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Theodore C White - One of the best experts on this subject based on the ideXlab platform.
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accumulation of azole drugs in the fungal plant pathogen magnaporthe oryzae is the result of facilitated diffusion influx
Frontiers in Microbiology, 2017Co-Authors: Ooke D Esquivel, Theodore C WhiteAbstract:Magnaporthe oryzae is an agricultural mold that causes disease in rice, resulting in devastating crop losses. Since rice is a world-wide staple food crop, infection by M. oryzae poses a serious global food security threat. Fungicides, including azole antifungals, are used to prevent and combat M. oryzae plant infections. The target of Azoles is CYP51, an enzyme localized on the endoplasmic reticulum (ER) and required for fungal ergosterol biosynthesis. However, many basic drug-pathogen interactions, such as how the azole gets past the fungal cell wall and plasma membrane, and is transported to the ER, are not understood. In addition, reduced intracellular accumulation of antifungals has consistently been observed as a drug resistance mechanism in many fungal species. Studying the basic biology of drug-pathogen interactions may elucidate uncharacterized mechanisms of drug resistance and susceptibility in M. oryzae and potentially other related fungal pathogens. We characterized intracellular accumulation of azole drugs in M. oryzae using a radioactively labeled fluconazole uptake assay to gain insight on whether Azoles enter the cell by passive diffusion, active transport, or facilitated diffusion. We show that azole accumulation is not ATP-dependent, nor does it rely on a pH-dependent process. Instead there is evidence for azole drug uptake in M. oryzae by a facilitated diffusion mechanism. The uptake system is specific for azole or azole-like compounds and can be modulated depending on cell phase and growth media. In addition, we found that co-treatment of M. oryzae with ‘repurposed’ clorgyline and radio-labeled fluconazole prevented energy-dependent efflux of fluconazole, resulting in an increased intracellular concentration of fluconazole in the fungal cell.
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Medically important fungi respond to azole drugs: an update
Future microbiology, 2015Co-Authors: Martin Zavrel, Theodore C WhiteAbstract:The increased numbers of patients with compromised immune systems in the last three decades have increased the chances of life-threatening fungal infections. Numerous antifungal drugs have been developed in the last 20 years to treat these infections. The largest group, the Azoles, inhibits the synthesis of fungal sterols. The use of these fungistatic Azoles has subsequently led to the emergence of acquired azole resistance. The most common mechanisms that result in azole resistance include the overexpression or mutation of the azole target enzyme, and overexpression of drug transporters that are responsible for azole efflux from cells. Additional, less-frequent mechanisms have also been identified. Understanding azole resistance mechanisms is crucial for current antifungal treatment and for the future development of new treatment strategies.
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azole drugs are imported by facilitated diffusion in candida albicans and other pathogenic fungi
PLOS Pathogens, 2010Co-Authors: Yce E Mansfield, Hanna N Oltea, Ia G Olive, Samantha J Hoo, Sarah Leyde, Lizbeth Hedstrom, Theodore C WhiteAbstract:Despite the wealth of knowledge regarding the mechanisms of action and the mechanisms of resistance to azole antifungals, very little is known about how the Azoles are imported into pathogenic fungal cells. Here the in-vitro accumulation and import of Fluconazole (FLC) was examined in the pathogenic fungus, Candida albicans. In energized cells, FLC accumulation correlates inversely with expression of ATP-dependent efflux pumps. In de-energized cells, all strains accumulate FLC, suggesting that FLC import is not ATP-dependent. The kinetics of import in de-energized cells displays saturation kinetics with a Km of 0.64 uM and Vmax of 0.0056 pmol/min/108 cells, demonstrating that FLC import proceeds via facilitated diffusion through a transporter rather than passive diffusion. Other Azoles inhibit FLC import on a mole/mole basis, suggesting that all Azoles utilize the same facilitated diffusion mechanism. An analysis of related compounds indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. Import of FLC by facilitated diffusion is observed in other fungi, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei, indicating that the mechanism of transport is conserved among fungal species. FLC import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered facilitated diffusion may be a previously uncharacterized mechanism of resistance to azole drugs.
