Optimizing Histatin 5: Effects of K13 and K17 Substitutions on Proteolytic Stability and Antifungal Activity

Human saliva contains a 24-amino acid peptide called histatin 5 (Hst5), which has activity against the fungal pathogen Candida albicans. 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 modifications at these sites to explore the influence of amino acid properties on Hst5's 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 Saps, and NIP revealed K17W as the most proteolytically stable variant overall. Additionally, we observed modest differences in stability in saliva, and the K17W variant was the only variant retaining more activity than Hst5 following incubation with saliva. We further assessed the K17W variant's ability to prevent biofilm formation and found that it was more effective than Hst5. Our findings highlight the interactions between the Hst5 K13 and K17 residues with Saps and saliva, providing a foundation for future Hst5 engineering efforts to improve proteolytic stability and antifungal efficacy in diverse proteolytic environments.