RESUMO
Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25 degrees C to 37 degrees C and is critical for pathogenicity. Intracellular Ca(2+) levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca(2+) with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca(2+) accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of P. brasiliensis. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca(2+) homeostasis and decreased mRNA transcription of the CCH1 gene for the plasma membrane Ca(2+) channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca(2+)/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca(2+) homeostasis in P. brasiliensis and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on P. brasiliensis reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis.
Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , Proteínas Fúngicas/metabolismo , Paracoccidioides/enzimologia , Paracoccidioides/crescimento & desenvolvimento , Calcineurina/genética , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Morfogênese/efeitos dos fármacos , Micélio/enzimologia , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Paracoccidioides/genética , Paracoccidioides/metabolismo , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/microbiologia , Tacrolimo/farmacologia , TemperaturaRESUMO
Paracoccidioides brasiliensis is a thermo-dimorphic fungus that is the causative agent of paracoccidioidomyicosis (PCM), a human systemic granulomatous mycosis found in Latin America. Dimorphic transition from mycelium to yeast is required for establishing pathogenicity. Dimorphism is marked by changes in mitochondrial physiology, including modulation of respiration rate. In this work, we present the identification of three P. brasiliensis nuclear genes PbCOX9, PbCOX12, and PbCOX16 that code for structural subunits and a putative assembly facilitator (PbCOX16) of the mitochondrial cytochrome c oxidase (COX), the terminal enzyme complex of the respiratory chain. We measured their expression pattern during the dimorphic transition from mycelium to yeast and back by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). Our results show that messages from these genes increase during the mycelium to yeast transition and decrease during the opposite conversion. This result supports active mitochondrial participation in the transition. Heterologous complementation of the corresponding Saccharomyces cerevisiae null mutant with the PbCOX9 gene was successfully obtained.
Assuntos
Paracoccidioides/enzimologia , Prostaglandina-Endoperóxido Sintases/genética , Clonagem Molecular , DNA Fúngico/genética , Teste de Complementação Genética , Isoenzimas , Micélio/enzimologia , Micélio/fisiologia , Paracoccidioides/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We report here the complete nucleotide sequence of the 30.9-kb mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum. All genes are encoded on the same DNA strand and include seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxireductase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), three subunits of cytochrome oxidase (cox1, cox2, and cox3), apocytochrome b (cob), three subunits of ATP synthase (atp6, atp8, and atp9), the small and large ribosomal RNAs (rns and rnl), and 25 tRNAs. A ribosomal protein gene (rps5) is present as an intronic ORF in the large ribosomal subunit. The genes coding for cob and cox1 carry one intron and nad5 carries two introns with ORFs. The mtDNA of E. floccosum has the same gene order as Trichophyton rubrum mtDNA, with the exception of some tRNA genes. Maximum likelihood phylogenetic analysis confirms T. rubrum as a close relative of E. floccosum. This is the first complete mitochondrial sequence of a species of the order Onygenales. This sequence is available under GenBank accession number AY916130.
Assuntos
Epidermophyton/classificação , Epidermophyton/genética , Genes Mitocondriais/genética , Genoma Fúngico/genética , Sequência de Bases , Códon/genética , Proteínas Fúngicas/genética , Ordem dos Genes , Genes Fúngicos , Genes de RNAr , Íntrons , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Filogenia , RNA de Transferência/genética , Análise de Sequência de DNARESUMO
To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged.
Assuntos
Biologia Computacional/métodos , DNA Complementar/análise , DNA Complementar/fisiologia , DNA de Plantas/análise , DNA de Plantas/fisiologia , Etiquetas de Sequências Expressas , Saccharum/genética , Saccharum/fisiologia , Biologia Computacional/estatística & dados numéricos , DNA Complementar/classificação , DNA de Plantas/classificação , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Peptídeos/classificação , Peptídeos/genética , Peptídeos/fisiologia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Polimorfismo Genético/genética , Estrutura Terciária de Proteína/genética , Saccharum/crescimento & desenvolvimento , Análise de Sequência de DNA/métodos , Transdução de Sinais/genéticaRESUMO
COX19, a nuclear gene of Saccharomyces cerevisiae, was cloned by transformation of a respiratory-deficient mutant from complementation group G188 of a pet mutant collection. The gene codes for an 11-kDa protein (Cox19p) required for expression of cytochrome oxidase. Because cox19 mutants are able to synthesize the mitochondrial and nuclear gene products of cytochrome oxidase, Cox19p probably functions post-translationally during assembly of the enzyme. Cox19p is present in the cytoplasm and mitochondria, where it exists as a soluble intermembrane protein. This dual location is similar to what was previously reported for Cox17p, a low molecular weight copper protein thought to be required for maturation of the CuA center of subunit 2 of cytochrome oxidase. The similarity in their subcellular distribution, combined with the presence of four cysteines in Cox19p that align with a subset of the cysteines in Cox17p, suggests that like the latter, Cox19p may function in metal transport to mitochondria.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Regulação Enzimológica da Expressão Gênica/genética , Mitocôndrias/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Humanos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Frações Subcelulares/enzimologiaRESUMO
A mitocôndria funciona como uma usina geradora metabólica por meio da fosforilaçäo oxidativa e tem sido alvo de um renovado interesse devido aos progressos no entendimento de sua biogênese e na descriçäo de novos papéis ligados à senescência, morte celular e montagem dos centros Fe/S. Uma análise global dos genes de planta ligados a esta organela é agora possível. A base de dados do projeto SUCEST foi examinada para detecçäo de ESTs com similaridade a genes nucleares relacionados às funções mitocondriais usando-se proteínas de Saccharomyces cerevisiae, Homo sapiens e Arabidopsis thaliana. Foram utilizadas 869 seqüências de proteínas para varrer o banco de ESTs do projeto SUCEST por meio do programa de busca de similaridade TBLASTN, sendo examinados 81.223 agrupamentos. Encontramos 367 agrupamentos com E-value>10-10 que representam os prováveis ortólogos em cana-de-açúcar dos genes correspondentes humanos, de levedura e de Arabidopsis. Encontramos produtos gênicos relacionados a todas as categorias funcionais ligados à atividade mitocondrial de maneira que este estudo serve de ponto de partida para a identificaçäo dos genes de cana-de-açúcar envolvidos na biogênese e funçäo da organela e para o estudo da estrutura e fisiologia destes genes.
