Histatin 5 is an antimicrobial peptide found in human saliva. The peptide has antifungal activity against the human fungal pathogen Candida albicans, which can cause oral and systemic infections in immunocompromised individuals. Although histatin 5 has potential as a therapeutic to treat or prevent infections caused by C. albicans, the fungus produces a family of proteases called secreted aspartic proteases (Saps) that degrade histatin 5 and reduce or eliminate its antifungal activity. Two of the Saps, Sap2 and Sap9, were previously shown to cleave histatin 5 at each of its four lysine residues. We introduced modifications to these lysine residues to study the interaction of histatin 5 with the Sap2 and Sap9 enzymes and to improve the resistance of histatin 5 to degradation by the proteases. Substitution to either leucine or arginine at the K17 position was particularly effective at reducing proteolytic degradation. Following incubation with Sap9, only 10% of K17L and 11% of K17R was degraded, while 52% of the parent peptide was degraded. Likewise, 35% of K17L and 31% of K17R were degraded by Sap9 compared to 64% of histatin 5. Surprisingly, the K11L, K13L, and K13R substitutions caused an increase in degradation by Sap9, with no detectable intact K13L remaining and over 87% of K11L and K13R degraded. The reduced degradation of the K17 variants and the increased degradation of the K13 mutants were also observed following incubation with C. albicans cells, illustrating the biological relevance of our results. Additionally, the cleavage products resulting from degradation of the variants with Sap9 and Sap2 exhibited substantial differences in antifungal activity. Peptides that were more degraded than histatin 5 had weaker antifungal activity against C. albicans cells compared to histatin 5, and peptides that were less degraded than histatin 5 showed improved antifungal activity compared to histatin 5. Mass spectrometry results revealed that the relative abundance of cleavage products resulting from degradation by the Sap enzymes was shifted in the histatin 5 variants compared to the parent histatin 5. Our results show that single amino-acid substitutions to histatin 5 can reduce degradation by Sap enzymes and suggest that the peptides could provide insight into the interaction of histatin 5 with the Sap enzymes. We are currently exploring the degradation kinetics and designing new histatin-5 variants to further improve proteolytic resistance.

Engineering histatin 5 for improved resistance to secreted aspartic proteases

253rd National Meeting (2017)

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