A Novel Metabolic Pathway Regulates Urinary Tract Infections in the Bladder

项目3：项目总结/摘要 尿路感染是世界范围内最常见的泌尿生殖系统异常，影响患者的多个器官。 泌尿道，包括尿道、膀胱、前列腺、输尿管和肾脏。为了确定尿路感染， 细菌必须获得营养以便进行连续复制。这些营养素来自尿液 和尿道上皮细胞。铁是细菌的“贵金属”，因为代谢过程包括 能量生产和细胞分裂需要每个细菌约100，000个铁原子。然而，这是最困难的 因为常见的形式，称为三价铁，不溶于水（Ksp=10- 12 M）。兰阴性 有机体已经设计出即使在这些消失的低浓度下也能获得铁的方法。他们通过 一系列被称为铁载体的小分子的产生，每一个都对铁有着天文般的亲和力。 一种称为肠螯铁蛋白的铁载体是最普遍的。然而，泌尿生殖道的上皮细胞 认识到这一威胁，并迅速产生大量称为NGAL（Lcn 2）的蛋白质。NGAL捕获 Ent：铁，并防止其铁到达细菌。我们发现，尿道和上至 肾脏表达NGAL。虽然我们的大多数研究都集中在NGAL：Ent：Fe，但必须有替代品。 营养途径和哺乳动物的替代防御。我们认为细菌血红素转运蛋白也会窃取 我们的铁和上皮细胞反过来捕获并代谢血红素。通过采用新的方法来分析 通过对新生RNA的“快照”，我们发现膀胱尿路上皮和集合管表达血红素捕获，血红素结合， 代谢，铁螯合和运输蛋白，与细菌竞争血红素。最有趣 我们的发现是，在感染时，尿路上皮的“血红素机器”被激活并释放血红素的副产品 代谢，称为一氧化碳，抑菌剂。而且，血红素机器是核心复合体 在这里，我们测试我们的假设的基本原则。我们仔细 记录血红素和铁的运输和代谢在泌尿系统，以测试的概念，泌尿系统和 细菌争夺血红素。我们认为这些机制被重新用于“营养免疫”， 防御”是从每天对穿过膀胱的红细胞溶解的日常防御而来的。调查这些 我们发明了新的RNA分离方法，检测细菌反应的成像工具， 一种检测和捕获体内CO、新型小鼠KO和从我们的UTI患者中分离的细菌的方法 携带铁载体和血红素途径的突变我们正与一位顶尖的微生物学家合作 （Uhlemann），动物遗传学家（Mendelsohn），UTI专家（Mysorekar）和血红素领域的领先科学家 生物学（Hamza）。总之，我们的研究表明，而不是一个被动的屏障，尿路上皮是一个 代谢活跃的细胞层，利用铁生物学来解毒血尿和保护自己免受UTI。

PROJECT 3: PROJECT SUMMARY/ABSTRACT Urinary tract infections are the most common urogenital abnormality worldwide affecting multiple organs of the urinary tract including the urethra, bladder, prostate, ureter, and kidney. To establish a urinary tract infection, bacteria must obtain nutrients in order to undergo continuous replication. These nutrients derive from the urine and the epithelia of the urinary tract. Iron is a “precious metal” for bacteria because metabolic processes including energy production and cell division require ~100,000 atoms of iron per bacterium. Yet it is the most difficult nutrient to obtain because the common form, called ferric iron, is insoluble in water (Ksp=10-12M). Gram-negative organisms have devised methods to obtain iron even with these vanishing low concentrations. They do this by the production of a series of small molecules known as siderophores, each with astronomical affinity for iron. One type of siderophore, called Enterochelin is the most prevalent. Yet, the epithelial cells of the urogenital tract recognize this threat and rapidly produce a protein called NGAL (Lcn2) in great abundance. NGAL captures Ent:Fe and prevents its iron from reaching bacteria. We discovered that the urothelium and the tracts up into the kidney expresses NGAL. While most of our studies have focused on NGAL:Ent:Fe, there must be alternative nutrient pathways and alternative mammalian defenses. We propose that bacterial heme transporters also steal our iron and that epithelia in turn capture and metabolize heme. By adapting a novel method to analyze “snapshots” of nascent RNA, we found that bladder urothelia and collecting ducts express heme capture, heme metabolism, and iron sequestration and transport proteins, which compete with bacteria for heme. Most intriguing is our finding that upon infection, the urothelial “heme machine” is activated and releases a byproduct of heme metabolism, called Carbon Monoxide, a bacteriostatic agent. Moreover, the heme machine is the core complex of the Circadian Clock, which is regulated by CO. Here we test the basic tenets of our hypothesis. We carefully document heme and iron transport and metabolism in the urothelium to test the notion of that the urothelium and bacteria compete for heme. We suggest that these mechanisms are re-purposed for “nutritional immune defense” from the daily defense against the lysis of RBC that traverse the bladder each day. To investigate these mechanisms, we have invented novel methods of RNA isolation, imaging tools to detect bacterial responses, a method to detect and capture CO in vivo, novel mouse ko’s, and bacteria isolated from our patients with UTI’s carrying mutations in siderophore and heme pathways. We are working with a leading microbiologist (Uhlemann), animal geneticist (Mendelsohn), UTI specialist (Mysorekar) and the leading scientist in heme biology (Hamza). Taken together, our studies demonstrate that rather than a passive barrier, the urothelium is a metabolically active cell layer, that uses iron biology to detoxify hematuria and defend itself from UTI.