J. G. M. Van Nistelrooy

Multidrug resistance in Aspergillus nidulans involves novel ATP-binding cassette transporters

Two single-copy genes, designated atrA and atrB (ATP-binding cassette transporter A and B), were cloned from the filamentous fungus Aspergillus nidulans and sequenced. Based on the presence of conserved motifs and on hydropathy analysis, the products encoded by atrA and atrB can be regarded as novel members of the ATP-binding cassette (ABC) superfamily of membrane transporters. Both products share the same topology as the ABC transporters PDR5 and SNQ2 from Saccharomyces cerevisiae and CDR1 from Candida albicans, which are involved in multidrug resistance of these yeasts. Significant homology also occurs between the ATP-binding cassettes of AtrA and AtrB, and those of mammalian ABC transporters (P-glycoproteins). The transcription of atrA and, in particular, atrB in mycelium of A. nidulans is strongly enhanced by treatment with several drugs, including antibiotics, azole fungicides and plant defense toxins. The enhanced transcription is detectable within a few minutes after drug treatment and coincides with the beginning of energy-dependent drug efflux activity, reported previously in the fungus for azole fungicides. Transcription of the atr genes has been studied in a wild-type and in a series of isogenic strains carrying the imaA and/or imaB genes, which confer multidrug resistance to various toxic compounds such as the azole fungicide imazalil. atrB is constitutively transcribed at a low level in the wild-type and in strains carrying imaA or imaB. Imazalil treatment enhances transcription of atrB to a similar extent in all strains tested. atrA, unlike atrB, displays a relatively high level of constitutive expression in mutants carrying imaB. Imazalil enhances transcription of atrA more strongly in imaB mutants, suggesting that the imaB locus regulates atrA. Functional analysis demonstrated that cDNA of atrB can complement the drug hypersensitivity associated with PDR5 deficiency in S. cerevisiae.

The ABC transporter AtrB from Aspergillus nidulans mediates resistance to all major classes of fungicides and some natural toxic compounds

This paper reports the functional characterization of AtrBp, an ABC transporter from Aspergillus nidulans. AtrBp is a multidrug transporter and has affinity to substrates belonging to all major classes of agricultural fungicides and some natural toxic compounds. The substrate profile of AtrBp was determined by assessing the sensitivity of deletion and overexpression mutants of atrB to several toxicants. All mutants showed normal growth as compared to control isolates. DeltaatrB mutants displayed increased sensitivity to anilinopyrimidine, benzimidazole, phenylpyrrole, phenylpyridylamine, strobirulin and some azole fungicides. Increased sensitivity to the natural toxic compounds camptothecin (alkaloid), the phytoalexin resveratrol (stilbene) and the mutagen 4-nitroquinoline oxide was also found. Overexpression mutants were less sensitive to a wide range of chemicals. In addition to the compounds mentioned above, decreased sensitivity to a broader range of azoles, dicarboximides, quintozene, acriflavine and rhodamine 6G was observed. Decreased sensitivity in overexpression mutants negatively correlated with levels of atrB expression. Interestingly, the overexpression mutants displayed increased sensitivity to dithiocarbamate fungicides, chlorothalonil and the iron-activated antibiotic phleomycin. Accumulation of the azole fungicide [(14)C]fenarimol by the overexpression mutants was lower as compared to the parental isolate, demonstrating that AtrBp acts by preventing intracellular accumulation of the toxicant. Various metabolic inhibitors increased accumulation levels of [(14)C]fenarimol in the overexpression mutants to wild-type levels, indicating that reduced accumulation of the fungicide in these mutants is due to increased energy-dependent efflux as a result of higher pump capacity of AtrBp.

The role of ABC transporters from Aspergillus nidulans in protection against cytotoxic agents and in antibiotic production.

