Laboratory of Phytopathology, Wageningen University, PO Box 8025, 6700 EE Wageningen, The Netherlands1
Institute of Plant Pathology, University of Naples Federico II, 80055 Portici, Naples, Italy2
Author for correspondence: Maarten A. De Waard. Tel: +31 317 483412. Fax: +31 317 483412. e-mail: maarten.dewaard{at}medew.fyto.wau.nl
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ABSTRACT |
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Keywords: multidrug resistance, efflux pumps, ABC transporters, AtrBp, Aspergillus nidulans
Abbreviations: ABC, ATP-binding cassette; CCCP, carbonyl cyanide m-chlorophenylhydrazone; EC50, effective concentration required for 50% growth inhibition; MDR, multidrug resistance; 4NQO, 4-nitroquinoline oxide; UTR, untranslated region
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INTRODUCTION |
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A common mechanism of MDR is the overexpression of energy-dependent multidrug efflux pumps, also known as multidrug transporter proteins or P-glycoproteins (P-gp). Overexpression of such proteins in cancer cells results in MDR to chemotherapeutic drugs and other hydrophobic pharmacological agents (Ambudkar et al., 1999 ). P-glycoproteins belong to the ubiquitous superfamily of ATP-binding cassette (ABC) transporters. Besides multidrug transporters, the family includes proteins involved in transmembrane transport of various substances such as ions, amino acids, peptides, sugars, vitamins, steroid hormones, bile acids and phospholipids (Higgins, 1992
, 1994
; van Helvoort et al., 1996
).
In filamentous fungi, MDR was first reported for laboratory-generated mutants of Aspergillus nidulans selected for resistance to azole fungicides, also described as sterol biosynthesis inhibitors (van Tuyl, 1977 ). Resistance to azoles in isogenic mutants is based on an energy-dependent efflux mechanism which results in decreased accumulation of compounds in fungal mycelium, similarly to the phenomenon observed in cancer cells (De Waard & Van Nistelrooy, 1979
, 1980
). This mechanism also operates in plant pathogens such as Penicillium italicum, Botrytis cinerea, Nectria haematococca, and probably Mycosphaerella graminicola (De Waard et al., 1995
; Joseph-Horne et al., 1996
). To date, at least five ABC transporters highly homologous to multidrug-efflux pumps from other organisms have been described for A. nidulans (Andrade et al., 1999
; Angermayr et al., 1999
; Del Sorbo et al., 1997
).
This paper describes the functional characterization of atrB, a previously described gene of A. nidulans (Del Sorbo et al., 1997 ). AtrBp displays a high degree of sequence homology to BcatrBp from B. cinerea, Mgatr5p from M. graminicola, Pmr1p from Penicillium digitatum, and Abc1p from Magnaporthe grisea (Goodall et al., 1999
; Nakaune et al., 1998
; Schoonbeek et al., 1998
; Urban et al., 1999
). A high degree of homology also exists with ABC proteins classified in subcluster I.1 from Saccharomyces cerevisiae (Decottignies & Goffeau, 1997
), Bfr1p from Schizosaccharomyces pombe (Nagao et al., 1995
), and the Cdr1p and Cdr2p proteins from Candida albicans (Prasad et al., 1995
; Sanglard et al., 1996
, 1997
). Most of these proteins have been characterized as multidrug-efflux pumps. Previously, we reported that heterologous overexpression of atrB in S. cerevisiae restores wild-type sensitivity to cycloheximide, tentatively indicating that AtrBp is also a multidrug-efflux protein. Here, we describe in detail the substrate specificity of the multidrug transporter AtrBp by phenotype characterization of knock-out and overexpression mutants of A. nidulans with respect to fungicide sensitivity.
atrB strains display increased sensitivity to several classes of fungicides and some natural toxic compounds. atrB overexpression mutants are less sensitive to a wide range of compounds. Interestingly, these overexpression mutants display at the same time increased sensitivity to some conventional fungicides and phleomycin, an iron-activated antibiotic. These results clearly indicate that AtrBp is a multidrug transporter involved in protection against natural toxins and xenobiotics and might play a role in iron metabolism.
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METHODS |
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Disruption constructs.
