Elevated gene transcription of the cyp51A gene is essential for azole drug resistance in Aspergillus fumigatus

University Of Iowa, Iowa City IA

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Abstract

Invasive Aspergillosis caused by azole resistant Aspergillus fumigatus (Afu) has a mortality rate exceeding 60%, making this a clinical problem of acute significance. Extensive analyses of resistant isolates of Afu have shown that the majority of resistance-causing mutations are linked to the cyp51A gene encoding the enzyme that is the target of azole drugs. The most common forms of resistant Afu contain two types of mutations: one that causes a substitution mutation within the coding sequence for the enzyme while a second alteration is a duplication of a short segment of the cyp51A promoter region. The most common allele is referred to as TR34 L98H cyp51A in which the TR34 designation indicates a tandem repeat of 34 bp of the promoter region. In the previous grant period, we identified a transcription factor called AtrR that binds to an element within the 34 bp region of cyp51A. AtrR binding is required for the elevated cyp51A expression driven by the TR34 promoter element as well as the enhanced voriconazole resistance this mutant gene provides. We provide genetic evidence that the function of the two 34 bp regions in the TR34 cyp51A promoter do not have similar functions and the appearance of the second 34 bp repeat triggers enhanced chromatin accessibility as measured by Assay for Transposase- Accessible Chromatin using sequencing (ATAC-seq). Our data indicate that the differential behavior of the 34 bp elements in cyp51A are essential for the unique elevated transcription supported by this mutant promoter. Previous models that the simple duplication of the effect of this 34 bp region was sufficient to explain its behavior are incorrect and the upstream 34 bp has now acquired a genetically distinct behavior that explains the elevation in cyp51A transcription. We believe that this behavior is likely reflective of the normal function of the 34 bp region and will test this idea using ATAC-seq on the wild- type promoter in the presence and absence of azole treatment. We will also examine the role of both the AtrR binding sites and the presence of AtrR in the observed increased chromatin accessibility seen in the TR34 cyp51A promoter. We have used biochemical purification of AtrR to identify proteins that associate with this factor and found that several of these are involved in chromatin modification. We will determine the role of these proteins in control of cyp51A promoter accessibility and expression of the TR34 cyp51A gene. We will purify the transcription factor SrbA that is also known to bind to the 34 bp element region and examine its role in chromatin accessibility and recruitment of coactivators. Finally, we will determine the role for cyp51A mutants in the azole response in a murine model using isogenic strains of Afu. Additionally, we will use an enrichment strategy to measure Afu transcription in infected lungs +/- azole drug challenge. This work will illuminate the function of a key region of the cyp51A promoter that is associated with 80% of azole resistant Afu isolates.