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the candida albicans lanosterol 14 α demethylase erg11 gene promoter is maximally induced after prolonged growth with antifungal drugs
Antimicrobial Agents and Chemotherapy, 2004Co-Authors: Jia L Song, Jo Beth Harry, Richard T Eastman, Brian G Oliver, Theodore C WhiteAbstract:The azole antifungal drugs that target lanosterol 14-α-demethylase, encoded by the ERG11 gene, are used to treat a variety of infections caused by Candida albicans. Azoles are known to induce expression of ERG11 mRNA. The ERG11 promoter was cloned 5′ of the luciferase-coding region, and the induction of ERG11 expression by Azoles was monitored by luciferase assays. Maximal induction of the ERG11 promoter by Azoles occurs not during logarithmic growth but after the diauxic shift and requires Azoles to be present throughout logarithmic growth. The effects of pH, carbon source, and aerobic or anaerobic growth on induction of the ERG11 promoter by Azoles were analyzed. Treatment with terbinafine and fenpropimorph, which target other enzymes in the ergosterol biosynthetic pathway, also resulted in a delayed induction of ERG11 promoter activity. Nascent sterol synthesis was shown to parallel ERG11 promoter activity, and total sterols were reduced coincident with the timing of ERG11 promoter activation. These results as a whole suggest that expression of the ERG11 promoter is regulated in response to sterol depletion.
Paul E. Verweij - One of the best experts on this subject based on the ideXlab platform.
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clinical implications of globally emerging azole resistance in aspergillus fumigatus
Philosophical Transactions of the Royal Society B, 2016Co-Authors: Jacques F Meis, Anuradha Chowdhary, Johanna Rhodes, Matthew C Fisher, Paul E. VerweijAbstract:Aspergillus fungi are the cause of an array of diseases affecting humans, animals and plants. The triazole antifungal agents itraconazole, voriconazole, isavuconazole and posaconazole are treatment options against diseases caused by Aspergillus However, resistance to Azoles has recently emerged as a new therapeutic challenge in six continents. Although de novo azole resistance occurs occasionally in patients during azole therapy, the main burden is the aquisition of resistance through the environment. In this setting, the evolution of resistance is attributed to the widespread use of azole-based fungicides. Although ubiquitously distributed, A. fumigatus is not a phytopathogen. However, agricultural fungicides deployed against plant pathogenic moulds such as Fusarium, Mycospaerella and A. flavus also show activity against A. fumigatus in the environment and exposure of non-target fungi is inevitable. Further, similarity in molecule structure between azole fungicides and antifungal drugs results in cross-resistance of A. fumigatus to medical Azoles. Clinical studies have shown that two-thirds of patients with azole-resistant infections had no previous history of azole therapy and high mortality rates between 50% and 100% are reported in azole-resistant invasive aspergillosis. The resistance phenotype is associated with key mutations in the cyp51A gene, including TR34/L98H, TR53 and TR46/Y121F/T289A resistance mechanisms. Early detection of resistance is of paramount importance and if demonstrated, either with susceptibility testing or through molecular analysis, azole monotherapy should be avoided. Liposomal amphotericin B or a combination of voriconazole and an echinocandin are recomended for azole-resistant aspergillosis.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.
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Azole Resistance in Aspergillus fumigatus: Can We Retain the Clinical Use of Mold-Active Antifungal Azoles?
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 2015Co-Authors: Paul E. Verweij, Willem J. G. Melchers, Anuradha Chowdhary, Jacques F MeisAbstract:Azole resistance in Aspergillus fumigatus has emerged as a global health problem. Although the number of cases of azole-resistant aspergillosis is still limited, resistance mechanisms continue to emerge, thereby threatening the role of the azole class in the management of diseases caused by Aspergillus. The majority of cases of azole-resistant disease are due to resistant A. fumigatus originating from the environment. Patient management is difficult due to the absence of patient risk factors, delayed diagnosis, and limited treatment options, resulting in poor treatment outcome. International and collaborative efforts are required to understand how resistance develops in the environment to allow effective measures to be implemented aimed at retaining the use of Azoles both for food production and human medicine.
