Influence of Histatin 5 on <i>Candida albicans</i> Mitochondrial Protein Expression Assessed by Quantitative Mass Spectrometry

Individual aspects of the mode of action of histatin 5, a human salivary antifungal protein, have been partially elucidated, but the mechanism likely involves a complex set of events that have not been characterized. Previous evidence points toward histatin-induced alterations in mitochondrial function. The purpose of the present study was to verify and quantify changes in the mitochondrial proteome of Candida albicans treated with histatin 5. Cell killing was determined by plating and differential protein expression levels in the mitochondrial samples were determined by quantitative proteomics approaches employing mTRAQ and ICAT labeling and Western blotting. Relative quantitation ratios were established for 144 different proteins. Up-regulated mitochondrial proteins were predominantly involved in genome maintenance and gene expression, whereas proteins that constitute the respiratory enzyme complexes were mostly down-regulated. The differential expression of ATP synthase gamma chain and elongation factor 1-alpha were confirmed by Western blotting by comparison to levels of cytochrome c which were unchanged upon histatin treatment. The mTRAQ and ICAT proteomics results suggest that key steps in the histatin 5 antifungal mechanism involve a bioenergetic collapse of C. albicans, caused essentially by a decrease in mitochondrial ATP synthesis.人类唾液抗真菌蛋白富组蛋白5（histatin 5）的作用模式的多个方面已得到部分阐明，但其功能发挥的机制可能涉及一系列尚未被表征的复杂事件。既往研究证据表明，富组蛋白可诱导线粒体功能发生改变。本研究旨在验证并定量分析经富组蛋白5处理的白色念珠菌（Candida albicans）的线粒体蛋白质组变化。研究通过平板菌落计数法测定细胞杀伤率，并采用同位素标记相对和绝对定量（mTRAQ）、同位素编码亲和标签（ICAT）标记以及蛋白质免疫印迹（Western blotting）等定量蛋白质组学方法，分析线粒体样品中的差异蛋白表达水平。最终确立了144种不同蛋白质的相对定量比值。上调表达的线粒体蛋白主要参与基因组维持与基因表达过程，而构成呼吸酶复合物的蛋白则大多呈现下调表达。通过与富组蛋白处理后水平未发生改变的细胞色素c（cytochrome c）进行对照，研究经蛋白质免疫印迹验证了ATP合酶γ亚基与延伸因子1-α的差异表达情况。同位素标记相对和绝对定量与同位素编码亲和标签蛋白质组学结果显示，富组蛋白5的抗真菌机制关键步骤涉及白色念珠菌的生物能崩溃，该过程本质上由线粒体ATP合成能力下降所引发。