Insertional Mutagenesis of Candida auris using Agrobacterium tumefaciens

抗药性可以说是传染病中最紧迫的问题之一。抗药性可能会发生 自然地，当生物体表现出天然的低水平抗生素敏感性时，或者它可以在 接触抗生素。真菌给抗生素的发展带来了特殊的问题，因为 用于治疗的抗生素选择。不幸的是，许多对真菌有毒的有希望的先导化合物 由于类似的真核细胞组织，对哺乳动物有毒。因此，抗真菌的发展 对于真菌来说一直都很难。金黄色念珠菌是一种新出现的真菌病原菌，表现出高度的 自然耐药的频率一线抗真菌治疗，但也已显示出发展 抵抗所有已知的抗逃逸药物。这种真菌在世界各地的传播速度极快，而且 死亡率(约30%-80%取决于国家)放大了系统性真菌病治疗中的已知问题： 缺乏针对真菌感染的抗真菌选择。我们所知道的关于抗真菌药物研究的大部分是 源自对非致病模式真菌的研究，如酿酒酵母，它们是 不适合作为泛真菌模型。为了解决这个问题，我们将开发一种快速审问 金黄色葡萄球菌基因组揭示了潜在的抗真菌靶点基因，其功能是 对细胞生存至关重要。这项研究的主要目标将是开发一种快速和 以低廉的成本确定这些目标。为了实现这一目标，我们将开发一种插入突变 以根癌农杆菌为基础的系统。第一个目标将是 提高现有农杆菌的转化效率，以获得足够的转化子 制作饱和插入突变图谱。接下来，我们将开发一种基于捕获-探测的浓缩 从占主导地位的非连接基因组DNA中恢复插入位点片段的方法 背景资料。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 和它相邻的侧翼基因组DNA，并最终组装成一个高密度插入图谱 用于识别对生存至关重要的基因，从而识别潜在的抗真菌靶点。

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.