(309b) Histatin 5 Modifications Impact Proteolytic Stability in the Presence of Fungal and Salivary Proteases

Candida albicans is an important human fungal pathogen that causes disease, including oral candidiasis, in immunocompromised patients. Small-molecule antifungal agents are currently used to treat candidiasis, but some can be toxic to patients and C. albicans infections can be resistant to them. To address the challenges of small-molecule antifungal agents, the antifungal peptide histatin 5 (Hst5) has been proposed as a new antifungal agent to target C. albicans. However, C. albicans produces a family of 10 secreted aspartyl proteases (Saps), some of which can cleave Hst5 and reduce its activity, and human saliva also contains human and microbial proteases that may degrade Hst5. Previous work showed the lysine residues in Hst5 play an important role in susceptibility to proteolysis by Sap2 and Sap9, with modification to arginine or leucine providing protection at the K17 residue and affecting proteolysis at other sites. In this work, we have designed variants of Hst5 with additional amino acid modifications at the lysine residues (K5, K11, K13, and K17) to further understand how the peptide sequence affects proteolytic stability in the presence of a wider range of Saps and human saliva.

To understand how lysine modification affect proteolysis by Saps and saliva, we mixed the Hst5 variants with purified Saps (Sap1, 2, 3, 5, 6, 9, and 10) or the supernatant of saliva, incubated at 37° C for 2 h, and quantified the degradation using gel electrophoresis and densitometry. In the presence of saliva, we found that K17W had improved proteolytic stability, while K11R had reduced stability. All other variants were similar to the parent peptide, suggesting the lysine sites do not play a major role in degradation by proteases present in saliva. Overall, we found that variants with substitutions at K17 were more resistant to proteolysis by Saps, while variants with substitutions at K11 and K13 were more susceptible to proteolysis. We tested the antifungal efficacy of degraded variants to see whether degradation reduced antifungal activity. We found that degraded K17 variants retained the most antifungal ability, while degraded K11 and K13 variants (especially K11L and K13E) had low antifungal activity. In general, variants with modifications at K17 had the most improved properties, likely because modifications at K17 lead to reduced proteolysis or because the degradation products retain antifungal activity . The K11 and K13 residues are in the region of Hst5 proposed to be necessary for antifungal activity, which could explain why mutations in this region reduce antifungal activity. Based on our results, we identified peptides with improved proteolytic stability and antifungal activity, with K17W being the most promising. We have used mass spectrometry to identify the fragments formed through degradation of the variants by saliva and Saps, and these results will lead to further modifications of Hst5 variants for improved proteolytic stability. By engineering Hst5 for improved proteolytic stability, we can potentially increase its half-life and improve its potential as an antifungal therapeutic.