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High-Level Pan-Azole-Resistant Aspergillosis
Journal of clinical microbiology, 2015Co-Authors: Jakko Van Ingen, Henrich A. L. Van Der Lee, A.j.m.m. Rijs, Eveline Snelders, Willem J. G. Melchers, Paul E. VerweijAbstract:High-level pan-azole-resistant Aspergillus fumigatus was recovered from four patients with chronic lung disease. In one patient, the development of progressive resistance followed long-term azole therapy and switching between antifungal Azoles. The high-level pan-azole-resistant phenotypes were not associated with a specific cyp51A gene mutation. New strategies that avoid the development of progressive azole resistance are needed.
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occurrence of azole resistant species of aspergillus in the uk environment
Journal of global antimicrobial resistance, 2014Co-Authors: Michael Bromley, Paul E. Verweij, Guus Van Muijlwijk, Marcin G Fraczek, Geoff Robson, David W Denning, Paul BowyerAbstract:The aim of this study was to survey environmental isolates of Aspergillus resistant to Azoles in azole-treated and naive areas to determine whether resistance could be related to azole treatment history. Aspergillus fumigatus was sampled from the centre of a large city and from fields with known azole history. Azole resistance was determined and sequencing was performed to identify strains and mutations in the cyp51A gene. Azole resistance was detected in azole-treated field isolates but not in urban isolates (P=0.038). In addition, an azole-resistant isolate of Neosartorya fischeri was isolated. These results support the hypothesis that agricultural azole use may lead to resistance in environmental fungi of clinical importance. We report the first environmental UK TR34/L98H isolate of A. fumigatus.
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azole resistance in aspergillus fumigatus a new challenge in the management of invasive aspergillosis
Future Microbiology, 2011Co-Authors: Eveline Snelders, Willem J. G. Melchers, Paul E. VerweijAbstract:Azole resistance is emerging in Aspergillus fumigatus isolates. The exact mechanism of evolution of azole resistance has not been fully elucidated yet but increasing evidence indicates a role for azole fungicide used in agriculture. Patients confronted with an invasive fungal infection from an azole-resistant A. fumigatus isolate will fail azole treatment. Azole resistance in A. fumigatus isolates impacts the management of invasive aspergillosis (IA) since the Azoles are the primary agents used for prophylaxis and treatment. Because A. fumigatus will always be present in our environment and also in the close vicinity of patients at risk for IA, there is an urgent need to understand the evolution of the increasing azole resistance in A. fumigatus. Thereby, induction of azole resistance or its spread can possibly be prevented to allow future treatment of A. fumigatus IA.
Steven L. Kelly - One of the best experts on this subject based on the ideXlab platform.
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impact of recently emerged sterol 14α demethylase cyp51 variants of mycosphaerella graminicola on azole fungicide sensitivity
Applied and Environmental Microbiology, 2011Co-Authors: H J Cools, J A Lucas, B A Fraaije, Jonathan G L Mullins, Josie E Parker, Diane E Kelly, Steven L. KellyAbstract:The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all Azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used Azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.
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Molecular basis of resistance to azole antifungals
Trends in molecular medicine, 2002Co-Authors: Antonella Lupetti, Romano Danesi, Mario Campa, Mario Del Tacca, Steven L. KellyAbstract:The increased incidence of invasive mycoses and the emerging problem of antifungal drug resistance has prompted investigations of the underlying molecular mechanisms, particularly for the azole compounds central to current therapy. The target site for the Azoles is the ERG11 gene product, the cytochrome P450 lanosterol 14alpha-demethylase, which is part of the ergosterol biosynthetic pathway. The resulting ergosterol depletion renders fungal cells vulnerable to further membrane damage. Development of azole resistance in fungi may occur through increased levels of the cellular target, upregulation of genes controlling drug efflux, alterations in sterol synthesis and decreased affinity of Azoles for the cellular target. Here, we review the adaptative changes in fungi, in particular Candida albicans, in response to inhibitors of ergosterol biosynthesis. The molecular mechanisms of azole resistance might help in devising more effective antifungal therapies.
