Functional analysis of apoptosis-inducing factor in the human fungal pathogen Candida albicans

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Journal Article

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Candida albicans, Apoptosis


Introduction, aims & objectives Apoptosis is a highly organized cellular process crucial for growth and development. Apoptosis has been demonstrated in unicellular yeast Saccharomyces cerevisiae, but little is known in opportunistic human fungal pathogen Candida albicans. Previous studies showed that typical apoptotic features could be induced in C. albicans. Mitochondria are bifunctional organelles: they are energy powerhouse; but also set a central stage that leads to cell demise. Mitochondrial dysfunction has been regarded as the onset of apoptosis, manifested by depolarization of mitochondrial membrane potential (MMP), massive elevation of ROS, and release of apoptosis-inducing factor (AIF) and cytochrome c. Translocation of AIF from mitochondrial intermembrane space to nucleus causes chromatin condensation, DNA cleavage and cell death. No C. albicans AIF has been identified so far. We previously revealed that purpurin causes MMP depolarization without significant increase in intracellular ROS levels in Candida fungi, suggesting an existence of a mitochondrial-mediated cell death pathway. Searching the published C. albicans genome using S. cerevisiae AIF, we have identified three putative AIF sequences (orf19.1438, orf19.2175 and orf19.2671). The objectives of this project were: 1. To clone and express the three putative C. albicans AIF sequences; and 2. To determine the cellular localization and function role of AIF in energy production and cell death. Materials & methods The three putative C. albicans AIF sequences were cloned by PCR, expressed and purified from Escherichia coli BL21 as His-tag fusion proteins (CaAifp). Chromosomal green fluorescent protein-tagged AIF was constructed to detect cellular localization using confocal microscopy. The NADH oxidase activity of the purified CaAifp was determined by spectrophotometric method; and the cell death-related functions of the CaAifp were evaluated by examining sub-cellular translocation after apoptotic insults, ability to degrade DNA, and complementation to S. cerevisiae ∆aif1 mutant. Results The three putative C. albicans AIF sequences were cloned, expressed and purified to homogeneity from E. coli BL21 (i.e. CaAifp-orf19.1438, CaAifp-orf19.2175 and CaAifp-orf19.2671). CaAifp-orf19.2175 and CaAifp-orf19.2671 possessed NADH oxidase activity. CaAifp-orf19.2175 degraded DNA and functionally complemented S. cerevisiae ∆aif1 mutant. Conclusions The collective data of the present study suggest that orf19.2175 encodes a bona fide C. albicans AIF and the AIF-mediated cell death mechanism in C. albicans shares phylogenetic conservation to that in S. cerevisiae. Deciphering C. albicans AIF paves way for the elucidation of additional effector molecules in the mitochondrial-mediated cell death pathway and also casts light on the design of anti-Candidal strategy by promoting cell suicide.

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Hong Kong Medical Journal

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