Engineering improved variants of the antifungal peptide histatin 5 with reduced susceptibility to Candida albicans secreted aspartic proteases and enhanced antimicrobial potency

Abstract

Candida albicans is an opportunistic fungal pathogen and a commensal organism that commonly colonizes mucosal surfaces, including those inside the human mouth. To help control C. albicans, human saliva contains the antifungal peptide histatin 5 (Hst-5), which has strong antifungal activity against C. albicans. However, the pathogen produces secreted aspartic proteases (Saps) that cleave Hst-5 at lysine residues and eliminate its antifungal properties. We designed variants of Hst-5 with its lysine residues substituted with arginine or leucine to evaluate the effect on proteolysis by Saps. We found site-, residue-, and Sap-dependent effects from single amino acid substitutions. The K17R and K17L modifications led to dramatic results, with over 77% and 100% intact peptide remaining after incubation with Sap9 and Sap2, respectively, compared to 47% and 61% of Hst-5. This decrease in proteolysis was accompanied by a reduction in cleavage on the C-terminal side of K17, suggesting the Saps prefer lysine at K17 for cleavage. Incubation with C. albicans cells and culture supernatant corroborated the results with purified Saps and highlighted their biological relevance. The modifications to Hst-5 do not diminish the antifungal activity of Hst-5, and, in fact, the K17R, K17L, and K11R peptides retained significantly more antifungal activity after treatment with Saps than Hst-5. Our results indicate that single amino acid modifications drastically impact both proteolysis at the modification sites and the overall level of proteolysis of the peptide, demonstrating the potential of designing peptides for resistance to proteolysis as a means for improving therapeutic efficacy.

<p>Antifungal Activity of ZnO Nanoparticles and Nystatin and Downregulation of SAP1-3 Genes Expression in Fluconazole-Resistant <em>Candida albicans</em> Isolates from Vulvovaginal Candidiasis</p>

“…17 ZnO improves durability and resistance to mildew; it has also been used in the pharmaceutical and cosmetic industries in powder and ointment forms. 18 ZnO increases the permeability of the cell wall and cytoplasmic membrane through binding to lipids and proteins and the destruction of protein's 3-D structure. 17 We conducted Fluconazole susceptibility testing on the clinical C. albicans isolates obtained from some young VVC women.…”

Phage display as a tool for vaccine and immunotherapy development

“…60 This fungus is especially dangerous to patients with comprised immune systems, where it has a high morbidity and mortality rate. 61 Secretory aspartyl proteinases (Saps) are considered the major antigenic determinant in CA infection, with Sap2 being the most prominent to cause virulence. In this study, a short peptide of Sap2, EPS, was chosen for inclusion in the vaccine because it was shown to be recognized by IgG antibodies from patients with CA infections.…”

Beyond function: Engineering improved peptides for therapeutic applications

“…Once the preferred degradation sequences for relevant enzymes are known, rational design can be used to reduce susceptibility to degradation. For example, histatin 5, a salivary peptide with antifungal potency for Candida albicans was engineered for improved proteolytic stability in the presence of secreted aspartic proteases (Sap2 and Sap9) produced by the fungus. The enzymes preferentially degrade histatin 5 at Lys residues, especially K17, and substitution of this residue to either Leu or Arg resulted in higher stability .…”

“…For example, histatin 5, a salivary peptide with antifungal potency for Candida albicans was engineered for improved proteolytic stability in the presence of secreted aspartic proteases (Sap2 and Sap9) produced by the fungus. The enzymes preferentially degrade histatin 5 at Lys residues, especially K17, and substitution of this residue to either Leu or Arg resulted in higher stability . In another example, the incorporation of Trp at four residues of the antimicrobial peptide cecropin A‐melittin resulted in significantly improved stability in the presence of digestive tract enzymes by improving its helical structure and avoiding protease recognition .…”