Abstract This paper describes the characterization of atrC and atrD (ABC transporters C and D), two novel ABC transporter-encoding genes from the filamentous fungus Aspergillus nidulans, and provides evidence for the involvement of atrD in multidrug transport and antibiotic production. BLAST analysis of the deduced amino acid sequences of AtrCp and AtrDp reveals high homology to ABC transporter proteins of the P-glycoprotein cluster. AtrDp shows a particularly high degree of identity to the amino acid sequence of AfuMdr1p, a previously characterized ABC transporter from the human pathogen A. fumigatus. Northern analysis demonstrates an increase in transcript levels of atrC and atrD in fungal germlings upon treatment with natural toxic compounds and xenobiotics. The atrC gene has a high constitutive level of expression relative to atrD, which suggests its involvement in a metabolic function. Single knock-out mutants for atrC and atrD were generated by gene replacement using pyrG from A. oryzae as a selectable marker. ΔatrD mutants display a hypersensitive phenotype to compounds such as cycloheximide, the cyclosporin derivative PSC 833, nigericin and valinomycin, indicating that AtrDp is involved in protection against cytotoxic compounds. Energy-dependent efflux of the azole-related fungicide fenarimol is inhibited by substrates of AtrDp (e.g. PSC 833, nigericin and valinomycin), suggesting that AtrDp plays a role in efflux of this fungicide. Most interestingly, ΔatrD mutants display a decrease in penicillin production, measured indirectly as antimicrobial activity against Micrococcus luteus. These results suggest that ABC transporters may be involved in secretion of penicillin from fungal cells.

An energy-dependent efflux mechanism for fenarimol in a wild-type strain and fenarimol-resistant mutants of Aspergillus nidulans

Abstract Uptake of [ 14 C]fenarimol (30 μ M ) by mycelium of wild-type Aspergillus nidulans was characterized by a rapid initial accumulation during the first 10 min of incubation with the fungicide and a subsequent gradual release with time. Uptake appeared to be the result of influx and efflux. Influx of fenarimol could not be inhibited by low temperature, anaerobiosis, starvation of mycelium, or incubation with several respiratory inhibitors and is, therefore, a passive process. Under identical test conditions efflux activity was severely inhibited and should, therefore, be regarded as an energy-dependent mechanism. After prolonged incubation (90 min) an equilibrium between influx and efflux was established, resulting in an energy-dependent permeability barrier, since uptake could instantaneously be enhanced by addition of oligomycin or N,N′ -dicyclohexylcarbodiimide. It also indicates that efflux activity is inducible; this hypothesis is supported by the observation that pretreatment of mycelium with unlabeled fungicide prevented subsequent uptake of [ 14 C]fenarimol. Uptake by fenarimol-resistant mutants J146, M193, and R264 of A. nidulans , all possessing the imaB gene for resistance, was relatively low and almost constant in time. In this case, uptake appeared to be considerably enhanced by low temperature, anaerobiosis, starvation of mycelium, and incubation with respiratory inhibitors. Low uptake by these mutants is ascribed to a higher energy-dependent efflux activity for fenarimol compared with the wild-type strain. Upon inhibition of the barrier activity, net uptake resulted from remaining passive influx, which in that case may be as high as in the wild-type strain. The results suggest that both wild-type and fenarimol-resistant mutants possess an energy-dependent efflux mechanism with different efficiencies to excrete fenarimol and probably other chemicals to which cross-resistance or collateral sensitivity is present.

Mechanism of resistance to fenarimol in Aspergillus nidulans

Abstract Mycelial uptake of [ 14 C]fenarimol (10 μg/ml) by 20 fenarimol-resistant mutants of Aspergillus nidulans was compared with uptake by wild-type strain 003. Uptake of the fungicide during the initial 10 min of incubation was significantly lower in all mutant strains than in the wild-type strain indicating that resistance is related with reduced uptake. Upon prolonged incubation a gradual decrease of accumulated radioactivity in the wild-type strain was observed. A few mutants displayed resistance to unrelated chemicals such as p -fluorophenylalanine or d -serine; this phenomenon appeared not to be due to a decreased uptake of the corresponding natural amino acids. Incorporation of [ 3 H]adenine and [ 14 C]leucine by mycelium of mutant M193 was hardly inhibited after 5 hr of incubation with the fungicide, whereas a distinct effect was found with the wild-type strain. At this time also fungitoxicity to the wild-type strain became apparent. Probably, this effect is indirectly caused by inhibition of ergosterol biosynthesis. Mycelium of mutant M193 incorporated [ 14 C]acetate slightly less effectively than the wild-type strain. After 2 hr of incubation with this radiochemical leakage of [ 14 C]acetate metabolites from mycelium of the mutant strain was observed. This indicates that resistance might be correlated with increased excretion of fungal metabolites, which in turn may be related with reduced fitness of fenarimol-resistant mutants.

