Pan-Fungal Essential Gene Discovery for Antifungal Targeting

 描述(申请人提供)：我们对抗真菌药物研究的大部分了解来自于对非致病模式真菌的研究，例如酿酒酵母，这些真菌可以很容易地插入高通量筛选平台，但不适合作为泛真菌替代品。或者，由于成本和研究投资缺乏市场回报，针对特定物种的抗真菌开发是不现实的。基因组序列的快速完成，以及对这些海量和不断增长的数据的不断进化的生物信息学操作，使抗真菌开发的新方法成为可能，现在可以从药物靶标开始，向后工作到药物开发--一种被称为“合理设计”的方法。这一策略有一个重要的要求：需要生物上基本的靶点来设计药物，使药物对病原体具有致命性。由于这一要求，这项研究的主要目标将是开发一种快速和廉价地确定这些目标的方法。为了实现这一目标，我们将利用两种分类上不同的酵母，新生隐球菌(担子菌)和白色念珠菌(子囊菌)，它们具有工作的根癌农杆菌(AT)转化系统。第一个目标是提高现有的AT转化效率，以产生足够的转化子来产生饱和的插入突变图谱。接下来，我们将开发一种基于捕获-探针的富集法，用于从占主导地位的非连接基因组DNA背景中恢复插入位点片段。最后，我们将对这些丰富的片段进行深度测序，以确定每个插入位点及其邻近的侧翼基因组DNA，并最终组装出高密度插入图谱，用于识别哪些基因是必需的。这项提议将使后续研究能够实现的未来长期目标将是识别所有真菌都拥有的一组核心必需基因。然后，这组核心基因可以作为广谱抗真菌开发的目标群体。

 DESCRIPTION (provided by applicant): Most of what we know about antifungal drug research is derived from studies of nonpathogenic model fungi, such as Saccharomyces cerevisiae, which can easily be inserted into high throughput screening platforms, but are unsuitable as pan-fungal surrogates. Alternatively, species-specific antifungal development is unrealistic due to cost and lack of market return on research investment. The rapid completion of genome sequences, and continually evolving bioinformatic manipulation of this vast and growing amount of data, has enabled new approaches to antifungal development that can now start with drug targets, and work backwards towards drug development-a method referred to as "rational design". This strategy has one important requirement; the need for biologically essential targets to design drugs against so that the drug is lethal to the pathogen. Because of this requirement, the major objective of this study will be to develop a way to rapidly and inexpensively identify these targets. To accomplish this goal, we will utilize two taxonomically diverged yeasts, Cryptococcus neoformans (a basidiomycete) and Candida albicans (an ascomycete) that have working Agrobacterium tumefaciens (AT) transformation systems. The first aim will be to improve the existing AT transformation efficiency to yield enough transformants to produce a saturated insertional mutagenesis map. We will next develop a capture-probe based enrichment method for recovering insertion site fragments from the predominating non-junctional genomic DNA background. Finally, we will apply deep sequencing to these enriched fragments to identify each insertion site and its neighboring flanking genomic DNA, and ultimately assemble a high density insertion map that will be used to identify which genes are essential. The future long-term goal that this proposal will enable with subsequent studies will be the identification of a core set of essential genes that all fungi have. This core set of genes can then serve as a target group for broad spectrum antifungal development.