ABC transporters and multidrug resistance in Aspergillus nidulans

The term multidrug resistance (MDR) stands for simultaneous cellular resistance to chemically unrelated toxicants and is often associated with overproduction of multidrug-efflux proteins of the A TP- b inding- c assette (ABC) superfamily. The ABC transporters comprise a large and multifunctional family of proteins. Besides multidrug transporters, the superfamily includes proteins involved in transmembrane transport of various substances such as ions, amino acids, peptides, sugars, vitamins, steroid hormones, bile acids, and phospholipids. An overview of the great variety of cellular functions that these proteins can perform in living cells is presented in Chapter 1. The aim of this thesis was to identify and characterize molecular mechanisms of drug resistance in Aspergillus nidulans , with special emphasis on drug-efflux proteins of the ABC-transporter superfamily. Using different approaches, we have identified seven A BC- tr ansporter genes ( atrA-G ) from A. nidulans . Heterologous screening of a genomic library from A. nidulans using a DNA probe from PDR5 , a well characterized multidrug transporter from Saccharomyces cerevisiae , yielded atrA and atrB , the first ABC-transporter genes isolated from a filamentous fungus (Chapter 2). The second approach, PCR with degenerate oligonucleotide primers based upon consensus sequences encoding ABC transporters from the subfamily of P-glycoproteins, resulted in the cloning of atrC and atrD (Chapter 3). The last approach, was based on a screening of an EST database of A. nidulans for sequences encoding proteins with homology to known fungal multidrug transporters, particularly involved in efflux of azole fungicides. With this approach, atrE , atrF and atrG were identified (Chapter 5). The proteins encoded by all seven atr genes isolated display high sequence homology to known multidrug-efflux proteins from other organisms. To investigate the role of the isolated atr genes from A. nidulans in multidrug transport, expression analysis of these genes in wild-type and MDR ( ima ) mutants of A. nidulans was performed, after treatment of germlings with toxicants. imaA and imaB are laboratory-generated mutants of A. nidulans selected for resistance to the azole fungicide imazalil and related compounds. In imaB mutants, resistance to the azole-like compound fenarimol is based on an increased energy-dependent efflux activity which results in decreased cytoplasmic drug accumulation. Therefore, these mutants were suitable to correlate azole resistance with expression levels of atr genes. Expression studies in a wild-type isolate demonstrated that the basal level of expression for most atr genes is low and can be strongly enhanced by treatment with unrelated toxicants (Chapters 2, 3, 5 and 6). Time course experiments indicated that within 5 min after treatment with a toxicant ( e.g. imazalil), enhanced transcript levels of atr genes can be observed (Chapter 2). Some compounds can specifically induce transcription of one particular atr gene while others may simultaneously affect transcription of several atr genes (Chapter 5). For instance, resveratrol specifically induces transcription of atrB , while treatment with fenarimol enhances transcription of several genes ( atrB-G ). Expression analyses in the ima mutants of A. nidulans shows that atrD , atrE , atrF , and atrG display a higher basal level of expression in imaB mutants than in the wild-type (Chapter 5). Treatment with fenarimol also enhances transcription of these atr genes in imaB mutants. Mutants in which atrB and atrD have been deleted display increased sensitivity to a number of unrelated toxicants. ∆ atrB mutants have increased sensitivity to different classes of fungicides, mutagens and natural toxic compounds. Δ atrD mutants display increased sensitivity to cycloheximide, the cyclosporin derivative PSC 833, nigericin and valinomycin. These results indicate that AtrBp and AtrDp from A. nidulans are multidrug transporters with different substrate specifities (Chapters 3 and 4). AtrBp has been further characterized by overexpression in A. nidulans and S. cerevisiae (Chapters 2, 4 and 6). Sensitivity to toxicants of a PDR5- deficient strain of S. cerevisiae was restored to wild-type levels, upon transformation with cDNA of atrB in a high copy number vector (Chapter 2). Mutants overexpressing atrB in A. nidulans also display decreased sensitivity to toxicants. These overexpression mutants display altered sensitivity to a wider range of compounds as compared to ∆atrB mutants (Chapter 4). These results indicate that the presence of additional drug-efflux pumps with affinity for the same compound prevent a change in phenotype of some deletion mutants. Redundancy of ABC transporters may explain, at least in part, the findings thatΔ atrA andΔ atrC mutants show no hypersensitive phenotype for any of the compounds tested (Chapters 3 and 5). However, the observation that atrA transcript levels were not influenced by any of the compounds tested and that atrA expression in S. cerevisiae does not confer drug resistance, suggest that AtrAp is not a multidrug transporter. ABC transporters which have overlapping substrate specificities may still have specific substrates. AtrBp has a distinctive specificity for the phenylpyrrole fungicide fludioxonil since both ∆ atrB and atrB overexpression mutants have altered sensitivity to this compound (Chapter 6). In addition, the increase in sensitivity to fludioxonil observed for ∆ atrB mutants correlates with the relatively high accumulation levels of this compund. This was not observed for fenarimol. Therefore, the ABC transporter AtrBp can be regarded as a major efflux pump of phenylpyrrole fungicides in A. nidulans . Unexpectedly, overexpression mutants of atrB displayed increased sensitivity to dithiocarbamate fungicides, chlorothalonil and the iron-activated antibiotic phleomycin (Chapter 4). This phenotype was most pronounced in the overexpression mutant with the highest levels of atrB expression. We hypothesize that this phenomenon could relate to involvement of AtrBp in iron metabolism. Δ atrD mutants display a decrease in penicillin production, indirectly measured as antimicrobial activity against Micrococcus luteus (Chapter 3). These results suggest that AtrDp has a role in penicillin production. In conclusion, data presented in this thesis demonstrated that some of the identified ABC transporters from A. nidulans function in protection against natural toxicants and xenobiotics. Deletion and overexpression mutants of specific atr genes display increased and decreased sensitivity to toxicants, respectively. A role for ABC transporters in production of fungal secondary metabolites has also been suggested. This may imply that strains overexpressing multidrug-transporter genes can show pleiotropic phenotypes with respect to production of secondary metabolites.