A modified recombineering protocol for the genetic manipulation of gene clusters in Aspergillus fumigatus.
A modified recombineering protocol for the genetic manipulation of gene clusters in Aspergillus fumigatus.
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DOI：
10.1371/journal.pone.0111875
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Bignell E
中科院分区：
文献类型：
--
作者：
Alcazar-Fuoli L;Cairns T;Lopez JF;Zonja B;Pérez S;Barceló D;Igarashi Y;Bowyer P;Bignell E
对真菌基因组结构的基因组分析揭示了物理上相连的基因组的存在，称为基因簇，其中编码基因产物的集体功能服务于共同的生物合成目的。在人类和植物的多种真菌病原体中，基因簇已被证明编码次生代谢物的生物合成途径，包括致病所需的代谢物。在人类的主要霉菌烟曲霉菌中，在小鼠感染的早期阶段，相对于实验室培养的等价物，多个协同上调的基因簇被鉴定为具有高转录丰度。这项研究的目的是开发和优化一种操纵基因簇结构的方法学，从而提供评估它们与真菌致病性的相关性的手段。为此，我们采用了一种重组工程方法，利用Lambda噬菌体介导的细菌DNA重组，产生基因簇缺失盒。通过利用已有的烟曲霉菌基因组克隆的细菌人工染色体(BAC)文库，我们能够在野生型和高度重组的烟曲霉菌分离株中实现端粒和端粒远端染色体位置上的单个或多个簇内基因替换事件。然后，我们应用该方法解决了产生杀线虫次级代谢物Pseurotin A的基因簇的边界，并研究了这种次级代谢物在昆虫和哺乳动物对烟曲霉菌挑战的反应中的作用。
Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.
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