A Novel Metabolic Pathway Regulates Urinary Tract Infections in the Bladder

项目3：项目摘要/摘要 尿路感染是世界上最常见的泌尿生殖系统异常，累及多个器官。 尿路包括尿路、膀胱、前列腺、输尿管和肾脏。以确定为尿路感染， 细菌必须获得营养才能进行持续复制。这些营养素来自尿液。 和尿路上皮细胞。铁是细菌的“贵金属”，因为新陈代谢过程包括 能量生产和细胞分裂需要每个细菌大约10万个铁原子。然而，这是最困难的 由于铁的常见形式，称为铁，不溶于水(KSP=10-12M)，因此无法获得营养。革兰氏阴性 生物体已经设计出了即使在这些低浓度消失的情况下也能获得铁的方法。他们这样做是通过 一系列被称为铁载体的小分子的产生，每个小分子对铁的亲和力都达到了天文数字。 一种称为肠球蛋白的铁载体是最普遍的。然而，泌尿生殖道的上皮细胞 认识到这种威胁，并迅速大量产生一种名为NGAL(Lcn2)的蛋白质。NGAL捕获 ENTER：铁，防止其铁接触细菌。我们发现尿路上皮和尿路通向 肾脏表达NGAL。虽然我们的大部分研究都集中在NGAL：ENT：Fe上，但肯定还有其他选择 营养途径和哺乳动物的另类防御。我们认为细菌的血红素转运体也会偷走 我们的铁和上皮细胞反过来捕捉和代谢血红素。采用一种新的方法来分析 “快照”的新生RNA，我们发现，膀胱尿路和集合管表达血红素捕获， 新陈代谢，以及铁的固存和运输蛋白，它们与细菌竞争血红素。最耐人寻味 我们的发现是，尿路上皮“血红素机器”在感染后被激活并释放出一种副产品--血红素 新陈代谢，称为一氧化碳，一种抑菌剂。此外，血红素机是核心综合体 昼夜节律时钟，由CO调节。在这里，我们测试我们假设的基本原理。我们小心翼翼 记录血红素和铁在尿路上皮中的运输和代谢，以测试尿路上皮和 细菌为争夺血红素而竞争。我们建议将这些机制重新用于“营养免疫”。 防御“来自日常防御的裂解的红细胞，每天横穿膀胱。为了调查这些 机制，我们发明了RNA分离的新方法，检测细菌反应的成像工具， 检测和捕获体内CO的方法、新的小鼠Ko‘s和从我们的UTI患者中分离的细菌 携带铁载体和血红素途径的突变。我们正在与一位领先的微生物学家合作 (Uhlemann)，动物遗传学家(Mendelsohn)，泌尿系感染专家(Mysorekar)，以及血红素方面的领先科学家 生物学(哈姆扎)。总之，我们的研究表明，尿路上皮不是一种被动的屏障，而是一种 代谢活跃的细胞层，使用铁生物来解毒血尿，防御尿路感染。

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.