Iron Acquisition by Mycobacterium tuberculosis Within Phagocytes

描述(由申请人提供)： 结核病(TB)的病理生理学与致病微生物结核分枝杆菌(M.TB)在人巨噬细胞内生长的能力有关。肺髓系树突状细胞是新近发现的肺部感染时结核分枝杆菌的储存库。结核分枝杆菌在巨噬细胞和树突状细胞中的代谢可能不同。铁(Fe)的获得对结核分枝杆菌的增长至关重要。在体内，大多数胞外铁与转铁蛋白(Tf)和乳铁蛋白(LF)螯合，减少了微生物对铁的获取。我们发现，人巨噬细胞中的结核分枝杆菌可以从Tf和Lf以及巨噬细胞的细胞质中获得铁。细胞外TF和LF对铁的摄取可能与铁进入细胞质有关。某些情况(如遗传性血色沉着症)会降低巨噬细胞内的铁，从而减少结核分枝杆菌铁的摄取。调节巨噬细胞铁代谢的其他因素(如铁蛋白、血红素加氧酶1(HO-1)、海普西丁)是否影响结核分枝杆菌铁的获取尚不清楚。同样未知的是，在树突状细胞中生长的结核分枝杆菌获取铁的能力是否与巨噬细胞中的不同。我们假设：1)结核分枝杆菌从巨噬细胞和树突状细胞的细胞内铁库(S)获得铁；2)结核分枝杆菌获取细胞外铁涉及到铁的初始移动；3)结核分枝杆菌铁摄取的动力学/途径因细胞外铁螯合物和所涉及的宿主细胞类型(巨噬细胞与树突状细胞)而异；4)调节巨噬细胞和/或树突状细胞胞浆铁的遗传或生理因素可供结核分枝杆菌利用；以及4)给予GA，扰乱结核分枝杆菌铁依赖的代谢，可能被证明是一种新的治疗方法。为了验证这些假设，我们将追求以下具体目标：1.描述和比较居住在人巨噬细胞和髓系树突状细胞中的结核分枝杆菌：A)从生理性细胞外螯合物(如Tf和LF)以及从巨噬细胞和树突状细胞的细胞质位置获取铁的能力；以及B)从细胞外螯合物获得铁在多大程度上涉及到初始Fe进入这些相同的细胞质位置。2.确定：A)结核分枝杆菌是否调节巨噬细胞铁的代谢，进而改变细菌对铁的获取和生长；以及B)巨噬细胞铁代谢的关键成分(如铁蛋白、HFe、HO-1、海普西丁)对结核分枝杆菌铁的获取和生长的作用。3.采用小鼠结核分枝杆菌感染模型，确定赤霉素A作为治疗药物对结核分枝杆菌的疗效。如果出现阳性结果，则进一步确定GA的抗结核活性的机制(S)。建议的研究使用人类巨噬细胞和树突状细胞以及完全毒力的结核分枝杆菌菌株来实现这些目标。采用的实验方法包括：测量59Fe摄取、siRNA、共聚焦显微镜、免疫印迹分析、RT-PCR和ELISA法，以及使用小鼠肺结核模型。对结核分枝杆菌铁代谢的深入了解可能会导致新的治疗方法，使退伍军人受益。 公共卫生相关性： 项目说明-与退伍军人事务部的任务相关：退伍军人患肺结核的风险很高。在治疗结核病方面，一个日益严重的问题是，引起结核病的微生物--结核分枝杆菌--对现有抗生素的抗药性越来越强。从受感染的宿主获取铁(Fe)的能力是结核分枝杆菌能够生长并因此引起感染所必需的。对结核分枝杆菌铁代谢的深入了解可能会导致开发新的方法来干扰有机体获得铁治疗的能力，这可能导致新类型抗生素的开发。这个应用程序将探索一种使用镓来实现这一目的的潜在机制。因此，对结核分枝杆菌在感染期间如何获得和使用铁的了解增加，可能会导致开发用于治疗结核病的新抗生素，这将对退伍军人大有裨益。

DESCRIPTION (provided by applicant): The pathophysiology of tuberculosis (TB) is linked to the ability of the causative organism, Mycobacterium tuberculosis (M.tb), to grow within human macrophages. Lung myeloid dendritic cells are a newly recognized reservoir of M.tb during lung infection. M.tb metabolism in macrophages and dendritic cells may differ. Iron (Fe) acquisition is critical for M.tb growth. In vivo, most extracellular Fe is chelated to transferrin (TF) and lactoferrin (LF), decreasing microbial access to Fe. We find that M.tb in human macrophages can acquire Fe from TF and LF, as well as the macrophage cytoplasm. Fe uptake from extracellular TF and LF may involve Fe moving into the cytoplasm. Some (e.g. hereditary hemochromatosis) conditions that lower intracellular Fe in macrophages, decrease M.tb Fe uptake. Whether other factors that regulate macrophage Fe metabolism (e.g. ferroportin, heme oxygenase 1 (HO-1), hepcidin) impact M.tb Fe acquisition is unknown. Similarly unknown is whether Fe acquisition by M.tb growing in dendritic cells is different from that occurring in macrophages. We hypothesize that: 1) M.tb acquires Fe from an intracellular Fe pool(s) in macrophages and dendritic cells; 2) acquisition of extracellular Fe by M.tb involves initial Fe movement into