Laboratory resistance to fungicides which inhibit ergosterol biosynthesis in Penicillium italicum

AbstractLaboratory isolates ofPenicillium italicum with varying levels of resistance to fenarimol were obtained via mass selection of conidia on fenarimol-amended PDA. All fenarimol-resistant isolates showed cross-resistance to other fungicides which inhibit ergosterol biosynthesis (bitertanol, etaconazole, fenapanil, and imazalil), but not to fenpropimorph. In contrast, all isolates with a relatively high degree of resistance to fenarimol, exhibited increased sensitivity to fenpropimorph (negatively correlated cross-resistance). The varying degrees of resistance to ergosterol biosynthesis inhibitors (EBIs) suggest that different mutations for resistance are involved. Isolates with a high degree of resistance were selected from conidial populations of isolates with a low resistance level. This indicates that in sich strains different mutations for resistance are present simultaneously.Somein vitro growth parameters of resistant isolates slightly differed from those of the wild type. Virulence of most resistant isolates on oranges was visually normal and in competition experiments with mixed inocula of wild-type and resistant isolates, the latter could still be isolated after five successive infection cycles on fungicide-free oranges. Nevertheless, the proportion of resistant conidia in the successive inocula gradually decreased.Decay of oranges inoculated with EBI-resistant isolates could still be controlled by a curative dip treatment with imazalil at dosage rates recommended in practice (500 μg ml−1). However, with the highly resistant isolates, decay control was not complete at half this dosage, indicating only a marginal control at the full dosage rate.On the basis of the results described it is assumed that at a high selection pressure of EBIs in practice, gradual accumulation of different mutations for resistance, together with selection of normal fitness may eventually lead to loss of control ofPenicillium decay. Therefore, desease control strategies with a low selection pressure of EBIs are advisable.SamenvattingLaboratoriumisolaten vanPenicillium italicum met uiteenlopende resistentieniveaus tegen fenarimol werden verkregen door massaselectie van conidiën op fenarimolbevattende PDA. Alle fenarimol-resistente isolaten vertoonden kruisresistentie tegen andere fungiciden die de ergosterolbiosynthese remmen (bitertanol, etaconazool, fenapanil, imazalil), maar niet tegen fenpropimorf. Alle isolaten met een relatief hoge graad van fenarimolresistentie waren zelfs gevoeliger voor dit laatste fungicide (negatief gecorreleerde kruisresistentie). De uiteenlopende graden van resistentie tegen ergosterolbiosynthese remmers (EBIs) suggereren dat verschillende mutaties een rol kunnen spelen. Isolaten met een hoge resistentiegraad werden geselecteerd in conidiënpopulaties van isolaten met een lage resistentiegraad. Dit duidt erop dat in dergelijke stammen verschillende mutaties gelijktijdig aanwezig zijn.Er werden kleine verschillen in parameters voorin vitro groei tussen resistente en gevoelige isolaten geconstateerd. De virulentie van vrijwel alle stammen op sinaasappels was normaal; in competitie-experimenten met mengpopulaties van gevoelige en resistente isolaten konden laatstgenoemde isolaten nog na vijf oppenvolgende infectiecycli op fungicide-vrije sinaasappels worden geïsoleerd. Desalniettemin nam het percentage resistente conidiën in de opeenvolgende inocula geleidelijk af. Penicillium-rot op sinaasappel, geïnoculeerd met EBI-resistente isolaten, kon nog worden bestreden door een curatieve dompelbehandeling met imazalil bij een dosering die in de praktijk wordt aanbevolen (500 μg ml−1). Bij een halvering van deze dosering werd echter op sinaasappels, geïnoculeerd met de hoog-resistente isolaten nog rot waargenomen, hetgeen erop duidt dat de bestrijding bij de volle dosering slechts marginaal is.Op grond van de beschreven resultaten kan worden verondersteld dat in de praktijk onder hoge selectiedruk van EBIs een geleidelijke accumulatie van verschillende mutaties, gepaard gaande met selectie van een normale fitheid, kan plaatsvinden, hetgeen uiteindelijk zou kunnen leiden tot onvoldoende bestrijding vanPenicillium-rot. Bestrijdingsstrategieën met een lage selectiedruk van EBIs zijn daarom wenselijk.

