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The Evolution of Azole Resistance in Candida albicans Sterol 14α-Demethylase (CYP51) through Incremental Amino Acid Substitutions



The Evolution of Azole Resistance in Candida albicans Sterol 14α-Demethylase (CYP51) through Incremental Amino Acid Substitutions



Antimicrobial Agents and ChemoTherapy 2019



Recombinant C. 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+-NTA agarose chromatogrpahy. Catalytic tolerance to azole antifungals was assessed by IC50 [concentration causing 50% enzyme inhibition] determination using CYP51 reconstitution assays. The greatest increase in IC50 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) compared to the wild type enzyme. The single substitutions K143R, D278N, S279F, S405F, G448E and G450E conferred at least two-fold increases in fluconazole IC50, and Y132F, F145L, Y257H, Y447H, V456I, G464S, R467K and I471T substitutions conferred increased residual CYP51 activity at high fluconazole concentrations. In vitro C. albicans testing of select CaCYP51 mutations showed that the Y132F, Y132H, K143R, F145L, S405F, G448E, G450E, G464S Y132F&K143R;, Y132F&F145L;, and D278N&G464S substitutions conferred at least a two-fold increase in fluconazole MIC. 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 the most resistant to changes in MIC as a result of CYP51 mutation compared to itraconazole, voriconazole, or fluconazole.

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Accession: 066512171

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PMID: 30783005

DOI: 10.1128/aac.02586-18


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