RESUMO
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 Afu Mdr1p, 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 attrD, 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. DeltatrD 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, (delta)atrD 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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/metabolismo , Aspergillus nidulans/genética , Resistência a Múltiplos Medicamentos/genética , Genes Fúngicos , Xenobióticos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Aspergillus nidulans/efeitos dos fármacos , Transporte Biológico Ativo , Sequência Conservada , Ciclosporinas/farmacologia , Proteínas Fúngicas/genética , Fungicidas Industriais/metabolismo , Regulação Fúngica da Expressão Gênica , Biblioteca Genômica , Ionóforos/farmacologia , Dados de Sequência Molecular , Nigericina/farmacologia , Pirimidinas/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Valinomicina/farmacologiaRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Aspergillus nidulans/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Genes Fúngicos , Transportadores de Cassetes de Ligação de ATP/química , Aspergillus nidulans/química , Aspergillus nidulans/genética , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Imidazóis/farmacologia , Mutação/genética , Fenótipo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Ativação Transcricional/genéticaRESUMO
The CYP51 gene encoding eburicol 14 alpha-demethylase (P450(14DM)) was cloned from a genomic library of the filamentous fungal plant pathogen Penicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14 alpha-demethylase from the yeast Candida 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 deduced P. italicum P450(14DM) protein and the P450(14DM) proteins from Candida albicans, C. tropicalis and Saccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of the CYP51 family. Multiple copies of a genomic DNA fragment of Pl italicum containing the cloned P450 gene were introduced into Aspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450(14DM) activity, indicating that the cloned gene encodes a functional eburicol 14 alpha-demethylase.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Genes Fúngicos , Lanosterol/análogos & derivados , Oxirredutases/genética , Penicillium/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Primers do DNA , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungicidas Industriais/farmacologia , Lanosterol/metabolismo , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/metabolismo , Penicillium/enzimologia , Pirimidinas/farmacologia , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Esterol 14-DesmetilaseRESUMO
Fungicides inhibiting sterol biosynthesis belong to chemically distinct classes such as imidazole, morpholine, pyridine, pyrimidine and triazole derivatives. Incubation of mycelium of Aspergillus nidulans for 90 min with representatives of these fungicides induced an efflux activity which prevented accumulation of fenarimol, a pyrimidine derivative, into the mycelium. Induction of this efflux activity reduced the fungitoxicity of fenarimol. Addition of oligomycin to mycelium in which fenarimol-efflux activity was induced immediately increased the uptake of fenarimol, indicating that the efflux activity is energy-dependent. Subsequent disruption of membrane permeability with sodium lauryl sulphate instantaneously caused leakage of fenarimol from the mycelium into the medium. The ability to induce fenarimol efflux is rather specific for inhibitors of sterol biosynthesis: except for pimaricin, fungicides with unrelated mechanisms of action did not have this ability.
