RESUMO
Aspergillus fumigatus is an aggressive opportunistic pathogen of humans as well as a major allergen. Environmental sensing and retrieving essential nutrients from the environment are general metabolic traits associated with the growth of this saprophytic fungus. Two important mediators of calcium signals in eukaryotic cells are the Ca(2+)-binding protein calmodulin and the Ca(2+)/calmodulin-dependent phosphatase calcineurin. Calcineurin is a heterodimer that consists of a catalytic subunit A and a Ca(2+)/calmodulin binding unit. We deleted the A. fumigatus calA gene, which encodes the calcineurin A catalytic subunit, and demonstrated that this gene is not essential in this fungus. The DeltacalA mutant strain has severe defects in growth extension, branching and conidial architecture. Furthermore, the A. fumigatus DeltacalA mutant strain has decreased fitness in a low dose murine infection and cannot grow in fetal bovine serum (FBS). After potassium phosphate was added to liquid FBS, the DeltacalA mutant strain could grow with the characteristic phenotype of the DeltacalA mutation. When A. fumigatus calcineurin is inhibited by tacrolimus in a phosphate depleted medium, there is a reduction in the inorganic phosphate transport and six putative phosphate transporter genes have altered mRNA levels. However, there is no effect on the acid phosphatase activity. These results suggest that calcineurin is involved in the regulation of the PHO pathway in A. fumigatus. Our work on calcineurin opens new venues for the research on sensing and nutrient acquisition in A. fumigatus.
Assuntos
Aspergillus fumigatus/enzimologia , Calcineurina/metabolismo , Proteínas Fúngicas/metabolismo , Fosfatase Ácida/metabolismo , Animais , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Calcineurina/genética , Domínio Catalítico/genética , Bovinos , Meios de Cultura/química , Meios de Cultura/farmacologia , Sangue Fetal/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Hifas/efeitos dos fármacos , Hifas/genética , Hifas/crescimento & desenvolvimento , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Mutação , Fosfatos/metabolismo , Fosfatos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esporos Fúngicos/genética , Esporos Fúngicos/ultraestrutura , Tacrolimo/farmacologia , Fatores de TempoRESUMO
Ataxia telangiectasia (A-T) is an inherited disorder characterized by progressive loss of motor function and susceptibility to cancer. The most prominent clinical feature observed in A-T patients is the degeneration of Purkinje motor neurons. Numerous studies have emphasized the role of the affected gene product, ATM, in the regulation of the DNA damage response. However, in Purkinje cells, the bulk of ATM localizes to the cytoplasm and may play a role in vesicle trafficking. The nature of this function, and its involvement in the pathology underlying A-T, remain unknown. Here we characterize the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we find that AtmA is also required for polarized hyphal growth. We demonstrate that an atmA mutant fails to generate a stable axis of hyphal polarity. Notably, cytoplasmic microtubules display aberrant cortical interactions at the hyphal tip. Our results suggest that AtmA regulates the function and/or localization of landmark proteins required for the formation of a polarity axis. We propose that a similar function may contribute to the establishment of neuronal polarity.
Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Hifas/crescimento & desenvolvimento , Morfogênese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Aspergillus nidulans/citologia , Aspergillus nidulans/genética , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular , Polaridade Celular , Clonagem Molecular , Deleção de Genes , Microtúbulos/metabolismo , FenótipoRESUMO
The ATM/ATR kinases and the Mre11 (Mre11-Rad50-Nbs1) protein complex are central players in the cellular DNA damage response. Here we characterize possible interactions between Aspergillus nidulans uvsB(ATR) and the Mre11 complex (scaA(NBS1)). We demonstrate that there is an epistatic relationship between uvsB(ATR), the homolog of the ATR/MEC1 gene, and scaA(NBS1), the homolog of the NBS1/XRS2 gene, for both repair and checkpoint functions and that correct ScaA(NBS1) expression during recovery from replication stress depends on uvsB(ATR). In addition, we also show that the formation of UvsC foci during recovery from replication stress is dependent on both uvsB(ATR) and scaA(NBS1) function. Furthermore, ScaA(NBS1) is also dependent on uvsB(ATR) for nuclear focus formation upon the induction of DNA double-strand breaks by phleomycin. Our results highlight the extensive genetic interactions between UvsB and the Mre11 complex that are required for S-phase progression and recovery from DNA damage.
