In situ evidence of an alternative oxidase and an uncoupling protein in the respiratory chain of Aspergillus fumigatus.
Aspergillus fumigatus is an unusual pathogen in immunocompetent individuals; its incidence has increased in the last decades in patients immunocompromised, like those with chronic granulomatosis disease and AIDS. The aim of this study was to identify differences between the respiratory chain of host and the fungus planning to use the later as a pharmacological target. We evaluated respiration, membrane potential and oxidative phosphorylation of mitochondria of the spheroplasts of A. fumigatus in situ, after permeabilization with digitonin. Firstly, a functional respiratory chain (complex I-V) was demonstrated: adenosine 5'-diphosphate (ADP) induced an oligomycin-sensitive transition from resting to phophorylating respiration in the presence of the oxidizable substrates malate, glutamate, alpha-ketoglutarate, pyruvate, dihydroorotate, succinate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and exogenous NADH. In addition, the ability of the fungus to oxidize exogenous NADH, as well as the insensitivity of its respiration to rotenone, in association with the sensitivity to flavone, indicate the presence of an alternative NADH-ubiquinone oxidoreductase; the partial sensitivity of respiration to antimycin A and cyanide, in association with the sensitivity to benzohydroxamic acid, indicates the presence of an alternative oxidase. The fatty acid-uncoupled respiration was partly reversed by bovine serum albumin (BSA) and guanosine 5'-triphosphate (GTP) and was insensitive to either carboxyatractyloside or ADP. These results, together with evidences obtained using antibodies raised against uncoupling protein (UCP) from potato, indicate in addition, the presence of an uncoupling protein in the respiratory chain of A. fumigatus.
Tudella
Valéria G
VG
Departament of Clinical, Toxicological and Bromatological Analysis, Faculty of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto 14040-903, SP, Brazil.
Curti
Carlos
C
Soriani
Frederico M
FM
Santos
Antonio C
AC
Uyemura
Sergio A
SA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology; D000968:Antimycin A / Q000494:pharmacology; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D003486:Cyanides / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D042967:Electron Transport Complex I / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006877:Hydroxamic Acids / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008564:Membrane Potentials; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D024101:Mitochondrial Proteins; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D010088:Oxidoreductases / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D010539:Permeability / Q000187:drug effects; D010940:Plant Proteins; D014475:Uncoupling Agents / Q000494:pharmacology; D000099188:Alternative Oxidase
D000244:Adenosine Diphosphate; D000935:Antifungal Agents; D000968:Antimycin A; D003486:Cyanides; D004072:Digitonin; D042967:Electron Transport Complex I; D004791:Enzyme Inhibitors; D005419:Flavonoids; D006877:Hydroxamic Acids; D007202:Indicators and Reagents; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D014475:Uncoupling Agents; D000099188:Alternative Oxidase; C006696:benzohydroxamic acid