Purification of a vesicle-vacuole fraction functionally linked to aflatoxin synthesis in Aspergillus parasiticus.
Purification of a vesicle-vacuole fraction functionally linked to aflatoxin synthesis in Aspergillus parasiticus.
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DOI：
10.1016/j.mimet.2009.03.014
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发表时间：
2009-07
影响因子：
2.2
通讯作者：
Linz, John E.
中科院分区：
文献类型：
--
作者：
Chanda, Anindya;Roze, Ludmila V.;Pastor, Alicia;Frame, Melinda K.;Linz, John E.
本实验室目前的研究表明，在丝状真菌寄生曲霉（Aspergillus parasiticus）中，囊泡、空泡和黄曲霉毒素生物合成之间存在功能性联系。在蔗糖酵母提取物液体培养基中，在黄曲霉毒素诱导条件下，A.寄生虫经历了从空泡生物发生到增加数量的囊泡的积累的转变，所述囊泡在大小和密度上表现出显著的异质性。作为第一步，在进行详细的分析，这些细胞器中的黄曲霉毒素合成的作用，我们开发了一种新的方法来纯化的囊泡和空泡部分，使用原生质体制备的细胞收获在黄曲霉毒素合成。该方法包括以下步骤：1）从在黄曲霉毒素诱导条件下生长36小时的菌丝体制备原生质体; 2）在Triton X-100存在下从纯化的原生质体释放囊泡和液泡;和3）使用“一步高密度垫”分级分离囊泡和液泡。囊泡-空泡部分显示α-甘露糖苷酶活性（空泡标记物）和不可检测的琥珀酸脱氢酶和乳酸脱氢酶活性（分别为线粒体和细胞质标记物）富集35倍。用液泡染料MDY-64和CMAC进行的共聚焦激光扫描显微镜表明，该馏分含有纯囊泡和液泡，并且没有膜碎片。透射电子显微镜（TEM）证实，没有线粒体或完整的原生质体污染的纯化馏分。纯化的细胞器表现出显着的大小异质性与在全细胞和原生质体中观察到的大小相似的范围。
Current studies in our laboratory demonstrate a functional link between vesicles, vacuoles and aflatoxin biosynthesis in the filamentous fungus, Aspergillus parasiticus. Under aflatoxin inducing conditions in liquid yeast-extract sucrose medium, A. parasiticus undergoes a shift from vacuole biogenesis to accumulation of an enhanced number of vesicles which exhibit significant heterogeneity in size and density. As a first step in conducting a detailed analysis of the role of these organelles in aflatoxin synthesis, we developed a novel method to purify the vesicle and vacuole fraction using protoplasts prepared from cells harvested during aflatoxin synthesis. The method includes the following steps: 1] preparation of protoplasts from mycelia grown for 36h under aflatoxin inducing conditions; 2] release of vesicles and vacuoles from purified protoplasts in the presence of Triton X-100; and 3] fractionation of the vesicles and vacuoles using a “one-step high density cushion”. The vesicle-vacuole fraction showed a 35 fold enrichment in alpha-mannosidase activity (vacuole marker) and non-detectable succinate dehydrogenase and lactate dehydrogenase activities (mitochondrial and cytoplasmic markers, respectively). Confocal laser scanning microscopy with the vacuole dyes MDY-64 and CMAC demonstrated that the fraction contained pure vesicles and vacuoles and was devoid of membranous debris. Transmission electron microscopy (TEM) confirmed that no mitochondria or unbroken protoplasts contaminated the purified fraction. The purified organelles exhibited significant size heterogeneity with a range of sizes similar to that observed in whole cells and protoplasts.
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