Pyomelanin and Legionella pneumophila Infection

描述（申请人提供）：嗜肺炎军团菌（Lp）是军团病的病原体。它普遍存在于自然和人造水系中，经气溶胶接种后感染人类。在水生环境中，Lp以生物膜和原生动物细胞内寄生虫的形式存活；在肺中，Lp以肺泡巨噬细胞和上皮细胞内寄生虫的形式存活。铁对Lp在细胞外和细胞内的生长及其致病能力至关重要。二十年来，我的实验室一直致力于破译Lp导致疾病的机制，其中十五年，一直在Lp铁获取研究中发挥领导作用。先前，我们证明Lp表达铁载体（legiobactin）介导的铁摄取和feob介导的亚铁同化，并且这些都是肺部最佳感染所必需的。在筛选与legiobactin产生有关的基因的过程中，我们确定Lp分泌的棕色色素，一种源自均质酸的脓黑素（即hga -黑色素），赋予铁还原酶活性。根据这些数据，我们假设i)分泌的hga -黑色素通过提供亚铁进入细菌细胞来促进Lp铁的获取，ii)这种脓黑素是受感染肺内Lp生长所必需的。对这一假设的追求将是对hga -黑色素介导的铁还原是否是细菌铁摄取的（一般）机制的第一次检验，也是对细菌黑色素（Lp或其他）在动物疾病模型中的重要性的第一次直接评估。在本R21中，我们将利用纯化的hga -黑色素和特异性缺乏hga -黑色素的Lp突变体来i)确定分泌的脓黑素是否可以介导铁获取并促进低铁条件下的细菌生长，ii)确定脓黑素对感染Lp的小鼠肺部细菌生长、炎症和组织损伤的重要性。由于多种原因，拟议研究的影响是重大的。首先，它将增加我们对Lp的了解，这是全世界一个重要的公共卫生问题。其次，由于许多其他重要的病原体以及其他重要的环境微生物分泌HGA-melanin，这将阐明铁同化和细菌发病机制的新范式。第三，它将潜在地为疾病诊断、治疗和/或预防提供一种新型靶点。

DESCRIPTION (provided by applicant): Legionella neumophila (Lp) is the agent of Legionnaires' disease. It is ubiquitous in natural and man-made water systems, infecting humans after aerosol inoculation. In aquatic habitats, Lp survives in biofilms and as an intracellular parasite of protozoa, and in the lung, it flourishes as an intracellular parasite of alveolar macrophages and epithelia. Iron is vital to Lp growth in extra- and intracellular niches and its ability to cause disease. For twenty years, my laboratory has been involved in deciphering mechanisms by which Lp causes disease and, for fifteen of those years, has served a leadership role in the study of Lp iron acquisition. Previously, we documented that Lp expresses both siderophore (legiobactin)-mediated ferric iron uptake and FeoB-mediated ferrous iron assimilation and that each of these is required for optimal infection of the lung. In the course of screening for genes involved in legiobactin production, we determined that the secreted brown pigment of Lp, a pyomelanin derived from homogentisic acid (i.e., HGA-melanin), confers ferric reductase activity. In light of these data, we hypothesize i) that secreted HGA-melanin promotes Lp iron acquisition by providing ferrous iron for import into the bacterial cell and ii) that this pyomelanin is required for Lp growth within the infected lung. The pursuit of this hypothesis would be the first test of whether HGA-melanin mediated iron reduction is a (general) mechanism for bacterial iron uptake as well as the first direct assessment of the importance of a bacterial melanin, Lp or otherwise, in an animal model of disease. In this R21 , we will utilize purified HGA-melanin and Lp mutants specifically lacking HGA-melanin to i) determine whether or not secreted pyomelanin can mediate iron acquisition and promote bacterial growth under low-iron conditions, ii) determine the importance of pyomelanin for bacterial growth, inflammation, and tissue damage within Lp-infected mouse lungs. The impact of the proposed studies is significant for multiple reasons. First, it will increase our understanding of Lp, which is an important public health concern throughout the world. Second, since many other important pathogens as well as other important environmental microbes secrete HGA-melanin, it will elucidate new paradigms for both iron assimilation and bacterial pathogenesis. Third, it will potentially provide a new type of target for disease diagnosis, treatment, and/or prevention.