Cloning and functional expression of the mitochondrial alternative oxidase ofAspergillus fumigatusand its induction by oxidative stress

Abstract

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.

Alternative oxidase: an inter-kingdom perspective on the function and regulation of this broadly distributed 'cyanide-resistant' terminal oxidase

“…heterologous expression systems. The AOX of the plant Sauromatum guttatum Schott (voodoo lily) is able to confer CN-resistant respiration in isolated mitochondria in the yeast Schizosaccharomyces pombe and the AOXs of the fungi Pichia (Hansenula) anomala and Aspergillus fumigatus are functional when expressed in S. cerevisiae (Crichton et al 2005;Mathy et al 2006;Magnani et al 2007). The AOX of the tunicate animal Ciona intestinalis has been shown to be functional when expressed in cultured human cells (Hakkaart et al 2006).…”

“…Fasting in the amoeba Chaos carolinensis also increases oxidative stress and the activation of AOX is seen as a protective mechanism (Deng et al 2002). In A. fumigatus AOX mRNA expression is induced by paraquat and menadione, two compounds that lead to cellular oxidative stress (Magnani et al 2007). AOX activation in durum wheat mitochondria also led to a decrease in the rate of superoxide anion generation (Pastore et al 2001).…”

“…The application of H 2 O 2 increases AOX1 expression in tobacco (Vanlerberghe and McIntosh 1996), AOX1a expression in corn (Polidoros et al 2005) and AOX expression in the fungus Magnaporthe grisea (Yukioka et al 1998). The ROS generator paraquat is capable of increasing AOX expression in the fungus Aspergillus fumigatus (Magnani et al 2007).…”

“…COX). The AOXs of the bacterium Novosphingobium aromaticivorans, the protist Cryptosporidium parvum, the protist Trypanosoma brucei brucei and the fungus Aspergillus fumigatus have all been demonstrated to be active quinol terminal oxidases when expressed in this E. coli system (Nihei et al 2003;Stenmark and Nordlund 2003;Suzuki et al 2004;Magnani et al 2007). Two yeast species that lack AOX have also been used as …”

Oxidative stress in fungal fermentation processes: the roles of alternative respiration

“…The effects of two ROS-generating agents, paraquat and H 2 O 2 were investigated in 12 fungal species, the results of which indicated exposure of fungal spores or mycelia to both agents stimulated CRR (Angelova et al 2005). In A. fumigatus, AOX activity and mRNA expression were both induced with menadione or paraquat, suggesting an important role of AOX under oxidative stress conditions (Magnani et al 2007). A recent study by our group showed that oxidative stress in batch cultures of A. niger caused by over-oxygenation led to a general decrease of ATP content in the cells (Li et al 2008b) which corroborates the engagement of alternative respiration under such conditions proposed by Bai et al (2003b).…”