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.