CYP51 Mutations Research Articles

Mutations in the CYP51 gene reduce DMI sensitivity in Parastagonospora nodorum populations in Europe and China.

Sterol demethylation inhibitors (DMIs) have been widely used to manage agronomically important fungal diseases in wheat, but reports of DMI-resistant pathogens continue to mount. Parastagonospora nodorum shows a wide range of sensitivity to DMIs, but until now no molecular mechanisms have been identified to explain these differences. The aim of this study was to correlate the DMI sensitivity of a global collection of P. nodorum isolates with mutations in the CYP51 gene that encodes the target of DMI fungicides. Two non-synonymous mutations connected to DMI resistance in other plant pathogenic fungi were detected for the first time in the CYP51 gene of P. nodorum. The two mutations occurred at amino acid position 144, which is homologous to position 137 in other pathogens. The Y144F mutation was detected in China, Denmark, Sweden and Switzerland while the Y144H mutation was found in China and Switzerland. Both mutations were correlated with significantly reduced sensitivity to the DMI fungicide propiconazole. CYP51 mutations conferred reduced sensitivity against DMIs in field populations of P. nodorum originating from China, Denmark, Sweden and Switzerland. © 2016 Society of Chemical Industry.

Multilocus resistance evolution to azole fungicides in fungal plant pathogen populations.

Evolution of fungicide resistance is a major threat to food production in agricultural ecosystems. Fungal pathogens rapidly evolved resistance to all classes of fungicides applied to the field. Resistance to the commonly used azole fungicides is thought to be driven mainly by mutations in a gene (CYP51) encoding a protein of the ergosterol biosynthesis pathway. However, some fungi gained azole resistance independently of CYP51 mutations and the mechanisms leading to CYP51-independent resistance are poorly understood. We used whole-genome sequencing and genome-wide association studies (GWAS) to perform an unbiased screen of azole resistance loci in Rhynchosporium commune, the causal agent of the barley scald disease. We assayed cyproconazole resistance in 120 isolates collected from nine populations worldwide. We found that mutations in highly conserved genes encoding the vacuolar cation channel YVC1, a transcription activator, and a saccharopine dehydrogenase made significant contributions to fungicide resistance. These three genes were not previously known to confer resistance in plant pathogens. However, YVC1 is involved in a conserved stress response pathway known to respond to azoles in human pathogenic fungi. We also performed GWAS to identify genetic polymorphism linked to fungal growth rates. We found that loci conferring increased fungicide resistance were negatively impacting growth rates, suggesting that fungicide resistance evolution imposed costs. Analyses of population structure showed that resistance mutations were likely introduced into local populations through gene flow. Multilocus resistance evolution to fungicides shows how pathogen populations can evolve a complex genetic architecture for an important phenotypic trait within a short time span.

Competitive Fitness of Phakopsora pachyrhizi Isolates with Mutations in the CYP51 and CYTB Genes.

Soybean rust (Phakopsora pachyrhizi) in Brazil is mainly controlled with applications of fungicides, including demethylation inhibitors (DMI) and quinone outside inhibitors (QoI). Isolates with less sensitivity to DMI and QoI have been reported, and these have been found to have mutations in the CYP51 and CYTB genes, respectively. There have been no reports of fitness costs in isolates with mutations in CYP51 and CYTB, and the aim of this work was to compare the competitive ability of isolates with lower DMI or QoI sensitivities with that of sensitive (wild-type) isolates. Urediniospores of sensitive wild-type isolates and isolates with different CYP51 or CYTB alleles were mixed and inoculated on detached soybean leaves. After 3 weeks, urediniospores were harvested and used as inoculum for the next disease cycle. Frequencies of relevant target site mutations were monitored using the pyrosequencing method over four disease cycles. Isolates with lower DMI sensitivity and different CYP51 alleles had competitive disadvantages compared with a DMI-sensitive, wild-type CYP51 isolate. In contrast, the isolate with the F129L mutation in the CYTB gene competed equally well with a QoI-sensitive, wild-type CYTB isolate under the conditions of this experiment. The CYP51 and CYTB alleles were stable in all isolates over four disease cycles when cultivated alone.

First Report of Resistance to DMI Fungicides in Australian Populations of the Wheat Pathogen Zymoseptoria tritici