Variation in sensitivity to fungicides which inhibit ergosterol biosynthesis in wheat powdery mildew

Fungicides which inhibit ergosterol biosynthesis have been in use for control of wheat powdery mildew (Erysiphe graminis f. sp.tritici) in the Netherlands since 1978. Mildew populations were tested for their variation in sensitivity to triadimefon from 1982 to 1984.In 1982 isolates from the province Limburg, with a triazole spray-regime history, were less sensitive to triadimefon than isolates from the provinces Gelderland and Noord-Brabant, where triazoles had not been used. In the following years isolates with reduced sensitivity were also detected in the latter provinces and other parts of the country. This spread correlates with the increased use of triazoles, both in frequency and space, from 1983 onwards. The reduced sensitivity can as a whole or in part be responsible for the decline in field performance of triazoles, observed during these years.Cross-sensitivity to the triazoles triadimefon and propiconazole was established, but not to triazoles and the morpholine fungicide fenpropimorph. Effectiveness of the latter compound was similar to all isolates from Limburg tested in 1984. Field performance of fenpropimorph, introduced in 1983, appeared to be normal. It is recommended to counteract further development of resistance by sequential use of fenpropimorph early in the season (May) and triazoles at the end (June–July).SamenvattingFungiciden die de ergosterolbiosynthese remmen worden in Nederland sinds 1978 gebruikt bij de bestrijding van tarwemeeldauw (Erysiphe graminis f. sp.tritici) Meeldauwpopulaties werden getoetst op hun variatie in gevoeligheid voor triadimefon van 1982 tot 1984.In 1982 bleken isolaten afkomstig uit de provincie Limburg, waar voordien triazolen werden toegepast, minder gevoelig te zijn voor triadimefon dan isolaten uit de provincies Gelderland en Noord-Brabant, waar nog geen triazolen werden gebruikt. In de daaropvolgende jaren werden isolaten met een verminderde gevoeligheid ook in laatstgenoemde provincies en in andere delen van het land gevonden. Deze uitbreiding is gecorreleerd met een toename in het gebruik van triazolen vanaf 1983 in areaal en frequentie. De afname in gevoeligheid kan geheel of gedeeltelijk verantwoordelijk zijn voor de verminderde meeldauwwerking van triazolen die gedurende deze jaren werd waargenomen.Kruisgevoeligheid werd vastgesteld voor de triazolen triadimefon en propiconazool, maar niet voor triazolen en het morfoline-derivaat fenpropimorf. De werking van dit middel was tegen alle isolaten uit Limburg die in 1984 werden getoetst, gelijk. De meeldauwwerking van fenpropimorf, dat in 1983 werd geïntroduceerd, was normaal. Aanbevolen wordt om verdere resistentie-ontwikkeling tegen te gaan door afwisselend gebruik van fenpropimorf vroeg in het groeiseizoen (mei) en een triazool aan het eind (juni-juli).

Isolation and molecular characterisation of the gene encoding eburicol 14α-demethylase (CYP51) from Penicillium italicum

TheCYP51 gene encoding eburicol 14α-demethylase (P45014DM) was cloned from a genomic library of the filamentous fungal plant pathogenPenicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14α-demethylase from the yeastCandida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORF) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a characteristic haem-binding domain, HR2, common to all P450 genes. The deducedP. italicum P45014DM protein and the P45014DM proteins fromCandida albicans, C. tropicalis andSaccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of theCYP51 family. Multiple copies of a genomic DNA fragment ofP. italicum containing the cloned P450 gene were introduced intoAspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P45014DM activity, indicating that the cloned gene encodes a functional eburicol 14α-demethylase.

