicrobial Properties of Histatin 8 against Wild-Type and Antibiotic-Resistant Pseudomonas aeruginosa

The unregulated and inappropriate use of antibiotics triggered the rapid spread of antimicrobial resistance in pathogenic microorganisms such as Pseudomonas aeruginosa. Antimicrobial peptides (AMPs) require investigations of their remarkable bioactivities and unique microbicidal mechanisms as potential replacement treatments for conventional antibiotics. Specifically, a naturally occurring antimicrobial dodecapeptide named Histatin 8 (HS8), from the Histatin family, has weak to moderate antimicrobial activities but is a promising bioactive molecule. In this study, three well-represented HS8-derived peptides with potential in silico bioactive propensities namely HS8-1 (Tyr12Phe), HS8-2 (Ser8Phe and Tyr12Phe), and HS8-3 (Gly11Phe) were explored. Results showed that HS8-1 was a significantly more active AMP than HS8-2 and HS8-3, displaying 3.0-fold and 3.56-fold decrease in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values, respectively, against wild-type P. aeruginosa. Furthermore, HS8-1 and HS8-2 displayed the lowest MIC and MBC values against the antibiotic-resistant P. aeruginosa. Both HS8-2 and HS8-1 are the more active peptides showing inhibitory activities against wild-type and antibiotic-resistant P. aeruginosa biofilms in vitro, respectively. HS8-1 and HS8-2, at their MICs, can both primarily induce surface roughening and corrugation suggesting possible disruptive effects on the P. aeruginosa membrane reflecting antimicrobial action. HS8-1 lyses human red blood cells (RBCs) in vitro at concentrations 250 µg/mL and below, suggesting it to be the least hemolytic peptide compared to HS8-2 and HS8-3 which strongly lyse RBCs at relatively lower concentrations. Overall, the results imply that the HS8-derived peptide bioactivities can be enhanced by site-specific phenylalanine (Phe) substitutions against P. aeruginosa.