Assuntos
Aspergillus nidulans/genética , Dano ao DNA/genética , Replicação do DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/fisiologia , Proteínas Quinases/genética , Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/efeitos da radiação , Dano ao DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Regulação Enzimológica da Expressão Gênica , Fenótipo , Proteínas Quinases/metabolismo , Fase S/genética , Raios UltravioletaRESUMO
The Mono carboxylate Transporter (MCT) is a family of membrane proteins from the Major Facilitator Superfamily (MFS) of transporters. Here, we report the first identified Aspergillus nidulans MCT homologue, AmcA. The amcA gene was isolated by high copy number suppression of the hydroxyurea (HU) sensitivity of an A. nidulans camptothecin-sensitive mutant. Expression of amcA is increased when hyphae are grown in media containing acetate or pyruvate as single carbon source, and after exposure to several unrelated drugs. Our results suggest that AmcA could be involved not only in monocarboxylate transport, but also in drug secretion. To our knowledge, this is the first report suggesting a possible involvement of MCT transporters with drug resistance in eukaryotic cells.
Assuntos
Aspergillus nidulans/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Dosagem de Genes , Transportadores de Ácidos Monocarboxílicos/genética , Mutação , Supressão Genética , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Transporte Biológico , Camptotecina/farmacologia , Ácidos Carboxílicos/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transcrição GênicaRESUMO
The Mre11-Rad50-Nbs1 protein complex has emerged as a central player in the cellular DNA damage response. Mutations in scaANBS1, which encodes the apparent homologue of human Nbs1 in Aspergillus nidulans, inhibit growth in the presence of the anti-topoisomerase I drug camptothecin. We have used the scaANBS1 cDNA as a bait in a yeast two-hybrid screening and report the identification of the A. nidulans Mre11 homologue (mreA). The inactivated mreA strain was more sensitive to several DNA damaging and oxidative stress agents. Septation in A. nidulans is dependent not only on the uvsBATR gene, but also on the mre11 complex. scaANBS1 and mreA genes are both involved in the DNA replication checkpoint whereas mreA is specifically involved in the intra-S-phase checkpoint. ScaANBS1 also participates in G2-M checkpoint control upon DNA damage caused by MMS. In addition, the scaANBS1 gene is also important for ascospore viability, whereas mreA is required for successful meiosis in A. nidulans. Consistent with this view, the Mre11 complex and the uvsCRAD51 gene are highly expressed at the mRNA level during the sexual development.
Assuntos
Aspergillus nidulans/fisiologia , Dano ao DNA , Reparo do DNA , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Complementar/genética , DNA Fúngico , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5' and 3' ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.
Assuntos
Candida albicans/genética , Candidíase/genética , Etiquetas de Sequências Expressas , Regulação Fúngica da Expressão Gênica/genética , Genoma Fúngico , Paracoccidioides/genética , Paracoccidioidomicose/genética , Sequência de Bases/genética , Candida albicans/enzimologia , Candida albicans/patogenicidade , Candidíase/enzimologia , Candidíase/fisiopatologia , DNA Complementar/análise , DNA Complementar/genética , Enzimas/biossíntese , Enzimas/genética , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Micélio/enzimologia , Micélio/genética , Micélio/crescimento & desenvolvimento , Paracoccidioides/enzimologia , Paracoccidioides/patogenicidade , Paracoccidioidomicose/enzimologia , Paracoccidioidomicose/fisiopatologia , RNA Mensageiro/genéticaRESUMO
A PCR assay based on the 5' nuclease assay using a fluorescent probe derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis. The assay could detect at least 10 copies of this DNA sequence, providing efficient accuracy to be useful for diagnosis of paracoccidioidomycosis.