Stepwise development of laboratory resistance to DMI-fungicides in Penicillium italicum.

Isolates ofPenicillium italicum with differential levels of resistance to imazalil were obtained via step-wise mass selection of conidia of the fenarimol-resistant isolate E300-3 on imazalilamended PDA. Three out of five selection steps were successful. The resistance level to imazalil of isolates acquired after the two last selection steps was on average 122 and 197. The differential level of resistance was also apparent in decay control on oranges by imazalil inoculated with the various isolates. The isolates showed a similar cross-resistance pattern to other fungicides which inhibit C-14 demethylation of sterols (DMIs), although the level of resistance to these fungicides was significantly higher. All isolates displayed negatively-correlated cross-resistance to tridemorph and dodine. Most isolates had a normal virulence on oranges. In competition experiments with mixed-inocula of the wild-type and a resistant isolate, the proportion of the wild-type increased in successive generations on untreated oranges and the proportion of the resistant isolate increased on imazalil-treated oranges. The lower competitive ability of the resistant isolate on untreated oranges may be due to a decrease in spore production as compared with the wild-type.Since isolate E300-3 was obtained in two selection steps on fenarimol-amended PDA, the isolates obtained in the last selection steps on imazalil-amended PDA may have at least five different genes for resistance to DMIs. This is consistent with resistance to DMIs being under polygenic control, with the genes involved having an additive interaction, although this is not the only possible explanation of the results.

Differential accumulation of fenarimol by a wild-type isolate and fenarimol-resistant isolates of Penicillium italicum

Accumulation of [14C] fenarimol by mycelium ofPenicillium italicum was studied with isolates having varying levels of laboratory resistance to fenarimol. All resistant isolates tested showed a significantly lower accumulation than the wild-type isolate.Various metabolic inhibitors enhanced accumulation to relatively high levels in both wildtype and resistant isolates. it indicates that accumulation is governed by two processes viz. a non-mediated influx and an energy-dependent efflux. A relatively high fenarimol efflux in resistant isolates probably accounts for low accumulation and for fenarimol resistance. One of the inhibitors which annihilated fenarimol efflux and enhanced fenarimol accumulation was sodium orthovanadate. The synergistic action of fenarimol and orthovanadate to both wildtype and resistant isolates in crossed-paper strip bioassays is probably related to the effect of the latter compound on fenarimol accumulation. Synergistic action between the chemicals in control ofPenicillium decay of oranges could not be detected.SamenvattingDe accumulatie van [14C]fenarimol door mycelium vanPenicillium italicum werd bestudeerd bij isolaten met een uiteenlopende graad van laboratorium-resistentie tegen fenarimol. Alle getoetste resistente isolaten vertoonden een lagere opname dan de wild-stam.Verschillende antimetabolieten verhoogden de accumulatie tot relatief hoge waarden die voor gevoelige en resistente isolaten ongeveer gelijk waren. Deze waarneming duidt erop dat accumulatie wordt bepaald door twee processen: ‘nonmediated’ influx en energie-afhankelijke efflux. Een hogere fenarimol efflux in resistente isolaten vormt waarschijnlijk de verklaring voor de lagere accumulatie en voor het resistentiemechanisme. Een van de remmers die de fenarimolefflux te niet doet, en de accumulatie van fenarimol verhoogt, is natriumorthovanadaat. De synergistische werking van fenarimol en orthovanadaat tegen zowel wild-type als resistente isolaten in ‘crossed-paper strip’ biotoetsen houdt waarschijnlijk verband met het effect van laatstgenoemde stof op de accumulatie van fenarimol. Synergistische werking van deze verbindingen bij de bestrijding vanPenicillium-rot op sinaasappels werd niet waargenomen.