Insertional Mutagenesis of Candida auris using Agrobacterium tumefaciens

耐药性可以说是传染病中最紧迫的问题之一。可能会出现阻力 自然地，当生物体表现出天然的低水平抗生素敏感性时，或者在 接触抗生素。真菌对抗生素的发展提出了特殊的问题，因为它们的抗生素非常有限， 抗生素治疗的选择。不幸的是，许多有希望的对真菌有毒的先导化合物， 由于类似的真核细胞组织而对哺乳动物有毒。因此，抗真菌药物的发展 对真菌来说一直很困难耳念珠菌是一种新兴的真菌病原体， 对一线抗真菌治疗的天然耐药频率，而且还显示出发展 对所有已知的抗逃逸药物都有抵抗力这种真菌在世界各地的传播速度非常快， 死亡率（约30-80%，取决于国家）扩大了治疗系统性真菌病的已知问题： 真菌感染缺乏抗真菌选择。我们对抗真菌药物研究的了解大多是 来源于非致病性模式真菌的研究，如酿酒酵母， 不适合作为泛真菌模型。为了解决这个问题，我们将开发一种方法， 梭耳基因组，以揭示基因是潜在的抗真菌靶点，因为它们的功能是 对细胞存活至关重要。这项研究的主要目标是开发一种方法， 廉价地识别这些目标。为了实现这一目标，我们将开发一种插入突变 系统基于细菌病原体根癌农杆菌（Agrobacterium tumefaciens）对C.耳。第一个目标是 提高现有农杆菌转化效率，获得足够的转化子， 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 从占优势的非连接基因组DNA中回收插入位点片段的方法 背景最后，我们将对这些富集片段进行深度测序，以确定每个插入位点 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， 用于识别对生存至关重要的基因，因此，潜在的抗真菌靶点。

Drug resistance is arguably one of the most pressing problems in infectious disease. Resistance can occur naturally, where organisms exhbit natural low-level antibiotic susceptibility or it can occur spontaneously after exposure to antibiotics. Fungi pose special problems to antibiotic development because there are very limited antibiotic choices for treatment. Many promising lead compounds that are toxic to fungi unfortunately are toxic to mamals due to the similar eukaryoctic cellular organization. Consequently antifungal development has always been difficult for fungi. Candida auris is an emerging fungal pathogen that displays a high frequency of natural resistance to first line antifungal treatment, but also has been shown to develop resistance to all know antifugals. The extremely rapid spread of this fungus around the world and high mortality rate (~30-80% depending on country) has amplified known problems in treating systemic mycosis: the lack of antifungal choices for fungal infections. Most of what we know about antifungal drug research is derived from studies of non-pathogenic model fungi, such as Saccharomyces cerevisiae, which are unsuitable as pan-fungal models. To address this issue, we will develop a method that rapidly interrogates the C. auris genome to reveal genes that are potential antifungal targets by virtue of their function being essential to cell survival. 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 develop an insertional mutagenesis system based on the bacterial pathogen, Agrobacterium tumefaciens for C. auris. The first aim will be to improve the existing Agrobacterium tumefaciens 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 genes that are essential for survival, and therefore, potential antifungal targets.