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The evolution of azole resistance in Candida albicans sterol 14α-demethylase (CYP51) through incremental amino acid substitutions

Warrilow, Andrew G., Nishimoto, Andrew T., Parker, Josie E., Price, Claire L., Flowers, Stephanie A., Kelly, Diane E., Rogers, P. David and Kelly, Steven L. 2019. The evolution of azole resistance in Candida albicans sterol 14α-demethylase (CYP51) through incremental amino acid substitutions. Antimicrobial Agents and Chemotherapy 63 (5) , e02586-18. 10.1128/AAC.02586-18

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Recombinant Candida albicans CYP51 (CaCYP51) proteins containing 23 single and 5 double amino acid substitutions found in clinical strains and the wild-type enzyme were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid agarose chromatography. Catalytic tolerance to azole antifungals was assessed by determination of the concentration causing 50% enzyme inhibition (IC50) using CYP51 reconstitution assays. The greatest increase in the IC50 compared to that of the wild-type enzyme was observed with the five double substitutions Y132F+K143R (15.3-fold), Y132H+K143R (22.1-fold), Y132F+F145L (10.1-fold), G307S+G450E (13-fold), and D278N+G464S (3.3-fold). The single substitutions K143R, D278N, S279F, S405F, G448E, and G450E conferred at least 2-fold increases in the fluconazole IC50, and the Y132F, F145L, Y257H, Y447H, V456I, G464S, R467K, and I471T substitutions conferred increased residual CYP51 activity at high fluconazole concentrations. In vitro testing of select CaCYP51 mutations in C. albicans showed that the Y132F, Y132H, K143R, F145L, S405F, G448E, G450E, G464S, Y132F+K143R, Y132F+F145L, and D278N+G464S substitutions conferred at least a 2-fold increase in the fluconazole MIC. The catalytic tolerance of the purified proteins to voriconazole, itraconazole, and posaconazole was far lower and limited to increased residual activities at high triazole concentrations for certain mutations rather than large increases in IC50 values. Itraconazole was the most effective at inhibiting CaCYP51. However, when tested against CaCYP51 mutant strains, posaconazole seemed to be the most resistant to changes in MIC as a result of CYP51 mutation compared to itraconazole, voriconazole, or fluconazole.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Publisher: American Society for Microbiology
ISSN: 0066-4804
Date of First Compliant Deposit: 8 July 2022
Date of Acceptance: 10 February 2019
Last Modified: 18 May 2023 01:03

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