Functional analysis of an ATP-binding cassette transporter protein from Aspergillus fumigatus by heterologous expression in Saccharomyces cerevisiae

Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Although A. fumigatus can be treated with many of the available antifungal drugs, including azole compounds, drug resistant isolates are being recovered at an increasing rate. In other fungal pathogens such as the Candida species, ATP-binding cassette (ABC) transporter proteins play important roles in development of clinically-significant azole resistance phenotypes. Central among these ABC transporter proteins are homologues of the Saccharomyces cerevisiae Pdr5 multidrug transporter. In this work, we test the two A. fumigatus genes encoding proteins sharing the highest degree of sequence similarity to S. cerevisiae Pdr5 for their ability to be function in a heterologous pdr5 Delta strain of S. cerevisiae. Expression of full-length cDNAs for these two Afu proteins failed to suppress the drug sensitive phenotype of a pdr5 Delta strain and no evidence could be obtained for their expression as green fluorescent protein (GFP) fusions. To improve the expression of one of these Afu ABC transporters (XP_755847), we changed the sequence of the cDNA to use codons corresponding to the major tRNA species in S. cerevisiae. This codon-optimized (CO Mu abcA) cDNA was efficiently expressed in pdr5 Delta cells and able to be detected as a GFP fusion protein. The CO Mu abcA did not correct the drug sensitivity of the pdr5 Delta strain and exhibited a high degree of perinuclear fluorescence suggesting that this fusion protein was localized to the S. cerevisiae ER. Interestingly, when these experiments were repeated at 37 degrees C, the CO Afu abcA was able to complement the drug sensitive phenotype of pdr5 Delta cells and exhibited less intracellular fluorescence. Additionally, we found that the CO Afu abcA was able to reduce resistance to drugs like phytosphingosine that act via causing mislocalization of amino acid permeases in fungi. These data suggest that the Mu abcA protein can carry out two different functions of Pdr5: drug transport and regulation of protein internalization from the plasma membrane. (C) 2013 Elsevier Inc. All rights reserved.