Primers for amplification of the atrB locus were designed in the 5' and 3' UTR (untranslated regions). Artificial EcoRI sites were included in the primers to allow further subcloning of the PCR product. Primer sequences were 5'-CGTGAATTCCTGGATGGTTCAGCTTA-3' and 5'-TAAGAATTCTTCAAGTTCGTCGAAGACG-3'. A 5·2 kb amplified PCR product using the lambda clone an2 (Del Sorbo et al., 1997 ) as template DNA was cloned in pGEM-T and coded pTB. This clone was checked by restriction analysis and sequencing. Furthermore, the 8·0 kb pTB clone was restricted with BamHI and a 5·15 kb DNA fragment was used to clone the pyrG from Aspergillus oryzae as a 3·8 kb BamHI insert from pAO4-2 restricted with BamHI (de Ruiter-Jacobs et al., 1989
). This construct was coded pAOB. The final transformation construct, a 5·95 kb EcoRI DNA fragment (DB), was obtained by restriction of pAOB with EcoRI. For generation of the control strains, the pAO4-2 clone was used for transformation (de Ruiter-Jacobs et al., 1989
).
Overexpression constructs.
The overexpression construct was made by restriction of pTB with EcoRI and a 5·2 kb DNA fragment containing the whole atrB locus was cloned in the EcoRI site of pPL6 (Oakley et al., 1987 ). The resulting 9·4 kb vector, coded pOB, was used for transformation. The control strains (PPL6) were obtained by transformation with the pPL6 vector.
Preparation of protoplasts and transformation.
Mycelial protoplasts were prepared as described by Wernars et al. (1985) with minor modifications. Liquid minimal medium (MM) supplemented with 2 g Casamino acids l-1, 0·5 g yeast extract l-1 and auxotrophic markers was inoculated with 106 conidia ml-1 and incubated overnight at 37 °C and 300 r.p.m. in an orbital incubator for 16 h. The germlings were harvested through Mira-Cloth, washed twice with sterile water and twice with STC buffer (1·0 M sorbitol, 10 mM Tris/HCl pH 7·5, 50 mM CaCl2) and squeezed between paper towels to remove excess of liquid. Protoplasts were released by incubation of 1 g mycelium at 30 °C and 100 r.p.m., resuspended in 20 ml of filter-sterilized iso-osmotic S0.8MC medium containing lytic enzymes (5 mg Novozym 234 ml-1, 0·8 M KCl, 50 mM CaCl2, 20 mM MES pH 5·8) for about 2 h. The protoplast suspension was filtered over glass wool, diluted (1:1, v/v) with STC buffer and incubated on ice for 10 min. Then, protoplasts were collected by centrifugation (10 min, 0 °C, 3000 r.p.m.) and washed twice with STC buffer. Transformation was performed as described by van Heemst et al. (1997)
using purified DNA of transformation constructs DB (3·5 µg) and pOB (5·0 µg) dissolved in sterile water (15 µl).
Toxicity assays.
Sensitivity of A. nidulans strains to toxicants was determined by measuring their EC50 values for inhibition of radial growth on MM plates (De Waard & Van Nistelrooy, 1979 ). Mycelial agar plugs of an overnight-grown confluent plate of each strain were placed upside down on MM plates amended with fungicides at different concentration of the compounds. Radial growth was assessed after 3 d incubation, at 37 °C. Carbendazim and sulfomethurom methyl were kindly provided by DuPont De Nemours, cilofungin by Eli Lilly, fenpiclonil, fludioxonil and trifloxystrobin by Novartis, kresoxim-methyl by BASF, fenarimol by Dow Elanco and imazalil nitrate and ketoconazole by Janssen Pharmaceuticals. All other chemicals tested were purchased from Sigma. For statistical analysis a radial growth test was performed in four replicates, at one concentration around the determined EC50 value of the compounds for the control strains. These concentrations were: azoxystrobin (0·05 µg ml-1), carbendazim (0·3 µg ml-1), cycloheximide (50 µg ml-1), cyprodinil (0·03 µg ml-1), eugenol (100 µg ml-1), fenarimol (3 µg ml-1), fenpiclonil (0·3 µg ml-1), fluazinam (0·3 µg ml-1), fludioxonil (0·1 µg ml-1), imazalil nitrate (0·05 µg ml-1), iprodione (5 µg ml-1), itraconazole (0·05 µg ml-1), kresoxim-methyl (0·05 µg ml-1), miconazole (0·5 µg ml-1), nigericin (3 µg ml-1), sodium o-phenylphenate (15 µg ml-1), nystatin (10 µg ml-1), 4-nitroquinoline oxide (1 µg ml-1), phleomycin (30 µg ml-1), prochloraz (0·1 µg ml-1), propiconazole (1 µg ml-1), pyrimethanil (0·3 µg ml-1), quintozene (10 µg ml-1), resveratrol (300 µg ml-1), rhodamine 6G (5 µg ml-1), thiabendazole (3 µg ml-1), trifloxystrobin (0·01 µg ml-1). The compounds were added from concentrated solutions in methanol. Amphotericin B (30 µg ml-1), camptothecin (10 µg ml-1), cilofungin (0·03 µg ml-1), chlorothalonil (3·0 µg ml-1), ferbam (30 µg ml-1) and thiram (30 µg ml-1), were added from concentrated solutions in DMSO. Acriflavine (3 µg ml-1) was dissolved in sterile water. The final concentration of the solvents in the agar was the same for all treatments and never exceeded 1%. Analysis of variance from two independent experiments was applied as described by Snedecor & Cochran (1989)
. Significant differences were obtained by comparing the mean values of colony size of control strains and mutants using Tukeys test (P<0·05).
