RESUMEN
Aspergillus fumigatus possesses a branched mitochondrial electron transport chain, with both cyanide-sensitive and -insensitive oxygen-consumption activities. Mitochondrial reactive oxygen species mediate signaling for alternative oxidase (AOX) expression. A 1173 bp-long Afaox gene encoding a 40 kDa protein has been cloned and identified. Recombinant constructs containing the Afaox ORF were transformed into Escherichia coli and Saccharomyces cerevisiae for heterologous expression. In A. fumigatus, AOX activity and mRNA expression were both induced with menadione or paraquat, suggesting an important role of AOX under oxidative stress. Therefore, positive transformants showed a cyanide-resistant and salicylhydroxamic acid-sensitive respiration, whereas in control cells the oxygen uptake was completely inhibited after KCN addition.
Asunto(s)
Aspergillus fumigatus/genética , Proteínas Fúngicas/genética , Estrés Oxidativo , Oxidorreductasas/genética , Secuencia de Aminoácidos , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/metabolismo , Western Blotting , Clonación Molecular , Cianuros/farmacología , Activación Enzimática/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteínas Mitocondriales , Datos de Secuencia Molecular , Oxidorreductasas/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Paraquat/farmacología , Proteínas de Plantas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Salicilamidas/farmacología , Homología de Secuencia de Aminoácido , Vitamina K 3/farmacologíaRESUMEN
The understanding of the controlling factors of calcium homeostasis in Aspergillus fumigatus is very poor, although this ion is involved in several important events of these particular cells. We have cloned, identified and expressed for functional complementation a PMR1-like Ca(2+)-ATPase gene from A. fumigatus. The Afpmr1 gene encodes a protein of 1061 deduced amino acids, containing all the conserved subdomains found in other P-type ATPases: the phosphatase region, phosphorylation site, FITC labelling site, ATP binding domain; E(386), N871, D875 amino acid residues for calcium ion interaction and Q880, a residue that alters ion selectivity in PMR1. The expressed AfPMR1 in S. cerevisiae K616 strain functionally complemented the deficient growth in EGTA (5-20 mM)- and MnCl2 (4 mM)-containing medium. These results demonstrate the first evidence of a Ca(2+)-ATPase in A. fumigatus and strongly suggest a role for this enzyme in calcium and manganese homeostasis.
