Optimizing histatin 5: Effects of K13 and K17 substitutions on proteolytic stability and antifungal activity

Abstract

Candida albicans is an opportunistic human fungal pathogen found in the oral cavity. Human saliva contains a 24-amino acid peptide called histatin 5 (Hst5) that has activity against C. albicans, but degradation of Hst5 by secreted aspartyl proteases (Saps) produced by C. albicans and by salivary proteases can reduce its antifungal efficacy. Building on our previous work that identified K13 and K17 as important residues for stability and activity of Hst5, we systematically investigated amino acid modification at these sites. Modifications explored the influence of hydrophobicity, charge, polarity, size, and aromaticity on Hst5s interaction with Saps and saliva. The K13R variant retained proteolytic stability and antifungal activity after incubation with Sap1, Sap2, Sap3, and Sap9, while other K13 variants generally had reduced stability and activity, emphasizing the importance of a positive charge at this position. At K17, substitutions generally enhanced proteolytic stability and improved antifungal activity after incubation with Saps. We introduced the normalized intact peptide (NIP) parameter as a tool for identifying Hst5 variants with improved stability in the presence of multiple Saps, and NIP revealed K17W as the most proteolytically stable variant overall. Additionally, we observed modest differences in peptide stability in saliva, and the K17W variant was the only variant that retained more activity than Hst5 following incubation with saliva. We further assessed the K17W variants ability to prevent biofilm formation and found it to be more effective than the parent peptide Hst5. Our findings highlight the interactions between the Hst5 K13 and K17 residues with Saps and saliva and provide a strong foundation for future Hst5 engineering efforts to improve proteolytic stability and antifungal efficacy in diverse proteolytic environments.