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molecular basis of resistance to azole antifungals
Trends in Molecular Medicine, 2002Co-Authors: Antonella Lupetti, Romano Danesi, Mario Campa, Mario Del Tacca, Steven L. KellyAbstract:Abstract The increased incidence of invasive mycoses and the emerging problem of antifungal drug resistance has prompted investigations of the underlying molecular mechanisms, particularly for the azole compounds central to current therapy. The target site for the Azoles is the ERG11 gene product, the cytochrome P450 lanosterol 14α-demethylase, which is part of the ergosterol biosynthetic pathway. The resulting ergosterol depletion renders fungal cells vulnerable to further membrane damage. Development of azole resistance in fungi may occur through increased levels of the cellular target, upregulation of genes controlling drug efflux, alterations in sterol synthesis and decreased affinity of Azoles for the cellular target. Here, we review the adaptative changes in fungi, in particular Candida albicans , in response to inhibitors of ergosterol biosynthesis. The molecular mechanisms of azole resistance might help in devising more effective antifungal therapies.
Michael A Pfaller - One of the best experts on this subject based on the ideXlab platform.
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analysis of global antifungal surveillance results reveals predominance of erg11 y132f alteration among azole resistant candida parapsilosis and candida tropicalis and country specific isolate dissemination
International Journal of Antimicrobial Agents, 2020Co-Authors: Mariana Castanheira, S A Messer, Lalitagauri M Deshpande, Paul R Rhomberg, Michael A PfallerAbstract:ABSTRACT This study evaluated the activity of echinocandins, Azoles and amphotericin B against Candida spp. isolates and other yeasts and characterised azole resistance mechanisms in Candida parapsilosis and Candida tropicalis. Invasive Candida spp. isolates (n = 2936) collected in 60 hospitals worldwide during 2016–2017 underwent antifungal susceptibility testing by broth microdilution. Azole-resistant C. parapsilosis and C. tropicalis were submitted to qPCR for ERG11, CDR1 and MDR1, and the whole genome sequence was analysed. Results of non-susceptibility to echinocandins ranged from 0.0–2.3%, being highest in Candida glabrata. More than 99.0% of the Candida albicans isolates were susceptible to both fluconazole and voriconazole. Fluconazole resistance in C. glabrata was 6.5% overall, being highest in the USA (13.0%). Resistance to voriconazole in Candida krusei was only noted in the USA (5.0%). Azoles inhibited 89.1–91.6% of C. parapsilosis isolates, with most resistant isolates noted in Europe (15.1%), including 36 isolates from Italy (three hospitals), of which 34 harboured Erg11 Y132F mutations and overexpressed MDR1. Azole non-wild-type C. tropicalis (7/227) were found in five countries: 3 isolates from Thailand had the same Erg11 Y132F alteration. Fluconazole non-wild-type isolates were noted among 3/77 (3.9%) Candida dubliniensis, 4/17 (23.5%) Candida guilliermondii, 4/47 (8.5%) Candida lusitaniae and other less common yeast species. Echinocandin use has been recommended over fluconazole for invasive Candida infections. However, Azoles are still active against the most common Candida spp. and resistance appears to be restricted to certain geographic regions and associated with Erg11 Y132 alterations in C. parapsilosis and C. tropicalis.
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analysis of global antifungal surveillance results reveals predominance of erg11 y132f alteration among azole resistant candida parapsilosis and candida tropicalis and country specific isolate dissemination
International Journal of Antimicrobial Agents, 2020Co-Authors: Mariana Castanheira, S A Messer, Lalitagauri M Deshpande, Paul R Rhomberg, Michael A PfallerAbstract:ABSTRACT This study evaluated the activity of echinocandins, Azoles and amphotericin B against Candida spp. isolates and other yeasts and characterised azole resistance mechanisms in Candida parapsilosis and Candida tropicalis. Invasive Candida spp. isolates (n = 2936) collected in 60 hospitals worldwide during 2016–2017 underwent antifungal susceptibility testing by broth microdilution. Azole-resistant C. parapsilosis and C. tropicalis were submitted to qPCR for ERG11, CDR1 and MDR1, and the whole genome sequence was analysed. Results of non-susceptibility to echinocandins ranged from 0.0–2.3%, being highest in Candida glabrata. More than 99.0% of the Candida albicans isolates were susceptible to both fluconazole and voriconazole. Fluconazole resistance in C. glabrata was 6.5% overall, being highest in the USA (13.0%). Resistance to voriconazole in Candida krusei was only noted in the USA (5.0%). Azoles inhibited 89.1–91.6% of C. parapsilosis isolates, with most resistant isolates noted in Europe (15.1%), including 36 isolates from Italy (three hospitals), of which 34 harboured Erg11 Y132F mutations and overexpressed MDR1. Azole non-wild-type C. tropicalis (7/227) were found in five countries: 3 isolates from Thailand had the same Erg11 Y132F alteration. Fluconazole non-wild-type isolates were noted among 3/77 (3.9%) Candida dubliniensis, 4/17 (23.5%) Candida guilliermondii, 4/47 (8.5%) Candida lusitaniae and other less common yeast species. Echinocandin use has been recommended over fluconazole for invasive Candida infections. However, Azoles are still active against the most common Candida spp. and resistance appears to be restricted to certain geographic regions and associated with Erg11 Y132 alterations in C. parapsilosis and C. tropicalis.
