Analysis of Promoter Function in Aspergillus fumigatus

Abstract

ABSTRACT The filamentous fungus Aspergillus fumigatus is an important opportunistic pathogen that can cause high mortality levels in susceptible patient populations. The increasing dependence on antifungal drugs to control A. fumigatus has led to the inevitable acquisition of drug-resistant forms of this pathogen. In other fungal pathogens, drug resistance is often associated with an increase in transcription of genes such as ATP-binding cassette (ABC) transporters that directly lead to tolerance to commonly employed antifungal drugs. In A. fumigatus , tolerance to azole drugs (the major class of antifungal) is often associated with changes in the sequence of the azole target enzyme as well as changes in the transcription level of this gene. The target gene for azole drugs in A. fumigatus is referred to as cyp51A . In order to dissect transcription of cyp51A transcription and other genes of interest, we constructed a set of firefly luciferase reporter genes designed for use in A. fumigatus . These reporter genes can either replicate autonomously or be targeted to the pyrG locus, generating an easily assayable uracil auxotrophy. We fused eight different A. fumigatus promoters to luciferase. Faithful behaviors of these reporter gene fusions compared to their chromosomal equivalents were evaluated by 5′ rapid amplification of cDNA ends (RACE) and quantitative reverse transcription-PCR (qRT-PCR) analysis. We used this reporter gene system to study stress-regulated transcription of a Hsp70-encoding gene, map an important promoter element in the cyp51A gene, and correct an annotation error in the actin gene. We anticipate that this luciferase reporter gene system will be broadly applicable in analyses of gene expression in A. fumigatus .