Dataset Information

Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus.

ABSTRACT: The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR₃₄, TR₄₆, and TR₅₃ Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

SUBMITTER: Bernal-Martinez L

PROVIDER: S-EPMC5700344 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus.

Bernal-Martínez L L Gil H H Rivero-Menéndez O O Gago S S Cuenca-Estrella M M Mellado E E Alastruey-Izquierdo A A

Antimicrobial agents and chemotherapy 20171122 12

The global emergence of azole-resistant <i>Aspergillus fumigatus</i> strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in <i>cyp51A</i> Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations ...[more]

PMID: 28893791

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Project description:Aspergillosis associated with azole-resistant Aspergillus fumigatus has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the cyp51A gene that encodes the target of azole drugs, lanosterol ?-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of cyp51A and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR). Our previous studies identified a positively acting transcription factor called AtrR that binds to the promoter of cyp51A as well as that of an important membrane transporter protein gene called abcG1 In this work, we characterize two different mutant alleles of atrR, either an overproducing or an epitope-tagged form, causing constitutive activation of this factor. Using an epitope-tagged allele of atrR for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), the genomic binding sites for AtrR were determined. Close to 900 genes were found to have an AtrR response element (ATRE) in their promoter regions. Transcriptome evaluation by RNA sequencing (RNA-seq) indicated that both alleles led to elevated transcription of a subset of target genes. An electrophoretic mobility shift assay and DNase I protection mapping localized the ATREs in both the abcG1 and cyp51A promoters. The ATRE in cyp51A was located within the 34-bp repeat element. Virulence in a murine model was compromised when AtrR was either deleted or overproduced, indicating that the proper dosage of this factor is key for pathogenesis.IMPORTANCE Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Infections associated with A. fumigatus are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene cyp51A as an important contributor to resistance in A. fumigatus Here, we demonstrate that the transcription factor AtrR binds to a region in the cyp51A promoter that is associated with alleles of this gene conferring clinically important azole resistance. Using high-throughput genomic technologies, we also uncover a large suite of target genes controlled by AtrR. These data indicate that AtrR coordinately regulates many different processes involved in drug resistance, metabolism, and virulence. Our new understanding of AtrR function provides important new insight into the pathogenesis of A. fumigatus.

Dataset Information

Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus.

Publications

Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus.

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