Histatin-5 is a highly charged antimicrobial peptide naturally found in human saliva, and it has been proposed as a potential therapeutic for treating infections caused by the fungal pathogen Candida albicans. C. albicans is a commensal organism that is part of the normal flora of the oral cavity, but it can cause disease, including infections of the oral mucosa, in immunocompromised patients. Although histatin-5 has strong and specific activity against C. albicans, the pathogen produces proteases that degrade the peptide and render it inactive. A family of secreted and cell-wall anchored enzymes called secreted aspartic proteases (Saps) are involved in this degradation. The Sap enzymes have been shown to cut at positively charged amino acid residues, particularly lysine residues. To improve the potential of histatin-5 as a therapeutic peptide and better understand its interactions with the Saps, we mutated the lysine residues in histatin-5 to leucine and arginine. All mutations resulted in antifungal activity similar to the parent histatin-5, illustrating the robust nature of the peptide’s mechanism of action, which involves interaction with intracellular targets. In contrast several mutations had significant effects on the susceptibility of the peptides to purified Sap2 and Sap 9. Mutation to either leucine or arginine at the K17 position led to nearly complete resistance to degradation by Sap2 under conditions where only 10% of the parent peptide is intact. Likewise, both K17L and K17R mutations increased the amount of intact peptide following incubation with Sap9 from 45% to 90%. Interestingly, the K13L and K13R mutations caused an increase in degradation by both enzymes, with almost complete degradation by Sap2. The reduced degradation of the K17 mutants and the increased degradation of the K13 mutants were also observed following incubation with C. albicans cells, showing that the mutations have the potential to improve our understanding of the interaction of the Saps with histatin-5 in a therapeutic context. We are currently using our results to design new histatin-5 mutants that will allow us to further dissect how the Sap enzymes recognize and degrade histatin-5.

Effect of histatin-5 mutations on proteolytic degradation by Candida albicans secreted aspartic proteases

251st National Meeting (2016)

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