The erosion of demethylation inhibitor (DMI) fungicides effectiveness over time in Europe has been attributed to mutation sites in the 14α-demethylase encoded by the nuclear gene CYP51 (Cools and Fraaije 2013). These mutations first appeared in Europe in the 1990s, became widespread over the next 20 years, and were also recently reported in North America (Estep et al. 2015; Lucas et al. 2015). Zymoseptoria tritici has been present and causing disease on wheat (Triticum aestivum) in Australia for many decades and the use of fungicides for its control has become more common over the past 15 years. However, no significant changes in the field performance of fungicides have been noted by growers. To investigate possible undetected changes, we sequenced the CYP51 gene of 18 isolates cultured from wheat leaves collected in a commercial field on 1 July 2012 at Inverleigh, Victoria (–38.086743° S; 143.935783° E), and 3 isolates cultured from wheat leaves collected from trial plots on 22 August 2002 at Wagga Wagga,NSW (–35.044544° S; 147.316318° E). The nucleotide sequence of Z. tritici strain ST1 eburicol 14 alpha-demethylase (CYP51) gene (GenBank Accession No. AY730587.1) was used to design a set of three forward and three reverse sequencing primers across the known mutations. The sequence reads were assembled into contigs and checked for complete sequence. To generate an alignment of mature protein sequences, the mature protein sequence of ST1 (CYP51) gene was downloaded from NCBI to use as a reference. CYP51 nucleotide sequences were translated into amino acid sequences using all six possible frame shifts. These were aligned to the mature protein sequence of ST1 (CYP51) to identify the correct frame-shift sequence, enable the removal of intron sequences, and finally generate an amino acid alignment. Nucleotide and amino acid sequences are deposited in GenBank with Accession Nos. KT201543 to KT201563. Sixteen of the isolates from Victoria were carrying the Y137F mutation, one isolate carried the L50S-Y461S mutation, and one the L50S-S188N-N513K. The three isolates from NSW collected in 2002 contained no mutations compared with the reference accession. Fungicide sensitivities for propiconazole were determined at 50% effective concentrations (EC50) using rates of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 mg/liter, and the resistance factor (RF) of each isolate was calculated as fold change in the EC50 compared with that of the wild-type strains as per Cools et al. (2011), four replicates per isolate were performed. Results of the phenotypic assay of a subset of nine isolates (four Y137F mutants, one L50S-Y461S, one L50S-S188N-N513K, and three Wild-type) confirmed the elevated EC50 values for the mutations known to cause reduced sensitivity. The Y137F and L50S-Y461S mutants showed EC50 values of 1.08 and 1.03 mg/liter and RF levels in the range of 3.07 and 2.93. respectively, while the wild-type and L50S-S188N-N513K mutant had EC50 of 0.352 and 0.367 mg/liter, respectively. The EC50 values observed for the isolates appear higher than those found by Cools et al. (2011); however, the RF values are lower for both mutations. Further exploration of CYP51 mutations occurring in Z. tritici within the Australian cropping regions will be necessary to establish how widespread the Y137F and L50S-Y461S mutants are, and if others such as the S524T or the V136A, which are associated with higher levels of resistance to DMIs, are also present.

First Detection of TR34 L98H and TR46 Y121F T289A Cyp51 Mutations in Aspergillus fumigatus Isolates in the United States.

Azole resistance in Aspergillus fumigatus is an increasing problem. The TR34 L98H and TR46 Y121F T289A mutations that can occur in patients without previous azole exposure have been reported in Europe, Asia, the Middle East, Africa, and Australia. Here, we report the detection of both the TR34 L98H and TR46 Y121F T289A mutations in confirmed A. fumigatus isolates collected in institutions in the United States. These mutations, other mutations known to cause azole resistance, and azole MICs are reported here.

Impact of DMI and SDHI fungicides on disease control and CYP51 mutations in populations of Zymoseptoria tritici from Northern Europe

Zymoseptoria tritici is a dominant pathogen in wheat causing Septoria leaf blotch (SLB), and sterol 14α-demethylation inhibitors fungicides (DMI) are commonly used for control in Northern Europe. In 14 winter wheat trials carried out in Denmark, Lithuania, and Sweden in the years 2011 to 2013, fungicides containing DMIs were investigated for their efficacy and impact on CYP51 mutations in Z. tritici populations. All fungicide treatments were applied twice – each time using 50 % of the standard rate, applied at GS 37 & 55. The single-agent DMIs, epoxiconazole and prothioconazole and a mixture of difenoconazole + propiconazole gave similar control and crop yields. The best solution varied among localities. Adding prochloraz to prothioconazole as well as using co-formulations of DMIs + SDHIs generally improved control compared with using DMIs alone. Yield responses were significant from all treatments, but co-formulations of DMIs plus SDHIs increased yield the most. Specific CYP51 mutations in Z. tritici were analysed by pyrosequencing and qPCR. Their frequency varied across sites and countries. The amount of I381V was high in all trials whereas the amount of A379G was moderate. Levels of D134G, V136C, and S524T were low to moderate across all sites. DMIs and mixtures of DMIs + SDHI selected differently for CYP51 mutations. Prochloraz increased selection for D134G and V136A and decreased selection for A379G and I381V. The lowest selection pressure towards D134G and V136A/C was recorded in the presence of the mixture difenoconazole and propioconazole. Mixtures of DMIs and SDHI tended to lower the frequency of V136A/C compared to DMIs used alone but had no measurable impact on the frequency of I381V and A379G. No responses were seen in relation to S524T, which only occurred at a very low level.

Molecular Modelling of the Emergence of Azole Resistance in Mycosphaerella graminicola

A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.

Azole resistance in aspergillosis: The next threat?

