Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans

Abstract

The respiration, membrane potential (∆ψ), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 µM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial ∆ψ respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 µM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 µM) inhibited respiration by 30% and 2 µM antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of ∆ψ induced by 5 mM ATP and 0.5% BSA, and ∆ψ decrease induced by 10 µM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death. Correspondence

Goa1p of Candida albicans Localizes to the Mitochondria during Stress and Is Required for Mitochondrial Function and Virulence

“…Safranine O is a dye whose fluorescence spectrum shifts as it binds to and stacks upon increasingly polarized inner mitochondrial membranes (1). The spectral shift is related to a developed mitochondrial membrane potential (⌬⌿ m ) up to at least 170 mV (1) and can be monitored by using a spectrofluorophotometer operating at excitation and emission wavelengths of 495 and 586 nm, respectively (9). Figure 9A shows that spheroplasts suspended in the stan-FIG.…”

Involvement of Candida albicans pyruvate dehydrogenase complex protein X (Pdx1) in filamentation

“…Cells were washed once in cold, sterile H 2 O and processed as previously described (Cavalheiro et al, 2004). BrieXy, cells were washed again in buVer A (1 M sorbitol, 10 mM MgCl 2 and 50 mM Tris-HCl, pH 7.8) and wet pellets were weighed, resuspended in buVer A (3 ml/g of cells) containing 30 mM dithiothreitol and incubated for 15 min at room temperature.…”

Plant Uncoupling Mitochondrial Protein and Alternative Oxidase: Energy Metabolism and Stress

“…During the last years, UCP gene sequences were also found in birds (Vianna et al, 2001;Hirabayashi et al, 2005;Talbot et al, 2004), ectothermic vertebrates, such as frog (Xenopus octopus; Klein et al, 2002), carp (Ciprinus carpio) and zebrafish (Danio rerio; Stuart et al, 1999), insects (Drosophila melanogaster; Fridell et al, 2004) and in the primitive eukaryotic organism Caenorhabditis elegans (CeUCP, Accession number AAB54239). UCP has also been identified in mitochondria from Acanthamoeba castellanii , Dictyostelium discoideum , Candida parapsilosis (CpUCP, Jarmuszkiewicz et al, 2000), Candida albicans (Cavalheiro et al, 2004), and in trophozoites of the malaria parasite Plasmodium Berghei (Uemura et al, 2000). The existence of UCPs in protozoa, fungi, plants, insects, and fishes suggests that uncoupling proteins emerged early during evolution as a distinct member of the mitochondrial anion carrier family (MACF), probably before the divergence of plant, animal, and fungi kingdoms.…”