Molecular mechanisms of uropathogenesis

尿路发病的分子机制

• 批准号: 10443713
• 负责人: HARRY L. MOBLEY
• 金额: $ 50.58万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10443713
• 关键词: Acute Cystitis; Adherence; Affect; Affinity; Agar; Amino Acids; Amino Sugars; Antimicrobial Resistance; Applied Research; Area; Bacteremia; Bacteria; Bacterial Adhesins; Bacterial Infections; Basic Science; Biological Assay; Birth; Bladder; Bladder Calculi; Body Surface; Categories; Catheters; Colon; Communicable Diseases; Complex; Data; Defect; Deformity; Development; Enterobacteriaceae; Epithelial; Escherichia coli; Funding; Future; Genes; Goals; Grant; Growth; Hemolysin; Hospitals; Human; Individual; Infection; Iron; Kidney; Kidney Calculi; Kidney Diseases; Kinetics; Libraries; Life; Measures; Mediating; Metabolism; Mission; Molecular; Mus; Mutate; National Institute of Allergy and Infectious Disease; Newborn Infant; Nutrient; Obstruction; Organism; Outcome; Peptide Hydrolases; Peptides; Persons; Phenotype; Physicians; Population; Productivity; Proteus mirabilis; Public Health; Radiolabeled; Reflux; Reporting; Research; Rest; Siderophores; Site; Structural defect; Surface; Symptoms; System; Testing; Time; Toxin; Tube; United States; United States National Institutes of Health; Urease; Urethra; Urinary tract; Urinary tract infection; Urine; Urologic Diseases; Uropathogen; Uropathogenic E. coli; Virulence; Virulence Factors; Virulent; Visit; Vulnerable Populations; Woman; Work; acute pyelonephritis; antimicrobial; ascending urinary tract infection; base; catheter associated UTI; cell motility; economic impact; fitness; heme-binding protein; in vivo; meetings; member; metabolome; mouse model; mutant; novel; pathogen; prevent; renal scarring; small molecule; transcriptome; transcriptome sequencing; transposon sequencing; uptake

Uropathogenic bacteria must acquire nutrients present in urine or those released by an inflamed or damaged epithelium to successfully colonize the urinary tract. Our long term goal is to understand the contribution of each virulence factor and core gene that allows uropathogens to colonize, persist, and damage the urinary tract. Our overall objective is to identify and study essential transporters required by uropathogenic Escherichia coli (UPEC), a cause of uncomplicated urinary tract infection (UTI), and Proteus mirabilis, a cause of catheter- associated UTI, for growth and survival in the human urinary tract. Both E. coli and P. mirabilis are members of the Enterobacteriaceae, are motile, produce numerous and distinct fimbriae with which they mediate adherence to the uroepithelium, secrete hemolysins and proteases, and elaborate siderophores and heme-binding proteins to capture iron from the host. While we have previously studied all of these classes of virulence determinants, we now propose to focus on transport systems as critical functions for the survival of these two pathogens. In this proposal, we will advance the central hypothesis that uropathogenic E. coli and P. mirabilis differentially employ transport systems, unique to each species, to acquire peptides/amino acids and sugars, respectively, to establish fitness advantages in the urinary tract. We will test this hypothesis by carrying out the following specific aims: 1) Identify specific transport systems required for the development of urinary tract infection by uropathogenic Escherichia coli and Proteus mirabilis. 2) Determine the kinetic parameters of unique and common transporters that contribute to the virulence of uropathogenic Escherichia coli and Proteus mirabilis in the urinary tract. In the first aim, we will identify genes from among 643 identified transporter genes of E. coli and 386 identified transport genes of P. mirabilis, that when mutated result in a fitness defect in the mouse model of ascending UTI. We will use a Tn-seq screen of ordered transposon libraries of both species in the mouse model of UTI, expression data from in vivo RNA-seq transcriptomes, transurethral cochallenge of mice with clean deletion mutants vs the wild type strain, and urine metabolomes to select transporters for further study. In the second aim, prioritized transporters will be characterized for growth in filter-sterilized human urine, minimal medium containing the known or suspected substrate, and Biolog assays to confirm substrates and identify transporters that share the same substrate. Kinetics of substrate uptake [KT (affinity for substrate) and Vmax (rate of transport)] will be measured using radiolabeled substrates. Finally, a hierarchy of transporters will be established for UPEC and P. mirabilis. The expected outcome of conducting these aims will be to characterize key transport systems and their substrates of our two most troublesome uropathogens. The positive impact will include identification of novel targets of antimicrobial therapy.

尿道病细菌必须获得尿液中存在的营养或发炎或受损的营养素 上皮成功定居尿路。我们的长期目标是了解每个人的贡献 毒力因子和核心基因，允许尿道病毒定植，持续和损害尿路。我们的 总体目的是识别和研究肝癌大肠杆菌所需的必需转运蛋白 （UPEC），这是引起尿路感染（UTI）的原因，而Proteus mirabilis是导管 - 相关的UTI，用于人类尿路的生长和生存。大肠杆菌和奇异疟原虫都是 肠杆菌科有动力，产生众多且独特的fimbriae介导的粘附 到巨像，分泌血素和蛋白酶，并精致的铁载体和血红素结合蛋白 从宿主那里捕获铁。尽管我们以前已经研究了所有这些类别的毒力决定因素，但 现在，我们建议将运输系统作为这两种病原体生存的关键功能。在 该提议，我们将提出一个中心假设，即尿液发病大肠杆菌和奇异疟原虫差异化 使用每个物种独有的运输系统，分别获得肽/氨基酸和糖的收购 在尿路中建立健身优势。我们将通过执行以下特定来检验该假设 目的：1）确定通过 尿道病性大肠杆菌和蛋白酶。 2）确定独特和 有助于尿路疾病大肠杆菌和蛋白酶mirabilis毒力的常见转运蛋白 尿路。在第一个目标中，我们将在643个鉴定的大肠杆菌和大肠杆菌的转运蛋白基因中识别基因 386鉴定出奇异假单胞菌的转运基因，当突变导致小鼠模型中的适应性缺陷时 上升的UTI。我们将使用鼠标模型中这两个物种有序的转座库的TN-Seq屏幕 UTI，来自体内RNA-seq转录组的表达数据，清洁小鼠的尿道胸骨。 缺失突变体与野生型菌株和尿液代谢组选择转运蛋白进行进一步研究。在 第二个目的，优先转运蛋白的特征是过滤过滤的人类尿液生长，最小 包含已知或怀疑底物的培养基，以及确认底物的生物学测定法 共享相同基材的转运蛋白。底物摄取的动力学[KT（基板的亲和力）和VMAX（速率） 传输）] [使用放射性标记的底物测量。最后，运输商的层次结构将是 为UPEC和P. mirabilis建立。进行这些目标的预期结果是表征 我们两个最麻烦的尿道病的关键运输系统及其底物。积极影响将 包括鉴定抗菌治疗的新靶标。