Dataset Information

Impact of Homologous Resistance Mutations from Pathogenic Yeast on Saccharomyces cerevisiae Lanosterol 14?-Demethylase.

ABSTRACT: Fungal infections frequently affect immunodeficient individuals and are estimated to kill 1.35 million people per annum. Azole antifungals target the membrane-bound cytochrome P450 monooxygenase lanosterol 14?-demethylase (CYP51; Erg11p). Mutations in CYP51 can render the widely used triazole drugs less effective. The Candida albicans CYP51 mutation G464S and the double mutation Y132F G464S (Y140F and G464S by Saccharomyces cerevisiae numbering) as well as the CYP51A G54E/R/W mutations of Aspergillus fumigatus (G73E/R/W by S. cerevisiae numbering) have been reproduced in a recombinant C-terminal hexahistidine-tagged version of S. cerevisiae CYP51 (ScErg11p6×His). Phenotypes and X-ray crystal structures were determined for the mutant enzymes. Liquid microdilution assays showed that the G464S mutation in ScErg11p6×His conferred no difference in the susceptibility of yeast to triazole drugs but in combination with the Y140F mutation gave a 4-fold reduction in susceptibility to the short-tailed triazole fluconazole. The ScErg11p6×His Y140F G464S mutant was unstable during purification and was not crystallized. The ScErg11p6×His G73E/R/W mutations conferred increased susceptibly to all triazoles tested in liquid microdilution assays. High-resolution X-ray crystal structures reveal two different conformations of the ligand itraconazole, including a previously unseen conformation, as well as interactions between the tail of this triazole and the E/W73 residue. This study shows that S. cerevisiae CYP51 adequately represents some but not all mutations in CYP51s of pathogenic fungi. Insight into the molecular mechanisms of resistance mutations in CYP51 will assist the development of inhibitors that will overcome antifungal resistance.

SUBMITTER: Sagatova AA

PROVIDER: S-EPMC5826108 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Impact of Homologous Resistance Mutations from Pathogenic Yeast on Saccharomyces cerevisiae Lanosterol 14α-Demethylase.

Sagatova Alia A AA Keniya Mikhail V MV Tyndall Joel D A JDA Monk Brian C BC

Antimicrobial agents and chemotherapy 20180223 3

Fungal infections frequently affect immunodeficient individuals and are estimated to kill 1.35 million people per annum. Azole antifungals target the membrane-bound cytochrome P450 monooxygenase lanosterol 14α-demethylase (CYP51; Erg11p). Mutations in CYP51 can render the widely used triazole drugs less effective. The <i>Candida albicans</i> CYP51 mutation G464S and the double mutation Y132F G464S (Y140F and G464S by <i>Saccharomyces cerevisiae</i> numbering) as well as the CYP51A G54E/R/W mutat ...[more]

PMID: 29263059

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Project description:Purpose: In order to identify genes for steroid 14α-hydroxylase P450lun and its associated redox partner CPRlun in filamentous fungus Cochliobolus lunatus ATCC#12017 Methods: Strand-specific RNA-seq libraries were constructed for two samples, including (I) Mycelia of C. lunatus treated with mock N, N-dimethylformamide (DMF); (II) Mycelia of C. lunatus treated with 170 mg/L 11-deoxycortisol 21-acetate (RSA) dissolved in N,N-dimethylformamide. For preparation of RNA samples, germinated young mycelia of C. lunatus after 22-24 pre-culture in liquid potato dextrose broth medium were harvested and transferred into a new 30 ml potassium phosphate buffer supplement with either 170 mg/L RSA dissolved in DMF, or an equal volume of DMF as mock. Additional two-hours cultivation was performed to induction of the P-450lun gene expression. Total RNA for samples were isolated using TRIzol reagent (Invitrogen, Grand Island, USA), then used for high-throughput RNA sequencing. The two 150-nt strand-specific paired-end RNA-seq libraries were generated commercially at Novogene Biotechnology Co. Ltd (Tianjin, China) by using Illumina’s Hiseq X Ten platform (Illumina, San Diego, USA). The clean reads were used to create a de novo transcriptome assembly using the Trinity pipeline (v2.4.0). The abundance for each of the resulted transcripts was calculated by RSEM (v1.2.0) using the Fragments Per Kilobase per Million (FPKM) method. Results: A total of 7.1 and 7.9 million 150-bp paired-end clean reads were separately generated from the DMF mock and RSA induced samples,and 23957 transcripts with length more than 600nt were assembled from the pooled reads of both samples using Trinity program. The translated sequences of these transcripts were predicted using TransDecoder, and then used as queries to search against the SWISS-PROT and the Pfam databases for functional annotation. Transcripts DN8493 and DN2353 were separately identified as C.lunatus P-450lun and CPRlun genes. Conclusions: The 14β-hydroxylase system of C.lunatus was deciphered by transcriptome analysis.

Dataset Information

Impact of Homologous Resistance Mutations from Pathogenic Yeast on Saccharomyces cerevisiae Lanosterol 14?-Demethylase.

Publications

Impact of Homologous Resistance Mutations from Pathogenic Yeast on Saccharomyces cerevisiae Lanosterol 14α-Demethylase.

Similar Datasets

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