Genetic Dissection of Mycobacterial Pathogenesis During Eicosanoid-Mediated Immunity

摘要 结核分枝杆菌(Mtb)是世界上最成功的病原体之一，由于其长期存在， 与人类的古老伙伴关系。尽管几十年来致力于预防和治疗，但结核分枝杆菌是 2019年由任何单一传染病病原体导致的头号死因。分枝杆菌的重要调节因子 感染是二十烷基类化合物：宿主产生的脂质信号分子，可以推动炎症和抗炎 炎症信号级联。使用斑马鱼-海洋分枝杆菌感染模型 分枝杆菌致病机制的重要方面，即特定的促炎和抗炎二十烷类化合物的平衡 被发现对控制感染至关重要。二十烷基类化合物合成中断，通过突变 白三烯A4水解酶缺陷(LTA4H-/-)，导致肉芽肿结构改变。肉芽肿为中心 结核分枝杆菌感染的特征，是免疫细胞的聚集，协调以限制细菌的生长。这是 寄主-病原体相互作用的主要场所。虽然肉芽肿是肺结核的关键部分 控制肉芽肿生物学的致病机制、宿主-病原体信号程序仍然难以捉摸。我们缺少的是 对肉芽肿的形成和信号的理解是由于研究肉芽肿的困难，因为 这种复杂的细胞组织只能在体内形成和观察到。斑马鱼--海洋分枝杆菌 模型，具有保守的毒力基因座和寄主-病原菌遗传程序，用于研究沙门氏菌的遗传学 分枝杆菌感染和肉芽肿结构。这项提议将在斑马鱼和斑马鱼中使用遗传方法- 海洋分枝杆菌肉芽肿模型和小鼠结核分枝杆菌感染模型检验假设有关键 二十烷类化合物介导的免疫和细菌反应的差异是总体上的组成部分 肉芽肿生物学。为此，第一张分枝杆菌肉芽肿的单细胞图谱，在实验中 将野生型和LTA4H-/-斑马鱼肉芽肿细胞组装成易于处理的系统。由此得出的结论是 DataSet提供了以下方法来询问宿主二十烷类信号对肉芽肿的影响 结构和分枝杆菌反应。目标1将定义以前未定义的人群的职能作用 依赖于LTA4H表达的肉芽肿细胞。目标2将使用Mtb TnSeq方法进行检查 缺乏二十烷类信号关键成分的小鼠对结核分枝杆菌的遗传需求。总而言之，这些目标 将增进我们对肉芽肿生物学和分枝杆菌反应的了解，并有助于指导 治疗结核病的治疗方法。

ABSTRACT Mycobacterium tuberculosis (Mtb) one of the most successful pathogen in the world due to its long-standing, ancient partnership with humans. Despite decades of effort put towards prevention and treatment, Mtb was the number one cause of death by any single infectious agent in 2019. An important regulator of mycobacterial infections are eicosanoids: host-produced, lipid signaling molecules that can drive both inflammatory and anti- inflammatory signaling cascades. Using a zebrafish-Mycobacterium marinum infection model that recapitulates important aspects of mycobacterial pathogenesis, the balance of specific pro- and anti-inflammatory eicosanoids was found to be critical to control of infection. Disruption of eicosanoid synthesis, through a mutation in leukotriene A4 hydrolase-deficient (lta4h-/-), results in altered granuloma structure. The granuloma is the central feature of Mtb infection and is an aggregation of immune cells coordinating to restrict bacterial growth. This is the principal site of host-pathogen interactions. Although the granuloma is a key part of tuberculosis pathogenesis, host-pathogen signaling programs that govern granuloma biology remain elusive. Our lack of understanding of granuloma formation and signaling is due to the difficulty in studying the granuloma because the complex cell organization can only be formed and observed in vivo. The zebrafish-Mycobacterium marinum model, with conserved virulence loci and host-pathogen genetic programs, is used to study the genetics of mycobacterial infection and granuloma structure. This proposal will use genetic approaches in both the zebrafish- M.marinum granuloma model and the mouse-Mtb model of infection to test the hypothesis that there are key differences in eicosanoid-mediated immunity and bacterial response that are integral to overall granuloma biology. To this end, the first single-cell map of mycobacterial granulomas, in an experimentally tractable system was assembled from wildtype and lta4h-/- zebrafish granuloma cells. The findings from this dataset informed the following approaches to interrogate the effects of host eicosanoid signaling on granuloma structure and mycobacterial response. Aim 1 will define the functional role of a previously undefined population of granuloma cells that are dependent on lta4h expression. Aim 2 will use Mtb TnSeq approaches to the examine the genetic requirements of Mtb in mice that lack key components of eicosanoid signaling. Together, these aims will enhance our understanding of granuloma biology and mycobacterial response and can help guide therapeutic approaches to treating tuberculosis.