Accumulation of [14C]fenarimol.
Experiments were performed with standard suspensions of germlings of A. nidulans at an initial external concentration of 30 µM [14C]fenarimol, as described before (De Waard & Van Nistelrooy, 1980 ).
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RESULTS |
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AtrB causes energy-dependent efflux of [14C]fenarimol
In genetically defined MDR mutants of A. nidulans, resistance to the azole fungicide fenarimol is based on increased energy-dependent efflux activity which results in decreased cytoplasmic drug accumulation (De Waard & Van Nistelrooy, 1979 , 1980
). We could not find any significant difference in [14C]fenarimol accumulation between the control PAO and the
atrB strains (Fig. 4a.
) However, initial [14C]fenarimol accumulation in atrB overexpression mutants was lower than in the control strain PPL6-1 (Fig. 4b
). In radial growth tests, mutants overexpressing atrB had decreased sensitivity to fenarimol.
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DISCUSSION |
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Deletion strains of atrB displayed increased sensitivity to different classes of agricultural fungicides: cyprodinil (anilinopyrimidine), ketoconazole, prochloraz and propiconazole (azoles), carbendazim (benzimidazole), fenpiclonil and fludioxonil (phenylpyrroles), fluazinam (phenylpyridianine) and azoxystrobin, kresoxim-methyl and trifloxystrobin (strobirulins). Increased sensitivity was also observed for other compounds such as 4-nitroquinoline oxide (mutagen), camptothecin (plant alkaloid) and the phytoalexin resveratrol (stilbene). These results indicate that these compounds are substrates of AtrBp.
Analogous to ABC transporters of yeast (Kolaczkowski et al., 1998 ; Sanglard et al., 1996
, 1997
), ABC transporters of A. nidulans may have distinct but overlapping substrate specificities. This makes it difficult to assess the substrate profile of an ABC protein using single knock-out mutants. To overcome this problem, the sensitivity of overexpression mutants to toxicants was also determined. This approach led to the characterization of additional substrates, such as fenarimol, imazalil and miconazole (azoles), pyrimethanil (anilinopyrimidine), iprodione and vinchlozolin (dicarboximides), quintozene (aromatic fungicides), acriflavine and rhodamine 6G. In all cases, an inverse correlation between levels of atrB expression in the overexpression mutants and sensitivity to toxicants was established. These results provide evidence that AtrBp pump activity is responsible for the decreased sensitivity to toxicants. The results also imply that the use of overexpression mutants avoids or minimizes the problem of redundancy of ABC transporters in characterization of the substrate specificity of AtrBp. Phenotype characterization of multiple deletion mutants is another approach that can be used to minimize the problem of redundancy. This approach was used to characterize the drug-resistance profile of the major ABC transporters of the PDR network from Saccharomyces cerevisae (Kolaczkowski et al., 1998
). The sensitivity of isogenic S. cerevisae strains deleted in PDR5, SNQ2 or YOR1, and multiple knock-outs in different combinations, was tested to 349 toxic compounds. Several fungicides, similar to the ones used in our study, appeared to be ABC-transporter substrates in that organism.
