Contributions of Aspergillus fumigatus ATP-binding cassette transporter proteins to drug resistance and virulence

Abstract

In yeast cells such as those of Saccharomyces cerevisiae, expression of ATP-binding cassette (ABC) transporter proteins has been found to be increased and correlates with a concomitant elevation in azole drug resistance. In this study, we investigated the roles of two Aspergillus fumigatus proteins that share high sequence similarity with S. cerevisiae Pdr5, an ABC transporter protein that is commonly overproduced in azole-resistant isolates in this yeast. The two A. fumigatus genes encoding the ABC transporters sharing the highest sequence similarity to S. cerevisiae Pdr5 are called abcA and abcB here. We constructed deletion alleles of these two different ABC transporter-encoding genes in three different strains of A. fumigatus. Loss of abcB invariably elicited increased azole susceptibility, while abcA disruption alleles had variable phenotypes. Specific antibodies were raised to both AbcA and AbcB proteins. These antisera allowed detection of AbcB in wild-type cells, while AbcA could be visualized only when overproduced from the hspA promoter in A. fumigatus. Overproduction of AbcA also yielded increased azole resistance. Green fluorescent protein fusions were used to provide evidence that both AbcA and AbcB are localized to the plasma membrane in A. fumigatus. Promoter fusions to firefly luciferase suggested that expression of both ABC transporter-encoding genes is inducible by azole challenge. Virulence assays implicated AbcB as a possible factor required for normal pathogenesis. This work provides important new insights into the physiological roles of ABC transporters in this major fungal pathogen.

Fig 1

ABCG transporter family in A. fumigatus. (A) The typical topology of a full-length ABCG transporter like Pdr5 is diagrammed here. The location of each set of 6 transmembrane domains is labeled “MSD” (for membrane spanning domains), while “NBD” is used to indicate the signature ATP-binding cassette motif called the nucleotide-binding domain. The region of the proteins used to raise antibodies is designated “Ab.” (B) Phylogenetic tree diagram of ABCG family in A. fumigatus. This alignment was generated using BLAST Tree View at the NCBI database (maximum sequence difference = 0.9).

Fig 2

Azole resistance phenotypes of mutants in ABC transporter-encoding genes. (A) The mutations shown here were produced in the wild-type strain Af293 (WT) using A. tumefaciens-mediated transformation. Each null allele lacked all coding sequences for the indicated ABC transporter and was marked with a hygromycin resistance cassette. Strains were grown to make fresh spore suspensions. These suspensions were diluted in PBST buffer, and ∼20 spores were placed on each spot. Plates were incubated at 37°C and then photographed. Increasing drug concentrations are indicated by increased bar width. (B) Same as panel A, but these strains are derivatives of A1151 (akuB^KU80Δ). (C) The strain used to generate these mutants is designated AfS35 (akuA^KU70Δ). Drug phenotypes were determined as for the other panels, but the final row of spots corresponds to a strain containing the hspA-abcA fusion gene that overproduces abcA. Panels A and C were photographed after 2 days, while panel B was photographed after 3 days.

Fig 3

MIC measurement for A. fumigatus strains lacking ABC transporter genes. Strains with the indicated genotypes were inoculated into 96-well microtiter plates containing serial 2-fold dilutions of either voriconazole (top panel) or itraconazole (bottom panel). Each plate was incubated at 37°C and evaluated after 24 or 48 h. The MIC endpoint was defined as the lowest concentration that produced complete inhibition of growth.

Fig 4

Expression of AbcA and AbcB. (A) Whole-cell protein extracts were prepared from A. fumigatus strains of the indicated genotypes. The hspA-abcA-containing fusion strain was either left unchallenged (−) or induced with 5% ethanol addition (+). Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were stained with Ponceau S dye to visualize total protein (top) or with a polyclonal antiserum directed against AbcA (bottom). Molecular mass markers, in kilodaltons, are indicated on the right side. (B) Whole-cell protein extracts prepared from the A. fumigatus strains of the indicated genotypes were processed as for panel A, except that the Western analyses were carried out using an antibody directed against recombinant AbcB.

Fig 5

Subcellular localization of AbcA and AbcB. (A) Isogenic wild-type (WT; A1151) or integrated abcA-GFP fusion strains were grown overnight in rich medium at 37°C. Mycelia were harvested by filtration, washed with water, and then visualized using Nomarski optics (DIC) or fluorescent light (GFP). (B) Same as panel A, but wild-type and abcB-GFP-containing strains were AfS35 cells.

Fig 6

Voriconazole induction of abcA and abcB expression. A. fumigatus transformants containing fusions between the indicated promoters and the firefly luciferase gene were grown to early log phase and then exposed to 20 ng/ml of voriconazole for 0, 45, or 90 min. Luciferase activity was assayed from representative aliquots and normalized to the untreated control. The error bars represent standard deviations in fold induction of luciferase activity upon drug treatment based on 3 independent experiments done on a representative strain.

Fig 7

Virulence effects of AbcA and AbcB. G. mellonella larvae were injected with the indicated A. fumigatus strains (2.5 × 10⁵ spores) or a buffer control (PBST). All these experiments employed the AfS35 background. These injections were done either without (A) or with (B) an initial injection with voriconazole to assess the ability of this azole drug to protect the larvae from infection. Survival of injected larvae was assessed every 24 h after injection.