Functional disruption of yeast metacaspase, Mca1, leads to miltefosine resistance and inability to mediate miltefosine-induced apoptotic effects
Functional disruption of yeast metacaspase, Mca1, leads to miltefosine resistance and inability to mediate miltefosine-induced apoptotic effects
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DOI：
10.1016/j.fgb.2014.04.003
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发表时间：
2014-06-01
影响因子：
3
通讯作者：
Djordjevic, Julianne T.
中科院分区：
文献类型：
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作者：
Biswas, Chayanika;Zuo, Xiaoming;Djordjevic, Julianne T.
米替福辛 (MI) 是一种新型、潜在的抗真菌剂，对某些酵母和丝状真菌病原体具有活性。我们之前在模型酵母酿酒酵母中证明，MI 通过与细胞色素 c 氧化酶 (COX) 的 Cox9p 亚基相互作用，导致线粒体膜电位破坏和细胞凋亡样细胞死亡。为了确定抗真菌作用的其他机制，通过暴露于诱变剂甲磺酸乙酯在酿酒酵母中诱导 MI 抗性，并研究了导致抗性的基因突变。创建了 MI 抗性单倍体菌株 (H-C101)。交配后，二倍体菌株（D-C101）保留了抗性，证实了显性遗传。对单个 D-C101 四分体的表型评估表明，只有一个突变基因导致 MI 抗性表型。为了鉴定该基因，对 H-C101 的基因组进行了测序，并鉴定了 17 个突变基因，包括编码元半胱天冬酶的 MCA1。 MCA1 突变导致第 164 位天冬酰胺 (N) 被天冬氨酸 (D) 取代 (MCA1(N164D))。发现 MI 抗性主要是由于 MCA1(N164D)，因为 MCA1(N164D) 的单拷贝附加型表达，而不是其他两个突变基因（FAS1(T14171) 和 BCK2(T104A)）导致野生型菌株中的 MI 抗性。此外，MCA1 缺失突变体 (mca1 Delta) 具有 MI 抗性。 MI处理导致MI抗性菌株（表达MCA1（N164D）和mca1 Delta）和MI敏感菌株（表达MCA1）中活性氧（ROS）积累，但未能激活MI抗性菌株中的Mca1，表明ROS积累对MI的杀菌作用没有贡献。总之，Mca1 的功能破坏会导致 MI 抵抗，并且无法介导 MI 诱导的细胞凋亡效应。因此，Mca1 介导的细胞凋亡是 MI 诱导的抗真菌作用的主要机制。 (C) 2014 Elsevier Inc. 保留所有权利。
Miltefosine (MI) is a novel, potential antifungal agent with activity against some yeast and filamentous fungal pathogens. We previously demonstrated in the model yeast, Saccharomyces cerevisiae, that MI causes disruption of mitochondrial membrane potential and apoptosis-like cell death via interaction with the Cox9p sub-unit of cytochrome c oxidase (COX). To identify additional mechanisms of antifungal action, MI resistance was induced in S. cerevisiae by exposure to the mutagen, ethyl methanesulfonate, and gene mutation(s) responsible for resistance were investigated. An MI-resistant haploid strain (H-C101) was created. Resistance was retained in the diploid strain (D-C101) following mating, confirming dominant inheritance. Phenotypic assessment of individual D-C101 tetrads revealed that only one mutant gene contributed to the MI-resistance phenotype. To identify this gene, the genome of H-C101 was sequenced and 17 mutated genes, including metacaspase-encoding MCA1, were identified. The MCA1 mutation resulted in substitution of asparagine (N) with aspartic acid (D) at position 164 (MCA1(N164D)). MI resistance was found to be primarily due to MCA1(N164D), as single-copy episomal expression of MCA1(N164D), but not two other mutated genes (FAS1(T14171) and BCK2(T104A)), resulted in MI resistance in the wild-type strain. Furthermore, an MCA1 deletion mutant (mca1 Delta) was MI-resistant. MI treatment led to accumulation of reactive oxygen species (ROS) in MI-resistant (MCA1(N164D)-expressing and mca1 Delta) strains and MI-susceptible (MCA1-expressing) strains, but failed to activate Mca1 in the MI-resistant strains, demonstrating that ROS accumulation does not contribute to the fungicidal effect of MI. In conclusion, functional disruption of Mca1, leads to MI resistance and inability to mediate MI-induced apoptotic effects. Mca1-mediated apoptosis is therefore a major mechanism of MI-induced antifungal action. (C) 2014 Elsevier Inc. All rights reserved.