Goa1p of Candida albicans Localizes to the Mitochondria during Stress and Is Required for Mitochondrial Function and Virulence
Goa1p of Candida albicans Localizes to the Mitochondria during Stress and Is Required for Mitochondrial Function and Virulence
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DOI：
10.1128/ec.00066-09
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发表时间：
2009-11-01
期刊：
影响因子：
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通讯作者：
Calderone, Richard
中科院分区：
文献类型：
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作者：
Bambach, Adrienne;Fernandes, Mariana P.;Calderone, Richard
利用白色念珠菌Tn7转座子文库，我们发现了一个突变体，在滴皿试验中对氧化剂如甲萘醌和过氧化氢表现出敏感性。为了验证突变基因在逆境适应中的作用，构建了零突变体并进行了表型表征。由于其在生长和氧化适应中的明显功能，我们将其命名为GOA1。Goa1p似乎是包括白色念珠菌在内的酵母菌纲的CTG亚支所特有的。缺乏goa1的白色念珠菌突变株（菌株GOA31）对6 mM H2O2和0.125 mM甲萘醌的敏感性高于野生型（wt）或基因重组菌株（GOA32）。与对照菌株相比，对氧化剂的敏感性与GOA31突变体在人中性粒细胞中的存活率和毒性降低有关。GOA31的其他表型包括在10%的血清、蜘蛛和SLAD琼脂培养基中生长和成丝减少，不能形成衣孢子。由于Goa1p有一个n端线粒体定位位点，我们还发现绿色荧光蛋白标记的Goa1p在氧化或渗透胁迫下表现出线粒体样分布。此外，GOA31无法在含有乳酸、乙醇或甘油作为唯一碳源的培养基中生长，这表明该突变体的线粒体存在缺陷。为了确定Goa1p如何影响线粒体功能，我们比较了wt、GOA32和GOA31菌株的线粒体电膜电位、呼吸和氧化磷酸化。我们现在表明，GOA31，而不是wt或GOA32，已经降低了呼吸和线粒体膜电位，使得突变细胞无法驱动氧化磷酸化。这是白色念珠菌中首次报道由线粒体膜电位丧失引起的呼吸缺陷。
Using a Tn7 transposon library of Candida albicans, we have identified a mutant that exhibited sensitivity in drop plate assays to oxidants such as menadione and hydrogen peroxide. To verify the role of the mutated gene in stress adaptation, null mutants were constructed and phenotypically characterized. Because of its apparent functions in growth and oxidant adaptation, we have named the gene GOA1. Goa1p appears to be unique to the CTG subclade of the Saccharomycotina, including C. albicans. Mutants of C. albicans lacking goa1 (strain GOA31) were more sensitive to 6 mM H2O2 and 0.125 mM menadione than the wild type (wt) or a gene-reconstituted (GOA32) strain. The sensitivity to oxidants correlated with reduced survival of the GOA31 mutant in human neutrophils and avirulence compared to control strains. Other phenotypes of GOA31 include reduced growth and filamentation in 10% serum, Spider, and SLAD agar media and an inability to form chlamydospores. Since Goa1p has an N-terminal mitochondrion localization site, we also show that green fluorescent protein-tagged Goa1p shows a mitochondrionlike distribution during oxidant or osmotic stress. Further, the inability of GOA31 to grow in medium containing lactate, ethanol, or glycerol as the sole carbon source indicates that the mitochondria are defective in the mutant. To determine how Goa1p contributes to mitochondrial function, we compared the wt, GOA32, and GOA31 strains for mitochondrial electrical membrane potential, respiration, and oxidative phosphorylation. We now show that GOA31, but not the wt or GOA32, had decreased respiration and mitochondrial membrane potential such that mutant cells are unable to drive oxidative phosphorylation. This is the first report in C. albicans of a respiratory defect caused by a loss of mitochondrial membrane potential.