During the past years, aspergilli less susceptible to antifungals have begun to emerge, and antifungal drug resistance may partially account for treatment failures. Resistance of Aspergillus fumigatus clinical isolates to itraconazole, voriconazole, and posaconazole has been reported with increasing frequency, although it is considered an uncommon phenomenon. Molecular biologists have begun to shed light on the mechanisms of A. fumigatus resistance to azoles. Several mechanisms of resistance have been described, such as point mutations of cyp51A and reduced concentrations of intracellular drug. The latter mechanism might be the result of either overexpression of efflux pumps or reduced drug penetration. The issue of cross-resistance between the newer triazoles is of concern and depends on cyp51 mutations. Fungal drug resistance is an issue because of the limited number of antifungal compounds. Patients receiving long-term azole treatment are at highest risk for developing multidrug-resistant A. fumigatus infections.

Evolutionary trace analysis of CYP51 family: implication for site-directed mutagenesis and novel antifungal drug design

Lanosterol 14alpha-demethylase (CYP51) is an essential enzyme in the fungal life cycle and also an important target for the antifungal drug development. Based on the multiple sequence alignments of CYP51 family, an evolutionary tree of the CYP51 family was constructed by the evolutionary trace (ET) method. The identified trace residues could provide a reliable and rational guide to the design of CYP51 mutations and give more information about the detailed mechanism of substrate (drug) recognition and binding. The reliability of ET analysis to identify residues of functional importance was validated by the reported site-directed mutagenesis studies of CYP51s. Several residues in the active site were also validated by our mutagenesis studies. Mapping the identified trace residues onto the active site of the modeled structure of Candida albicans CYP51 (CACYP51) may provide useful information for the design of novel antifungal agents.

Evolution of the CYP51 gene in Mycosphaerella graminicola: evidence for intragenic recombination and selective replacement

Recent findings are consistent with a slow but constant shift towards reduced sensitivity of Mycosphaerella graminicola to azole fungicides, which target the CYP51 gene. The goal of this study was to elucidate the evolutionary mechanisms through which CYP51-based mutations associated with altered sensitivity have evolved in M. graminicola over space and time. To accomplish this, we sequenced and compared a portion of the CYP51 gene encompassing the main mutations associated with altered sensitivity towards demethylation inhibitor fungicides. The CYP51 gene showed an extraordinary dynamic shift consistent with a selective haplotype replacement both in space and in time. No mutations associated with increased resistance to azoles were found in non-European populations. These mutations were also absent in the oldest collections from Europe, whereas they dominated in the recent European populations. Intragenic recombination was identified as an important evolutionary process in populations affected by high fungicide selection, suggesting the creation of novel alleles among existing mutations as a potential source of novel resistance alleles. We propose that CYP51 mutations giving resistance in M. graminicola arose only locally (perhaps in Denmark or the UK) and were then spread eastward across Europe through wind-dispersed ascospores. We conclude that recurring cycles of recombination coupled with selection due to the widespread use of azole fungicides will increase the frequency of novel mutants or recombinants with higher resistance. Long-distance gene flow due to wind dispersal of ascospores will move the resulting new alleles to new areas following the prevailing wind directions. A selective replacement favouring haplotypes with various coding mutations at the target site for azole fungicides during the last 5-10 years is the most likely cause of the decrease in sensitivity reported for many azole fungicides in the same period.

Substrate recognition sites in 14α-sterol demethylase from comparative analysis of amino acid sequences and X-ray structure of Mycobacterium tuberculosis CYP51

The crystal structure of 14α-sterol demethylase from Mycobacterium tuberculosis (MTCYP51) [Proc. Natl. Acad. Sci. USA 98 (2001) 3068–3073] provides a template for analysis of eukaryotic orthologs which constitute the CYP51 family of cytochrome P450 proteins. Putative substrate recognition sites (SRSs) were identified in MTCYP51 based on the X-ray structures and have been compared with SRSs predicted based on Gotoh’s analysis [J. Biol. Chem. 267 (1992) 83–90]. While Gotoh’s SRS-4, 5, and 6 contribute in formation of the putative MTCYP51 substrate binding site, SRS-2 and 3 likely do not exist in MTCYP51. SRS-1, as part of the open BC loop, in the conformation found in the crystal can provide only limited contacts with the sterol. However, its role in substrate binding might dramatically increase if the loop closes in response to substrate binding. Thus, while the notion of SRSs has been very useful in leading to our current understanding of P450 structure and function, their identification by sequence alignment between distant P450 families will not necessarily be a good predictor of residues associated with substrate binding. Localization of CYP51 mutation hotspots in Candida albicans azole resistant isolates was analyzed with respect to SRSs. These mutations are found to be outside of the putative substrate interacting sites indicating the preservation of the protein active site under the pressure of azole treatment. Since the mutations residing outside the putative CYP51 active side can profoundly influence ligand binding within the active site, perhaps they provide insight into the basis of evolutionary changes which have occurred leading to different P450s.