The evolution of Azole resistance in Candida albicans Sterol 14-demethylase (CYP51) through incremental amino acid substitutions

Andrew G. Warrilow, Andrew T. Nishimoto, Josie E. Parker, Claire L. Price, Stephanie A. Flowers, Diane E. Kelly, Phillip Rogers, Steven L. Kelly

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Abstract

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 Ni 2 -nitrilotriacetic acid agarose chromatography. Catalytic tolerance to azole antifungals was assessed by determination of the concentration causing 50% enzyme inhibition (IC 50 ) using CYP51 reconstitution assays. The greatest increase in the IC 50 compared to that of the wild-type enzyme was observed with the five double substitutions Y132FK143R (15.3-fold), Y132HK143R (22.1-fold), Y132FF145L (10.1-fold), G307SG450E (13-fold), and D278NG464S (3.3-fold). The single substitutions K143R, D278N, S279F, S405F, G448E, and G450E conferred at least 2-fold increases in the fluconazole IC 50 , 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, Y132FK143R, Y132FF145L, and D278NG464S 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 IC 50 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.

Original languageEnglish (US)
Article numbere02586-18
JournalAntimicrobial Agents and Chemotherapy
DOIs
StatePublished - May 1 2019

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Sterol 14-Demethylase
Azoles
Amino Acid Substitution
Candida albicans
Fluconazole
Itraconazole
Mutation
Enzymes
Nitrilotriacetic Acid
Agarose Chromatography
Triazoles
Proteins
Escherichia coli

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

Cite this

Warrilow, A. G., Nishimoto, A. T., Parker, J. E., Price, C. L., Flowers, S. A., Kelly, D. E., ... Kelly, S. L. (2019). The evolution of Azole resistance in Candida albicans Sterol 14-demethylase (CYP51) through incremental amino acid substitutions. Antimicrobial Agents and Chemotherapy, [e02586-18]. https://doi.org/10.1128/AAC.02586-18

The evolution of Azole resistance in Candida albicans Sterol 14-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, Phillip; Kelly, Steven L.

In: Antimicrobial Agents and Chemotherapy, 01.05.2019.

Research output: Contribution to journalArticle

Warrilow, Andrew G. ; Nishimoto, Andrew T. ; Parker, Josie E. ; Price, Claire L. ; Flowers, Stephanie A. ; Kelly, Diane E. ; Rogers, Phillip ; Kelly, Steven L. / The evolution of Azole resistance in Candida albicans Sterol 14-demethylase (CYP51) through incremental amino acid substitutions. In: Antimicrobial Agents and Chemotherapy. 2019.
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