The transient accumulation of [14C]fenarimol in the atrB mutants and control strains is similar. In constrast, the overexpression mutants have a lower initial level of [14C]fenarimol accumulation. These results indicate that AtrBp can act as a fenarimol efflux pump. However, results also suggest that A. nidulans has (an) additional efflux pump(s) accepting fenarimol as substrate. In
atrB mutants, such additional efflux pumps may compensate for the absence of AtrBp, resulting in similar patterns of [14C]fenarimol accumulation. Such compensating efflux pumps are still unknown but it might be one of the many ABC-transporter-candidate genes present in the expressed sequence tag (EST) database of A. nidulans (Roe et al., 1998
).
Restoration of wild-type levels of [14C]fenarimol accumulation in the overexpression mutant OB35 after addition of the respiratory inhibitors (CCCP and oligomycin) and an inhibitor of membrane ATPases (orthovanadate), demonstrates that the [14C]fenarimol efflux is energy-dependent. This may be due to a direct effect of the inhibitor on the AtrB protein (vanadate), an effect on ATP synthesis in mitochondria (CCCP, oligomycin), and indirectly via dissipation of the proton-motive force (CCCP). Furthermore, identified substrates in the toxicity assays, such as kresoxim-methyl and iprodione, also stimulate accumulation of [14C]fenarimol, suggesting that these compounds are competitive inhibitors of [14C]fenarimol efflux. Interestingly, a different pattern of inhibition for the two compounds was observed. First, the iprodione concentration (300 µM) required to increase [14C]fenarimol accumulation was ten times higher than the one used for fenarimol (30 µM). Kresoxim-methyl showed this effect at equimolar concentrations (30 µM). This suggests that AtrBp has a higher affinity for kresoxim-methyl than for iprodione. Altered sensitivity to iprodione was only detected in the overexpression mutants whilst altered sensitivity to kresoxim-methyl was detected in both deletion and overexpression mutants of atrB. These results also suggest that AtrBp has a relative high affinity to kresoxim-methyl.
Similarly to the yeast ABC-transporter proteins of subcluster I.1 (Decottignies & Goffeau, 1997 ), AtrBp has the (NBF-TMD)2 configuration. The majority of ABC transporters involved in MDR from yeast, such as Pdr5p, Snq2p and Pdr12p, are grouped in this subcluster. Genes encoding proteins with very high homology to AtrBp have been described for at least two important plant pathogens, B. cinerea and M. graminicola (Goodall et al., 1999
; Schoonbeek et al., 1998
). A BLAST analysis with the AtrBp sequence reveals that BcatrBp from B. cinerea is its closest homologue with an overall identity of 70%. Most interestingly, the predicted transmembrane domains of both proteins are also highly conserved. This suggests that BcatrBp from B. cinerea may have similar substrates as AtrBp from A. nidulans.
Wild-type sensitivity to cycloheximide was restored to the PDR5-deficient strain, upon transformation with the cDNA of atrB in a high-copy-number vector (Del Sorbo et al., 1997 ). In the present work, neither
atrB nor overexpression mutants of A. nidulans displayed altered sensitivity to cycloheximide as compared to the control strains. It has been demonstrated for the human MDR1 protein that lipid composition of membranes can affect its substrate specificity and ATPase activity (Doige et al., 1993
; Romsicki & Sharom, 1998
; Sharom, 1997
). Hence, differences in membrane composition of yeast as compared to A. nidulans could explain these results.
Most interestingly, the overexpression mutants of atrB displayed increased sensitivity to dithiocarbamate fungicides, chlorothalonil and the iron-activated antibiotic phleomycin. The increase in sensitivity of the overexpression mutants negatively correlated with the levels of atrB expression in the different mutants. We hypothesize that the explanation for the increased sensitivity displayed by the overexpression mutants could relate to iron metabolism, as the toxicity of phleomycin is directly correlated with intracellular iron contents (Haas et al., 1999 ). Therefore, it might be that atrB is also involved in iron uptake or secretion of siderophores.
A better understanding of the role of AtrBp in sensitivity and resistance to toxicants may elucidate additional functions of AtrBp. This is of general relevance, since it might help to design strategies to overcome MDR in practice. This is already exemplified by our observation that dithiocarbamate fungicides and other compounds showed increased activity against overexpression mutants of atrB, with an MDR phenotype.
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ACKNOWLEDGEMENTS |
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Received 21 February 2000;
revised 27 April 2000;
accepted 5 May 2000.