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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.
H J Cools - 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.
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.
Stefan G. R. Wirsel - One of the best experts on this subject based on the ideXlab platform.
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Fungal cytochrome P450 sterol 14α-demethylase (CYP51) and Azole resistance in plant and human pathogens
Applied Microbiology and Biotechnology, 2012Co-Authors: Rayko Becher, Stefan G. R. WirselAbstract:Azoles have been applied widely to combat pathogenic fungi in medicine and agriculture and, consequently, loss of efficacy has occurred in populations of some species. Often, but not always, resistance was found to result from amino acid substitutions in the molecular target of Azoles, 14α-sterol demethylase (CYP51 syn. ERG11). This review summarizes CYP51 function, evolution, and structure. Furthermore, we compare the occurrence and contribution of CYP51 substitutions to Azole resistance in clinical and field isolates of important fungal pathogens. Although no crystal structure is available yet for any fungal CYP51, homology modeling using structures from other origins as template allowed deducing models for fungal orthologs. These models served to map amino acid changes known from clinical and field isolates. We conclude with describing the potential consequences of these changes on the topology of the protein to explain CYP51-based Azole resistance. Knowledge gained from molecular modeling and resistance research will help to develop novel Azole structures.