HISTATIN 5 MODIFICATIONS IMPACT PROTEOLYTIC STABILITY IN THE PRESENCE OF FUNGAL AND SALIVARY PROTEASES

Abstract

Candida albicans, found in the oral cavities of 30-50% of the global population, can lead to oral candidiasis, particularly in immunocompromised individuals like those with HIV or diabetes. The current treatments, small-molecule antifungals, often fall short due to drug resistance and toxicity. To address these challenges, histatin 5 (Hst5), a 24-amino-acid peptide naturally present in human saliva, has been studied as a potential antifungal therapy. Hst5, however, is susceptible to degradation by secreted aspartyl proteases (Saps) produced by C. albicans and salivary enzymes, limiting its potential efficacy as a therapeutic. We have engineered Hst5 variants utilizing rational design in order to understand the interactions with Saps and Saliva. We have also made advancements in developing a novel screening method utilizing the directed evolution technique yeast surface display. Our study employed rational design to modify Hst5, at its lysine residues (K5, K11, K13, and K17), substituting them with leucine or arginine to examine their influence on interactions with Saps (Sap1, Sap2, Sap3, Sap5, Sap6, Sap9, and Sap10). Sap5, Sap6, and Sap10 did not degrade Hst5 at the tested conditions, while Sap1, Sap2, Sap3, and Sap9 did. Some modifications, such as K13L, are particularly susceptible to proteolysis by Sap1, Sap2, Sap3, and Sap9. In contrast, K17L substantially increases the stability and antifungal activity of Hst5 in the presence of Saps. Additionally, although the K11RK17L variant was degraded more than the K17L variant, their antifungal activities were largely similar. The proteolysis products of were also identified by mass spectrometry identifying the [4-24], [1-17], and [14-24] Sap proteolysis products. We also evaluated the proteolytic stability of these variants in saliva. Both K17L and K5R showed improved stability; however, the enhancements were modest, suggesting that further engineering is required to achieve significant improvements. Further experiments evaluated how additional amino acid substitutions at K13 and K17 affect the peptide’s proteolytic stability in the presence of Saps (with and without zinc). Our findings suggest that the positive charge at K13 is important for the proteolytic stability of Hst5, as all other variants tested except K13R reduce overall proteolytic stability. Furthermore, many substitutions at K17, including tryptophan, significantly enhance proteolytic resistance and antifungal activity following incubation with Saps. The K17W variant showed improved stability and antifungal efficacy, maintaining its function even in the presence of zinc and exhibiting stronger antibiofilm activity than the parent Hst5. In addition to the rational design work, we have advanced the development of a directed evolution yeast surface display platform for screening peptides for proteolytic stability. This would allow for the expression of large peptide libraries on the surface of Saccharomyces cerevisiae. Through optimization of expression and display conditions, we determined an induction media at 30°C with a pH of 3.5 and devoid of glucose improved the expression and display of Hst5 peptides on the surface of S. cerevisiae. We also optimized the degradation conditions for Sap2 37°C, a pH not exceeding 7.4, and a Sap2 concentration of 0.78 µg/mL led to the best discrepancy between proteolytically stable variants. Additionally, we found that a 40 amino acid linker between the peptide and the yeast surface provided the best observing proteolytic degradation. Using the optimized system, we showed that yeast surface display can be used to discriminate between peptide variants with different levels of proteolytic stability. This lays the foundation for future work to screen large libraries of peptides for proteolytic stability. From these results, we have gained a deeper understanding of the interactions between Hst5 and Saps, showing that modification at different lysine residues greatly impacts the proteolytic stability of Hst5. Furthermore, we have shown that the yeast surface display platform can be used to screen the proteolytic stability of peptides. Looking forward, this peptide should be engineered for proteolytic stability in saliva. Furthermore, mock screens should be made before screening a library of peptides using the yeast surface display platform.