Applied and Environmental Microbiology ( IF 4.4 ) Pub Date : 2020-03-18 Zhang, Y., Wei, W., Fan, J., Jin, C., Lu, L., Fang, W.
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) and AfERG10B (AFUB_083570), were discovered in the opportunistic fungal pathogen Aspergillus fumigatus. Despite the essentiality of AfERG10B having been previously validated, the biological function of AfERG10A remains unclear. In this study, we have characterized recombinant AfERG10A 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 that Aferg10A is essential for the survival of A. fumigatus. The reduced expression of Aferg10A leads 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 of AfERG10A 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 mitochondrial AfERG10A is essential for A. fumigatus cell viability and could be a potential drug target to feed the antifungal drug development pipeline.
IMPORTANCE A 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 are AfErg10A and AfErg10B. However, the biological function of AfErg10A is yet to be investigated. In this study, we showed that AfErg10A is localized in the mitochondria and is essential for A. fumigatus survival and morphological development. In combination with structural studies, we validated AfErg10A as a potential drug target that will facilitate the development of novel antifungals and improve the efficiency of existing drugs.
中文翻译:
烟曲霉线粒体乙酰辅酶A乙酰转移酶作为抗真菌靶标
麦角固醇在维持真菌中细胞膜固醇稳态方面起着重要作用,因此,它被认为是抗真菌化学疗法的有效靶标。在酵母中,乙酰辅酶A(CoA)乙酰转移酶(ERG10)在麦角固醇生物合成途径中催化两个乙酰辅酶A分子的Claisen缩合为乙酰乙酰辅酶A,据报道对于细胞生存力至关重要。使用酵母ERG10进行比对,在机会性真菌病原体烟曲霉中发现了两个直向同源物Af ERG10A(AFUB_000550)和Af ERG10B(AFUB_083570)。尽管本性的房颤ERG10B先前已经被验证,生物功能房颤ERG10A仍不清楚。在这项研究中,我们已将重组Af ERG10A表征为功能性乙酰辅酶A乙酰转移酶,可催化合成和降解反应。出乎意料的是,Af ERG10A定位于烟曲霉的线粒体,如C端绿色荧光蛋白(GFP)标签融合所示。敲除和诱导的启动子策略都证明Aferg10A对烟曲霉的生存至关重要。Aferg10A的表达降低会导致严重的形态缺陷并增加对氧化和细胞壁应力的敏感性。尽管乙酰辅酶A乙酰转移酶家族的催化机制是高度保守的,但Af ERG10A的晶体结构及其与辅酶A的配合物得以解析,揭示了辅酶A结合位点内与人类直系同源物不同的四个取代。两者合计,我们的遗传和结构研究的结合表明,线粒体Af ERG10A对烟曲霉细胞的生存能力是必不可少的,并且可能成为抗真菌药物开发管道的潜在药物靶标。
重要全世界越来越多的人患有由人类机会性真菌病原烟曲霉引起的侵袭性曲霉病。当前的治疗选择依赖于有限的抗真菌剂。麦角固醇是真菌细胞膜的重要组成部分,也是目前抗真菌药的目标。麦角固醇的生物合成涉及大约20种酶,其中乙酰-CoA乙酰转移酶(ACAT)是第一种酶。烟曲霉中的两个ACAT为Af Erg10A和Af Erg10B。但是,Af Erg10A的生物学功能尚待研究。在这项研究中,我们证明了AfErg10A位于线粒体中,对于烟曲霉的生存和形态发育至关重要。结合结构研究,我们验证了Af Erg10A是潜在的药物靶标,它将促进新型抗真菌药的开发并提高现有药物的效率。