Characterization of Aspergillus fumigatus mitochondrial acetyl-CoA acetyltransferase as an antifungal target
Characterization of Aspergillus fumigatus mitochondrial acetyl-CoA acetyltransferase as an antifungal target
复制标题
烟曲霉线粒体乙酰辅酶A乙酰转移酶作为抗真菌靶点的表征
DOI：
10.1128/aem.02986-19
复制
发表时间：
2020
影响因子：
4.4
通讯作者：
Fang Wenxia
中科院分区：
文献类型：
--
作者：
Zhang Yuanwei;Wei Wenfan;Fan Jialu;Jin Cheng;Lu Ling;Fang Wenxia
麦角甾醇在维持真菌细胞膜类固醇动态平衡方面起着重要作用，因此被认为是抗真菌化疗的有效靶点。在酵母中，乙酰辅酶A(CoA)乙酰转移酶(ERG10)催化麦角甾醇生物合成途径中两个乙酰辅酶A分子的Claisen缩合反应，被认为是细胞存活的关键。以酵母菌ERG10为模板，在条件致病菌烟曲霉中发现了两个同源基因AfERG10A(AFUB_000550)和AfERG10B(AFUB_083570)。尽管AfERG10B的重要性已被证实，但AfERG10A的生物学功能仍不清楚。在本研究中，我们鉴定了重组人AfERG10A是一种功能性乙酰辅酶A乙酰转移酶，可催化合成反应和降解反应。通过C端绿色荧光蛋白(GFP)标签融合显示，AfERG10A定位于烟曲霉菌的线粒体。基因敲除和诱导型启动子策略都证明了Aferg10A对于烟曲霉的生存是必不可少的。Aferg10A的表达减少会导致严重的形态缺陷，并增加对氧化和细胞壁压力的敏感性。虽然乙酰辅酶A乙酰转移酶家族的催化机制是高度保守的，但AFERG10A及其与辅酶A的络合物的晶体结构被解析，揭示了辅酶A结合部位的四个不同于人类同源基因的取代。综上所述，我们的遗传和结构研究相结合表明，线粒体AfERG10A对烟曲霉菌细胞的生存是必不可少的，并可能成为喂养抗真菌药物开发管道的潜在药物靶点。目前的治疗选择依赖于有限的抗真菌药物。麦角甾醇是真菌细胞膜的重要组成部分，也是目前抗真菌药物的靶点。大约有20种酶参与麦角甾醇的生物合成，其中乙酰辅酶A乙酰转移酶(ACAT)是第一种。烟曲霉菌中的两个ACAT分别为AfErg10A和AfErg10B。然而，AfErg10A的生物学功能还有待研究。在本研究中，我们发现AfErg10A定位于线粒体中，对烟曲霉菌的存活和形态发育是必不可少的。结合结构研究，我们验证了AfErg10A作为一个潜在的药物靶点，将促进新的抗真菌药物的开发，并提高现有药物的效率。
Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, it is considered an effective target in antifungal chemotherapy. In yeast, the enzyme acetyl-coenzyme A (CoA) acetyltransferase (ERG10) catalyzes the Claisen condensation of two acetyl-CoA molecules to acetoacetyl-CoA in the ergosterol biosynthesis pathway and is reported as being critical for cell viability. Using yeast ERG10 for alignment, two orthologues,AfERG10A (AFUB_000550) andAfERG10B (AFUB_083570), were discovered in the opportunistic fungal pathogen Aspergillus fumigatus. Despite the essentiality ofAfERG10B having been previously validated, the biological function ofAfERG10A remains unclear. In this study, we have characterized recombinantAfERG10A as a functional acetyl-CoA acetyltransferase catalyzing both synthetic and degradative reactions. Unexpectedly,AfERG10A localizes to the mitochondria in A. fumigatus, as shown by C-terminal green fluorescent protein (GFP) tag fusion. Both knockout and inducible promoter strategies demonstrate thatAferg10Ais essential for the survival of A. fumigatus. The reduced expression ofAferg10Aleads to severe morphological defects and increased susceptibility to oxidative and cell wall stresses. Although the catalytic mechanism of acetyl-CoA acetyltransferase family is highly conserved, the crystal structure ofAfERG10A and its complex with CoA are solved, revealing four substitutions within the CoA binding site that are different from human orthologues. Taken together, our combination of genetic and structural studies demonstrates that mitochondrialAfERG10A is essential for A. fumigatus cell viability and could be a potential drug target to feed the antifungal drug development pipeline.IMPORTANCEA growing number of people worldwide are suffering from invasive aspergillosis caused by the human opportunistic fungal pathogen A. fumigatus. Current therapeutic options rely on a limited repertoire of antifungals. Ergosterol is an essential component of the fungal cell membrane as well as a target of current antifungals. Approximately 20 enzymes are involved in ergosterol biosynthesis, of which acetyl-CoA acetyltransferase (ACAT) is the first enzyme. Two ACATs in A. fumigatus areAfErg10A andAfErg10B. However, the biological function ofAfErg10A is yet to be investigated. In this study, we showed thatAfErg10A is localized in the mitochondria and is essential for A. fumigatus survival and morphological development. In combination with structural studies, we validatedAfErg10A as a potential drug target that will facilitate the development of novel antifungals and improve the efficiency of existing drugs.