Characterization of pathogen nutrient acquisition and transport systems required during UTI

UTI 期间所需的病原体营养获取和运输系统的特征

• 批准号: 10064959
• 负责人: Allyson Shea
• 金额: $ 6.53万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 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 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064959
• 关键词: Address; Affect; Amino Acid Transporter; Amino Acids; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Arginine; Bacteria; Biological Assay; Biology; Bladder; Carbon; Clinic Visits; Communicable Diseases; Consumption; Data; Databases; Defect; Development; Disaccharides; Energy-Generating Resources; Environment; Escherichia coli; Expression Profiling; Financial Hardship; Gene Expression; Genes; Glycolysis; Goals; Growth; Growth Factor; Healthcare Systems; Host Defense Mechanism; Hour; Human; Individual; Infection; Intestines; Iron; Isoleucine; Knock-out; Knowledge; Libraries; Metabolic; Metabolic Pathway; Metabolism; Methionine; Modeling; Mus; Nutrient; Organism; Outcome; Pathway interactions; Patients; Peptides; Phenotype; Prevalence; Preventive Medicine; Process; Recurrence; Recurrent disease; Regimen; Research; Resources; Ribose; Role; Source; System; Techniques; Testing; Therapeutic; United States; Uracil; Urethra; Urinary tract; Urinary tract infection; Urine; Uropathogenic E. coli; Validation; Valine; Virulence Factors; Woman; Work; antimicrobial; ascending urinary tract infection; cost; experience; fitness; fitness test; flexibility; follow-up; gastrointestinal; improved; in vivo; insight; metabolomics; mouse model; mutant; novel; novel therapeutics; pathogen; pathogenic bacteria; receptor; sugar; transcriptome sequencing; transposon sequencing; treatment strategy; uptake

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.

项目摘要 尿路感染（UTI）代表着美国的重大负担 诊所就诊，每年耗资35亿美元。肝病大肠杆菌（UPEC）是病因 对于目前用抗生素治疗的简单的UTI病例中有80％；它变得越来越 显然需要其他治疗。这些治疗对400万美国尤其重要 遭受经常性UTI并获得长期抗生素方案的妇女，这反过来燃料增加 抗生素抗性。目前，了解UPEC如何获得营养的差距很大 需要快速复制主机内部，以及对促进的特定增长化合物的了解 成功的殖民化。尿路是一个苛刻而营养的限制环境。因此，细菌 病原体必须调整其营养摄取和相应的代谢途径才能最佳利用可用的 资源。与肠道中的大肠杆菌相反，膀胱中的UPEC被认为是利用氨基酸作为一种 原始碳源。在初步数据的指导下，这项拟议的研究将致力于长期目标 对UPEC生物学有了更好的了解，以鼓励开发改进的UTI治疗方法。 我的中心假设是，UTI期间需要特定的UPEC运输系统来促进代谢 适应宿主尿路环境，并允许发生感染。该假设将进行检验 通过执行两个具体目标：1）描述至关重要的感染特异性适应性的运输系统 人类尿液生长的因素，以及2）确定在UTI期间作为适应性因素的运输系统 体内。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 取决于在感染过程中运输的底物。我们以前已经构建了 确定并订购了本研究所需的所需的转座子突变体。在第一个目标下，运输蛋白 突变体将在人类尿液和营养丰富的培养基中生长，以比较，然后消除突变体 具有普遍的生长缺陷，以识别尿液中只有生长缺陷的人。另外，基因 将比较表达曲线，以识别当必需营养物质时发生的代谢转移 从环境中淘汰。在第二个目标下，已建立了良好的CBA/J鼠升级uti模型 将用于识别在体内UTI期间用作宿主特异性适应性因素的运输系统（例如， 在UTI期间需要，但不需要人类尿液生长）。单个运输者的贡献将 通过利用QPCR到的新型共同挑战技术以公正的方式评估和排名 在类似突变体的小亚群中量化突变细菌的水平。表型测定将是 在精选的传输突变体上进行，以阐明有助于体内适应性的作用机制 缺陷。拟议的研究很重要，因为它将提供有关UPEC如何获取和利用的见解 重要的营养素允许成功引起UTI所需的代谢灵活性。