B A Fraaije - One of the best experts on this subject based on the ideXlab platform.
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update on mechanisms of azole resistance in mycosphaerella graminicola and implications for future control
Pest Management Science, 2013Co-Authors: H J Cools, B A FraaijeAbstract:This review summarises recent investigations into the molecular mechanisms responsible for the decline in sensitivity to azole (imidazole and triazole) fungicides in European populations of the Septoria leaf blotch pathogen, Mycosphaerella graminicola. The complex recent evolution of the azole target sterol 14a-demethylase (MgCYP51) enzyme in response to selection by the sequential introduction of progressively more effective Azoles is described, and the contribution of individual MgCYP51 amino acid alterations and their combinations to azole resistance phenotypes and intrinsic enzyme activity is discussed. In addition, the recent identification of mechanisms independent of changes in MgCYP51 structure correlated with novel azole cross-resistant phenotypes suggests that the further evolution of M. graminicola under continued selection by azole fungicides could involve multiple mechanisms. The prospects for azole fungicides in controlling European M. graminicola populations in the future are discussed in the context of these new findings. Copyright (C) 2012 Society of Chemical Industry
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overexpression of the sterol 14α demethylase gene mgcyp51 in mycosphaerella graminicola isolates confers a novel azole fungicide sensitivity phenotype
Pest Management Science, 2012Co-Authors: H J Cools, C Bayon, Sarah Atkins, J A Lucas, B A FraaijeAbstract:BACKGROUND: The recent evolution towards resistance to azole fungicides in European populations of the wheat pathogen Mycosphaerella graminicola has been caused by the progressive accumulation of mutations in MgCYP51 gene, encoding the azole target sterol 14a-demethylase. Particular combinations of mutations have been shown specifically to affect the interaction of the MgCYP51 protein with different members of the azole class. Although additional mechanisms, including increased MgCYP51 expression and enhanced active efflux, have been proposed, the genetic changes underlying these mechanisms are unknown. RESULTS: Analysis of the azole sensitivities of recent M. graminicola isolates identified a novel phenotype, seemingly independent of changes in MgCYP51 coding sequence. Characterised by a 7-16-fold reduction in in vitro sensitivity to all Azoles tested and by growth on seedlings at higher doses of Azoles in glasshouse tests compared with isolates carrying the same MgCYP51 variant (L50S, S188N, I381V, ?Y459/G460, N513K), isolates with this phenotype constitutively overexpress MgCYP51 by between 10- and 40-fold compared with the wild type. Analysis of sequences upstream of the predicted MgCYP51 translation start codon identified a novel 120 bp indel, considered to be an insertion, in isolates overexpressing MgCYP51. CONCLUSIONS: The identification of an insertion in the predicted MgCYP51 promoter in azole-resistant isolates overexpressing MgCYP51 is the first report of a genetic mechanism, other than changes in target-site coding sequence, affecting sensitivity to multiple Azoles in field isolates of M. graminicola. The identification of recent isolates overexpressing MgCYP51 confirms the ongoing evolution and diversification of resistance mechanisms in European populations of M. graminicola. Copyright (c) 2012 Society of Chemical Industry
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impact of recently emerged sterol 14α demethylase cyp51 variants of mycosphaerella graminicola on azole fungicide sensitivity
Applied and Environmental Microbiology, 2011Co-Authors: H J Cools, J A Lucas, B A Fraaije, Jonathan G L Mullins, Josie E Parker, Diane E Kelly, Steven L. KellyAbstract:The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all Azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used Azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.
