Enzymatic Dysfunction of Mitochondrial Complex I of the Candida albicansgoa1Mutant Is Associated with Increased Reactive Oxidants and Cell Death

Abstract

ABSTRACTWe have previously shown that deletion ofGOA1(growth andoxidantadaptation) ofCandida albicansresults in a loss of mitochondrial membrane potential, ATP synthesis, increased sensitivity to oxidants and killing by human neutrophils, and avirulence in a systemic model of candidiasis. We established that translocation of Goa1p to mitochondria occurred during peroxide stress. In this report, we show that thegoa1Δ (GOA31), compared to the wild type (WT) and a gene-reconstituted (GOA32) strain, exhibits sensitivity to inhibitors of the classical respiratory chain (CRC), including especially rotenone (complex I [CI]) and salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway (AOX), while potassium cyanide (KCN; CIV) causes a partial inhibition of respiration. In the presence of SHAM, however, GOA31 has an enhanced respiration, which we attribute to the parallel respiratory (PAR) pathway and alternative NADH dehydrogenases. Interestingly, deletion ofGOA1also results in a decrease in transcription of the alternative oxidase geneAOX1in untreated cells as well as negligibleAOX1andAOX2transcription in peroxide-treated cells. To explain the rotenone sensitivity, we measured enzyme activities of complexes I to IV (CI to CIV) and observed a major loss of CI activity in GOA31 but not in control strains. Enzymatic data of CI were supported by blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments which demonstrated less CI protein and reduced enzyme activity. The consequence of a defective CI in GOA31 is an increase in reactive oxidant species (ROS), loss of chronological aging, and programmed cell death ([PCD] apoptosis)in vitrocompared to control strains. The increase in PCD was indicated by an increase in caspase activity and DNA fragmentation in GOA31. Thus,GOA1is required for a functional CI and partially for the AOX pathway; loss ofGOA1compromises cell survival. Further, the loss of chronological aging is new to studies ofCandidaspecies and may offer an insight into therapies to control these pathogens. Our observation of increased ROS production associated with a defective CI and PCD is reminiscent of mitochondrial studies of patients with some types of neurodegenerative diseases where CI and/or CIII dysfunctions lead to increased ROS and apoptosis.