Characterization of pathogen nutrient acquisition and transport systems required during UTI

项目摘要 尿路感染（UTI）是美国的一个重大负担， 诊所访问，每年花费35亿美元。尿路致病性大肠杆菌（UPEC）是致病微生物 对于目前用抗生素治疗的80%的无并发症的UTI病例， 显然，需要其他治疗。这些治疗对400万美国人来说尤其重要。 患有复发性UTI并接受长期抗生素治疗的女性，这反过来又会增加 抗生素耐药性目前在了解UPEC如何获得营养素方面存在重大差距 需要在宿主体内快速复制，以及促进生长的特定生长化合物的知识 成功的殖民泌尿道是一个苛刻的和营养有限的环境;因此，细菌 病原体必须调整其营养吸收和相应的代谢途径，以最好地利用现有的 资源与E.大肠杆菌在肠道中，膀胱中的UPEC被认为是利用氨基酸作为 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： 更好地理解UPEC生物学，以鼓励开发改进的UTI治疗方法。 我的中心假设是，在UTI期间需要特定的UPEC转运系统来促进代谢 适应宿主泌尿道环境并允许发生感染。这一假设将得到检验 通过进行两个特定的目标：1）描绘运输系统是至关重要的感染特异性的健身 人尿中生长的因素，以及2）确定在UTI期间作为健身因素的运输系统， vivo.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 依赖于在感染过程中被转运的底物。我们以前建造的， 确定并订购了本研究所需的转座子突变体。在第一个目标下，运输机 突变体将在人尿和营养丰富的培养基中生长，以比较并随后消除突变体 以鉴定那些仅在尿液中具有生长缺陷的患者。此外，基因 将比较表达谱，以确定必需营养素时发生的代谢变化 从环境中消失。在第二个目标下，建立了良好的上行性UTI的CBA/J小鼠模型。 将用于鉴定在体内UTI期间充当宿主特异性适应因子的转运系统（例如， 在UTI期间需要，但在人尿中生长不需要）。个人运输者的贡献将 通过利用qPCR的新型联合攻毒技术以无偏倚的方式进行评估和排名， 在类似突变体的小亚群中定量突变细菌的水平。表型分析将 在选择的转运突变体上进行，以阐明有助于体内适应性的作用机制 缺陷拟议的研究是重要的，因为它将提供洞察如何UPEC获取和利用 重要的营养素，允许成功引起UTI所需的代谢灵活性。

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.