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Regulation and subcellular localization of aflatoxin biosynthesis in Aspergillus parasiticus.

机译:寄生曲霉中黄曲霉毒素生物合成的调控和亚细胞定位。

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摘要

The filamentous fungus, Aspergillus parasiticus grows on a variety of susceptible food crops and produces aflatoxin, a carcinogenic secondary metabolite that is both a health and economic threat. Aflatoxin biosynthesis is one of the most well-characterized eukaryotic secondary metabolic pathways. Understanding how aflatoxin biosynthesis initiates and where aflatoxin is made are critical to control synthesis and export of fungal metabolites. A previous study conducted in the Linz laboratory using electrophoretic mobility shift analysis (EMSA) and chromatin immunoprecipitiation (ChIP) found that the bZIP transcription factor AtfB binds to promoters of specific aflatoxin and stress response genes. Once aflatoxin gene expression initiates, our lab showed that A. parasiticus performs synthesis, compartmentalization, and export of aflatoxin in subcellular organelles called toxisomes. This dissertation is dedicated to further characterizing the role of transcriptional regulation and subcellular localization of aflatoxin biosynthesis specifically through two cellular targets, AtfB and Vps34. In order to study function of these two targets, we exploited a novel and endogenous CRISPR/cas9-like system in A. parasiticus to down-regulate function of AtfB and Vps34. This system is an easy, rapid, and highly efficient co-transformation method; 1 out of every 3 niaD + (the gene encoding nitrate reductase [niaD] is the selectable marker) transformants exhibited an atypical phenotype that we later showed was associated with the presence and expression of the disruption construct. The disruption of AtfB resulted in a consistent phenotype of low aflatoxin production, lower conidiospore numbers, and lower levels of spore pigmentation. Down-regulation of AtfB targets involved in aflatoxin biosynthesis and stress response showed that the level of expression of target genes is consistent with the observed phenotype of the disruption strains. Of particular importance, global expression data and computer-based network analysis also suggested the AtfB network extends beyond mycotoxin biosynthesis and stress response. Interestingly, the disruption of Vps34 resulted in the opposite phenotype compared to disruption of AtfB. Vps34 transformants exhibited high aflatoxin production, high conidiospore numbers, and high levels of spore pigmentation. We conducted an independent experiment using 3-methyladenine, a biochemical inhibitor of Vps34. Treatment of A. parasiticus SU-1 with 3-methyladenine caused a 10-fold increase in aflatoxin levels detected in aflatoxin-inducing medium. This biochemical approach supports the phenotype of high aflatoxin levels observed in Vps34 disruption strains. We further propose that Vps34 negatively regulates the transport of toxisomes to the vacuoles. Vps34 also positively regulates the export pathway directing early endosomes carrying aflatoxin for export out of the cell. Fungal targets like AtfB and Vps34 that differentially regulate aflatoxin biosynthesis serve as useful tools to understand the molecular mechanisms that contribute to fungal virulence. From a public health perspective, our research goal is to use practical and sustainable natural inhibitors that target fungal specific cellular targets to block toxin production in the field and in storage.
机译:丝状真菌寄生曲霉(Aspergillus parasiticus)在多种易感的粮食作物上生长,并产生黄曲霉毒素(一种致癌的次生代谢产物,对健康和经济均构成威胁)。黄曲霉毒素的生物合成是最典型的真核次级代谢途径之一。了解黄曲霉毒素的生物合成是如何开始的以及在何处产生黄曲霉毒素对于控制真菌代谢物的合成和输出至关重要。在Linz实验室中进行的一项先前研究使用电泳迁移率变动分析(EMSA)和染色质免疫沉淀(ChIP)研究发现,bZIP转录因子AtfB与特定黄曲霉毒素和应激反应基因的启动子结合。一旦黄曲霉毒素基因表达开始,我们的实验室就表明,寄生曲霉可以在称为毒素的亚细胞细胞器中进行黄曲霉毒素的合成,区分开和输出。本论文致力于进一步表征黄曲霉毒素生物合成的转录调控和亚细胞定位的作用,特别是通过两个细胞靶点AtfB和Vps34。为了研究这两个靶标的功能,我们利用寄生虫曲霉中的新型内源性CRISPR / cas9样系统来下调AtfB和Vps34的功能。该系统是一种简单,快速且高效的共转换方法。每3个niaD +中有1个(编码硝酸还原酶[niaD]的基因为选择标记)的转化子表现出非典型表型,我们后来证明该表型与破坏构建体的存在和表达有关。 AtfB的破坏导致了低黄曲霉毒素产量,较低的分生孢子数和较低的孢子色素沉着的一致表型。下调黄曲霉毒素生物合成和应激反应的AtfB靶标表明,靶标基因的表达水平与观察到的破坏菌株表型一致。尤其重要的是,全球表达数据和基于计算机的网络分析还表明,AtfB网络已超出了霉菌毒素的生物合成和应激反应。有趣的是,与AtfB的破坏相比,Vps34的破坏导致了相反的表型。 Vps34转化体表现出高的黄曲霉毒素产量,高的分生孢子数量和高水平的孢子色素沉着。我们使用Vps34的生化抑制剂3-甲基腺嘌呤进行了独立实验。用3-甲基腺嘌呤处理寄生曲霉SU-1导致在诱导黄曲霉毒素的培养基中检测到的黄曲霉毒素水平增加了10倍。这种生化方法支持在Vps34破坏菌株中观察到高黄曲霉毒素水平的表型。我们进一步提出,Vps34负调控毒素向液泡的运输。 Vps34还积极调节出口途径,引导携带黄曲霉毒素的早期内体从细胞中输出。像AtfB和Vps34这样的真菌靶标可不同地调节黄曲霉毒素的生物合成,成为了解有助于真菌毒性的分子机制的有用工具。从公共卫生的角度来看,我们的研究目标是使用针对真菌特定细胞靶标的实用且可持续的天然抑制剂来阻断田间和储存中的毒素产生。

著录项

  • 作者

    Wee, Josephine.;

  • 作者单位

    Michigan State University.;

  • 授予单位 Michigan State University.;
  • 学科 Food science.;Microbiology.;Environmental science.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 209 p.
  • 总页数 209
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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