Molecular Determinants of Intracellular Survival and Replication in Leishmania

说明(申请人提供)：铁和含铁的血红素是执行关键生物学功能的反应中的基本辅因子，但过量是有毒的。因此，这些营养素的可获得性受到严格控制。利什曼原虫在细胞内复制，不能合成血红素，因此必须从宿主细胞获取铁和血红素。直到最近，人们还不知道这是如何实现的。上一次赠款周期的工作确定了三种与铁和血红素摄取有关的膜蛋白，它们是亚马孙链霉菌细胞内生长和毒力所必需的：铁铁还原酶LFR1，亚铁转运蛋白LIT1，以及长期寻找的血红素转运蛋白LHR1。缺乏这些分子的寄生虫菌株的可获得性以及用于询问宿主细胞血红素-铁代谢的强大工具现在将使我们能够将这项工作带入下一个层次：研究寄生虫和宿主细胞之间的血红素-铁稳态途径之间的相互关系。了解这一过程是至关重要的，因为利什曼原虫寄生巨噬细胞，巨噬细胞在维持生物体的血红素-铁代谢中发挥关键作用。网状内皮巨噬细胞以惊人的速度从吞噬衰老的红细胞中回收铁。在哺乳动物中，大约5x106个红细胞或5x1015个血红素分子每秒在巨噬细胞吞噬酶体内降解，释放铁，通过严格调控的膜转运蛋白铁蛋白进行身体重新分配。我们将利用巨噬细胞中血红素-铁运输领域令人兴奋的最新进展：(1)阐明巨噬细胞的铁输出机制如何影响亚马逊利什曼原虫获取铁并在细胞内复制的能力；(2)确定巨噬细胞中血红素运输的动态如何影响亚马逊利什曼原虫对细胞内血红素的摄取；以及(3)阐明血红素作为亚马逊利什曼原虫铁源的重要性。我们的结果将有助于未来的药物开发，因为我们将确定亚马孙乳杆菌在哪里拦截巨噬细胞复杂的血红素-铁运输机制，并揭示血红素到达寄生虫的液泡是否代表寄生虫的主要铁源。重要的是，该项目还将推动我们对人类对利什曼病易感性的理解。缺铁是最常见的营养紊乱，识别新的宿主因素可以抵消寄生虫获取血红素-铁的机制，这对感染风险的评估具有重要影响。

DESCRIPTION (provided by applicant): Iron and iron-containing heme are essential cofactors in reactions that perform critical biological functions, but are toxic in excess. Hence, the availability of these nutrients is tightly controlled. Leishmania parasites replicate intracellulary and can't synthesize heme, so must acquire iron and heme from the host cell. Until recently, it was not known how this was achieved. Work from the previous grant cycle identified three membrane proteins involved in iron and heme uptake that are essential for the intracellular growth and virulence of L. amazonensis: The ferric iron reductase LFR1, the ferrous iron transporter LIT1, and the long- sought heme transporter, LHR1. The availability of parasite strains deficient in these molecules and powerful tools to interrogate host cell heme-iron metabolism will now allow us to take this work to the next level: An investigation of the functiona interrelationship between parasite and host cell pathways for heme-iron homeostasis. Understanding this process is critical because Leishmania parasitizes macrophages, cells that perform a key role in the maintenance of organismal heme-iron metabolism. Reticuloendothelial macrophages recycle iron from phagocytosed senescent red blood cells at a staggering rate. In mammals, about 5x106 red blood cells or 5x1015 heme molecules are degraded each second inside macrophage phagolysosomes, releasing iron for body redistribution through a tightly regulated membrane transporter, ferroportin. We will take advantage of exciting recent advances in the field of heme- iron traffic in macrophages to: (1) Elucidate how the iron export machinery of macrophages impacts the ability of Leishmania amazonensis to acquire iron and replicate intracellularly; (2) Determine how the dynamics of heme traffic in macrophages affects intracellular heme uptake by L. amazonensis; and (3) Elucidate the importance of heme as a source of iron for L. amazonensis. Our results will facilitate future drug development by defining where L. amazonensis intercepts the elaborate heme-iron traffic machinery of macrophages, and by revealing whether heme reaching parasitophorous vacuoles represents a major source of iron for the parasites. Importantly, this project will also propel forward our understanding of human susceptibility to leishmaniasis. Iron deficiency is the most prevalent nutritional disorder, and identification of novel host factors that counteract the parasite's machinery for heme-iron acquisition can have a significant impact in the assessment of infection risk.