Aspergillus fumigatus is the major human filamentous fungal pathogen. Azole drugs represent the gold standard in treatment of aspergillosis and are the only agent that can be used without hospitalization. Problematic findings in Western Europe have shown that azole resistant forms of A. fumigatus can arise and spread frequently. These studies have demonstrated that the primary cause of azole resistance is a compound mutation in the gene encoding the target enzyme for azole drugs, cyp51A. These linked mutations consist of an alteration in the promoter sequence (tandem duplication of 34 base pairs: TR34) coupled with an amino acid replacement in the coding sequence (L98H) in cyp51A. Mutant strains containing this TR34 L98H cyp51A allele are highly azole drug resistant and appear to have no fitness defect, leading to the high frequency of resistant infections. We have discovered a new transcription factor called AtrR that binds to this TR34 element and is required for normal cyp51A expression. Importantly, both we and others have found that atrR? null mutants are avirulent in a mouse inhalation model of infection. Here we propose to use a chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) dataset that we have generated to identify genes under control of AtrR that impact virulence. We will use a mouse infection model to determine AtrR-regulated genes that exhibit transcriptional responses in the infected mouse lung. Nanostring technology will be employed to allow measurement of fungal gene expression in the high background of mammalian RNA. AtrR target genes will be rank ordered by their in vivo expression profile. We will use CRISPR technology and existing disruption collections to assess the role of up to 40 AtrR target genes for their effect on an in vitro epithelial cell damage assay. Our goal will be to prioritize these genes based on their in vivo expression profile and impact on epithelial cell damage. From these analyses, we will select up to 8 target gene disruption mutants to screen for an effect on virulence using our mouse inhalation model. This work will provide the first examination of the molecular basis of AtrR-mediated virulence factors that are critical for pathogenesis in this animal model of infected lungs.
Aspergillus fumigatus is the major human filamentous fungal pathogen, with mortality reaching 90% during infections caused by drug resistant forms of this fungus. This work will analyze the contribution of a newly identified, globally-acting transcription factor to disease pathogenesis in this organism.
