Characterization of pathogen nutrient acquisition and transport systems required during UTI

项目摘要 尿路感染(UTI)在美国是一个沉重的负担，造成了1100多万人 诊所就诊，每年花费35亿美元。尿路致病性大肠杆菌(UPEC)是引起尿路感染的病原菌 对于目前使用抗生素治疗的80%不复杂的尿路感染病例；它正在变得越来越多 显然，还需要其他治疗方法。这些治疗方法对400万美国人来说尤其重要。 患有反复尿路感染并接受长期抗生素治疗的女性，这反过来又会加剧 抗生素耐药性。目前，对UPEC如何获得营养物质的理解存在很大差距 需要在宿主内快速复制，以及促进 成功的殖民统治。尿路是一个严酷和营养受限的环境；因此，细菌 病原体必须调整他们的营养吸收和相应的代谢途径，以最大限度地利用可用的 资源。与肠道中的大肠杆菌不同，膀胱中的UPEC被认为利用氨基酸作为一种 主要碳源。在初步数据的指导下，这项拟议的研究将朝着以下长期目标努力 更好地了解UPEC生物学，以鼓励开发改进的尿路感染治疗方法。 我的中心假设是，在尿路感染期间需要特定的UPEC转运系统来促进新陈代谢 适应宿主的尿路环境，并允许发生感染。这一假设将得到检验。 通过实施两个具体目标：1)描述对感染特异性健康至关重要的运输系统 人类尿液增长的因素，以及2)确定在尿路感染期间充当健康因素的运输系统 活着。这项拟议的研究将表征关键的UPEC运输系统，并确定代谢途径 它们依赖于在感染过程中运输的底物。我们之前已经建造了， 鉴定并订购本研究所需的转座子突变体。在第一个目标下，运输机 突变株将在人类尿液和营养丰富的培养基中培养，以比较并随后消除突变株。 用全身性生长缺陷来识别那些只有尿液生长缺陷的人。此外，基因 将比较表达谱，以确定当必要的营养物质 从环境中被淘汰了。在第二个目标下，已建立的CBA/J小鼠尿路感染上升模型 将被用来识别在体内UTI期间充当宿主特定的健康因素的运输系统(例如， 在尿路感染过程中需要，但在人体尿液生长时不需要)。个别运输商的贡献将 通过利用qPCR的新的共同挑战技术以公正的方式进行评估和排名 量化相似突变的小亚群中突变细菌的水平。表型分析将是 在选定的运输突变体上进行实验，以阐明有助于体内适应性的作用机制 缺陷。拟议的研究具有重要意义，因为它将为UPEC如何获得和利用提供洞察 重要的营养物质，允许新陈代谢的灵活性，成功地引起尿路感染。

Project Abstract Urinary tract infection (UTI) represents a substantial burden in the United States generating over 11 million clinic visits and costing $3.5 billion annually. Uropathogenic Escherichia coli (UPEC) is the causative organism for 80% of uncomplicated UTI cases that are currently treated with antibiotics; it is becoming increasingly evident that other treatments are needed. These treatments will be especially important for the 4 million U.S. women who suffer recurrent UTI and are given long term antibiotic regimens, which in turn fuels increasing antibiotic resistance. There is currently a major gap in the understanding of how UPEC obtain the nutrients needed to rapidly replicate inside the host, as well as knowledge of specific growth compounds that promote successful colonization. The urinary tract is a harsh and nutrient-restricted environment; therefore, bacterial pathogens must adapt their nutrient uptake and corresponding metabolic pathways to best utilize available resources. In contrast to E. coli in the intestinal tract, UPEC in the bladder is thought to utilize amino acids as a primary carbon source. Guided by preliminary data, this proposed study will work toward the long-term goal of achieving a better understanding of UPEC biology to encourage the development of improved UTI treatments. My central hypothesis is that specific UPEC transport systems are required during UTI to facilitate metabolic adaptation to the host urinary tract environment and allow for infection to occur. This hypothesis will be tested by conducting two Specific Aims: 1) delineate transport systems that are crucial infection-specific fitness factors for growth in human urine, and 2) identify transport systems that serve as fitness factors during UTI in vivo. This proposed study will characterize critical UPEC transport systems and identify metabolic pathways that are dependent on the substrate being transported during infection. We have previously constructed, identified, and ordered the required transposon mutants needed for this study. Under the first aim, transporter mutants will be grown in human urine and nutrient-rich media to compare, and subsequently eliminate, mutants with generalized growth defects to identify those with only growth defects in urine. Additionally, gene expression profiles will be compared to identify the metabolic shifts that occur when essential nutrients are eliminated from the milieu. Under the second aim, the well-established CBA/J murine model of ascending UTI will be utilized to identify transport systems that serve as host-specific fitness factors during UTI in vivo (e.g., required during UTI but not required for growth in human urine). The contribution of individual transporters will be assessed and ranked in an unbiased manner through a novel co-challenge technique utilizing qPCR to quantify levels of mutant bacteria among small subpopulations of similar mutants. Phenotypic assays will be performed on select transport mutants to elucidate the mechanisms of action contributing to in vivo fitness defects. The proposed research is significant because it will provide insight into how UPEC acquire and utilize vital nutrients that allows for the metabolic flexibility needed to successfully cause UTI.