Pan-Fungal Essential Gene Discovery for Antifungal Targeting

 描述（由申请人提供）：我们所知道的关于抗真菌药物研究的大部分内容来自非致病性模式真菌的研究，例如酿酒酵母，其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物。或者，由于成本和研究投资缺乏市场回报，物种特异性抗真菌药物的开发是不现实的。基因组序列的快速完成，以及对这一庞大且不断增长的数据量的不断发展的生物信息学操作，已经使抗真菌开发的新方法成为可能，这种方法现在可以从药物靶点开始，并向后进行药物开发，这种方法被称为“理性设计”。这种策略有一个重要的要求;需要生物学上的基本目标来设计药物，以便药物对病原体是致命的。由于这一要求，本研究的主要目标将是开发一种快速和廉价地识别这些目标的方法。为了实现这一目标，我们将利用两种分类学上不同的酵母，新型隐球菌（一种担子菌）和白色念珠菌（一种子囊菌），它们具有根癌农杆菌（AT）转化系统。第一个目标将是提高现有的AT转化效率，以产生足够的转化体来产生饱和插入诱变图谱。我们接下来将开发一种基于捕获探针的富集方法，用于从占主导地位的非连接基因组DNA背景中回收插入位点片段。最后，我们将对这些富集片段进行深度测序，以识别每个插入位点及其相邻的侧翼基因组DNA，并最终组装一个高密度插入图谱，用于识别哪些基因是必需的。这项建议将使后续研究的未来长期目标将是确定所有真菌都具有的一组核心必需基因。这组核心基因可以作为广谱抗真菌药物开发的目标群体。

 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.