Insertional Mutagenesis of Candida auris using Agrobacterium tumefaciens

使用根癌农杆菌插入诱变耳念珠菌

• 批准号: 9808697
• 负责人: BRIAN WICKES
• 金额: $ 23.06万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808697
• 关键词: Address; Ammonium; Antibiotic susceptibility; Antibiotics; Antifungal Agents; Azoles; Candida; Candidate Disease Gene; Cell Survival; Cells; Centers for Disease Control and Prevention (U.S.); Characteristics; Clinic; Clinical; Communicable Diseases; Country; Data; Development; Disinfection; Drug Targeting; Drug resistance; Essential Genes; Exposure to; Formulation; Frequencies; Fungi Model; Furniture; Future; Generations; Genes; Genome; Genomic DNA; Genomics; Goals; Human; Infection; Insertional Mutagenesis; Lead; Libraries; Mammalian Cell; Maps; Mattresses; Medical; Methods; Modeling; Modification; Morbidity - disease rate; Multi-Drug Resistance; Mycoses; Natural Products; Natural Resistance; Nature; Organism; Patients; Pharmaceutical Preparations; Poison; Probability; Public Health; Refractory; Reporting; Research; Resistance; Resistance development; Rhizobium radiobacter; Risk; Saccharomyces cerevisiae; Site; Sodium Chloride; Speed; Structure; System; Testing; Untranslated RNA; base; cell transformation; deep sequencing; density; drug candidate; drug discovery; exhaust; fungus; improved; knockout gene; mortality; next generation; next generation sequencing; novel sequencing technology; novel strategies; pathogenic bacteria; pathogenic fungus; rapid technique; response; screening; small molecule libraries; transmission process; treatment choice

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.

耐药性可以说是传染病中最紧迫的问题之一。可以发生阻力 自然，有机体驱除天然低级抗生素敏感性，或者可以自发发生 暴露于抗生素。真菌为抗生素开发带来特殊问题，因为有非常有限的 治疗的抗生素选择。不幸的是，许多对真菌有毒的有前途的铅化合物是 由于类似的真核细胞组织，对妈妈有毒。因此，抗真菌发育 真菌一直很困难。念珠菌是一种新兴的真菌病原体，显示出高度 对第一线抗真菌治疗的自然电阻频率，但也已证明会发展 对所有人都知道抗药性。这种真菌在世界各地的迅速传播和高 死亡率（约30-80％取决于国家）在治疗全身膜细胞增多方面的已知问题： 缺乏真菌感染的抗真菌选择。我们对抗真菌药物研究的了解的大部分是 源自非致病模型真菌的研究，例如酿酒酵母，是 不适合作为泛膜模型。为了解决这个问题，我们将开发一种迅速询问的方法 C. auris基因组以揭示其功能的基因，这些基因是潜在的抗真菌靶标 对细胞存活至关重要。这项研究的主要目的是开发一种快速和 廉价地确定这些目标。为了实现这一目标，我们将开发插入诱变 基于细菌病原体的系统，用于奥里斯梭菌的农杆菌。第一个目标是 提高现有的农杆菌转化效率，以产生足够的转化子 产生饱和的插入诱变图。接下来，我们将开发基于捕获的探针的丰富 从主要的非结基因组DNA中恢复插入位点片段的方法 背景。最后，我们将对这些丰富的片段进行深入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 用于鉴定生存至关重要的基因，因此是潜在的抗真菌靶标。