Impact of Homologous Resistance Mutations from Pathogenic Yeast on Saccharomyces cerevisiae Lanosterol 14α-Demethylase (original) (raw)
Related papers
1998
The cytochrome P-450 lanosterol 14␣-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378-2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1 genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1 gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.
Microbiology (Reading, England), 1999
The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G4655). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole suscepti...
Antimicrobial Agents and Chemotherapy, 1998
The cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378–2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was ...
Journal of Biological Chemistry, 1997
Sterol 14␣-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance. We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae to probe a model structure for the enzyme. The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site. This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-fold azole resistance in transformants of S. cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.
Journal of Fungi
Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray c...
Antimicrobial Agents and Chemotherapy, 2008
Inhibition of sterol-14α-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of Saccharomyces cerevisiae that was humanized with respect to the amino acids encoded at the CYP51 ( ERG11 ) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to >1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from...
Formation of Azole-Resistant Candida albicans by Mutation of Sterol 14-Demethylase P450
Antimicrobial Agents and Chemotherapy, 1999
The sterol 14-demethylase P450 (CYP51) of a fluconazole-resistant isolate of Candida albicans, DUMC136, showed reduced susceptibility to this azole but with little change in its catalytic activity. Twelve nucleotide substitutions, resulting in four amino acid changes, were identified in the DUMC136 CYP51 gene in comparison with a reported CYP51 sequence from a wild-type, fluconazole-susceptible C. albicans strain. Seven of these substitutions, including all of those causing amino acid changes, were located within a region covering one of the putative substrate recognition sites of the enzyme (SRS-1). Polymorphisms within this region were observed in several C. albicans isolates, and some were found to be CYP51 heterozygotes. Among the amino acid changes occurring in this region, only an alteration of Y132 was common among these fluconazole-resistant isolates, which suggests the importance of this residue to the fluconazole resistance of the target enzyme. DUMC136 and another fluconazole-resistant isolate were homozygotes with respect to CYP51, although the typical wild-type, fluconazole-susceptible C. albicans was a CYP51 heterozygote. These findings suggest that part of the fluconazole-resistant phenotype of C. albicans DUMC136 was acquired through a mutation-prone area of CYP51, an area which might promote the formation of fluconazole-resistant CYP51, along with a mechanism(s) which allows the formation of a homozygote of this altered CYP51 in this diploid pathogenic yeast. Candida albicans is one of the major pathogenic fungi causing systemic infection among immunocompromised hosts. Azole antifungal agents, such as fluconazole and itraconazole, are commonly prescribed to treat systemic and mucocutaneous candidiasis. However, emergence of fluconazole-resistant C. albicans strains in patients receiving triazole treatment has become a serious problem, particularly in the treatment of oral candidiasis in AIDS patients (15, 23, 24). Fluconazole resistance has been proposed to occur through several mechanisms: (i) failure of cells to accumulate the agent (1, 4, 27); (ii) alteration of the ergosterol biosynthetic pathway through a defect in sterol ⌬ 5,6-desaturase (16); (iii) an increase in the cellular content of sterol 14-demethylase P450 (CYP51), the primary target of the azole (30); and (iv) a decrease in the affinity of CYP51 for fluconazole (19, 26, 31). It has now been shown that these distinct mechanisms can develop in single Candida isolates in a stepwise process over the period of antifungal treatment (30). The most common mechanism of azole resistance appears to involve the enhancement of efflux pumps that remove azole compounds from the cytoplasm (1, 4, 27). Expression of the multidrug efflux transporters of the ATP-binding cassette superfamily, such as CDR1, and of the major facilitator class, such as MDR1, has been frequently reported in fluconazole-resistant strains. Also, alterations in the cell membrane through changes in sterol and/or lipid con
Genes, 2020
Infections caused by Aspergillus species are being increasingly reported. Aspergillus flavus is the second most common species within this genus causing invasive infections in humans, and isolates showing azole resistance have been recently described. A. flavus has three cyp51-related genes (cyp51A, cyp51B, and cyp51C) encoding 14-α sterol demethylase-like enzymes which are the target of azole drugs. In order to study triazole drug resistance in A. flavus, three strains showing reduced azole susceptibility and 17 azole susceptible isolates were compared. The three cyp51-related genes were amplified and sequenced. A comparison of the deduced Cyp51A, Cyp51B, and Cyp51C protein sequences with other protein sequences from orthologous genes in different filamentous fungi led to a protein identity that ranged from 50% to 80%. Cyp51A and Cyp51C presented several synonymous and non-synonymous point mutations among both susceptible and non-susceptible strains. However, two amino acid mutatio...
Formation of azole-resistant Candida albicans by mutation of sterol 14-demethylase P450
Antimicrobial agents and chemotherapy, 1999
The sterol 14-demethylase P450 (CYP51) of a fluconazole-resistant isolate of Candida albicans, DUMC136, showed reduced susceptibility to this azole but with little change in its catalytic activity. Twelve nucleotide substitutions, resulting in four amino acid changes, were identified in the DUMC136 CYP51 gene in comparison with a reported CYP51 sequence from a wild-type, fluconazole-susceptible C. albicans strain. Seven of these substitutions, including all of those causing amino acid changes, were located within a region covering one of the putative substrate recognition sites of the enzyme (SRS-1). Polymorphisms within this region were observed in several C. albicans isolates, and some were found to be CYP51 heterozygotes. Among the amino acid changes occurring in this region, only an alteration of Y132 was common among these fluconazole-resistant isolates, which suggests the importance of this residue to the fluconazole resistance of the target enzyme. DUMC136 and another flucona...