this pool; 3) the kinetics/route of M.tb Fe uptake varies with the extracellular Fe chelate and host cell type (macrophage vs. dendritic cell) involved; 4) genetic or physiological factors that modulate macrophage and/or dendritic cell cytoplasmic Fe alter Fe available for use by M.tb; and 4) administration of Ga, which disrupts M.tb Fe-dependent metabolism could prove to be a novel therapy for TB. In order to test these hypotheses we will pursue the following specific aims: 1. Delineate and compare the ability of M.tb residing within human macrophages and myeloid dendritic cells to: A) acquire Fe from physiologic extracellular chelates (e.g. TF and LF) and from cytoplasmic sites in the macrophage and dendritic cell; and B) to what extent Fe acquisition from extracellular chelates involves initial Fe movement into these same cytoplasmic sites. 2. Determine: A) if M.tb modulates macrophage Fe metabolism, which in turn alters Fe acquisition and growth by the bacteria; and B) the role of key components of macrophage Fe metabolism (e.g. ferroportin, HFE, HO-1, hepcidin) on M.tb Fe acquisition and growth. 3. Determine the efficacy of Ga as a therapeutic agent against M.tb using a murine model of M.tb infection. If positive results are seen, further ascertain the mechanism(s) of Ga's anti-tuberculous activity. The proposed studies use human macrophages and dendritic cells along with fully virulent M.tb strains to accomplish these aims. Experimental methods employed include: measuring uptake of 59Fe, siRNA, confocal microscopy, immunoblot analysis, RT-PCR, and ELISA and use of a murine model of pulmonary TB. Insight into M.tb Fe metabolism could lead to novel treatments that would benefit veterans. PUBLIC HEALTH RELEVANCE: Project Narrative - Relevance to VA Mission: Veterans are at high risk for pulmonary TB. There is a growing problem in treating tuberculosis in that the causative organism, Myocobacterium tuberculosis, is becoming increasingly resistant to available antibiotics. The ability to acquire iron (Fe) from the infected host is required for M. tuberculosis to be able to grow and therefore cause infection. Insight into M. tuberculosis Fe metabolism could lead to development of novel approaches to interfere with the organism's ability to acquire Fe treatments that could lead to the development of new kinds of antibiotics. This application will explore one potential mechanism of doing this using gallium. Thus, increased understanding of how M. tuberculosis acquires and uses Fe during infection could lead to the development of new antibiotics to be used in treating TB, something that would be of great benefit to veterans.