Genetic Basis of Mycobacterial Invasion

描述(申请人提供)：结核病是由结核分枝杆菌引起的，是世界范围内导致疾病和死亡的最大传染病原因之一。近年来，耐药菌株的出现和同时出现的艾滋病毒流行加剧了这一问题。目前还没有针对结核病的可靠疫苗，而且越来越多的证据表明，自然感染和疾病不能防止随后的再次感染。因此，宿主免疫在预防和消除感染方面只有部分效果。为了加强我们对致病分枝杆菌和宿主免疫之间基本相互作用的了解，我们将使用一种密切相关的致病物种海洋分枝杆菌。海洋分枝杆菌在体外会引起一种全身性肺结核样疾病，在人类身上会引起一种称为鱼缸肉芽肿的外周肉芽肿疾病。海洋分枝杆菌是研究结核病的有力模型，因为它与结核分枝杆菌的遗传和致病相似，生长相对较快，并且存在作为动物模型的多种自然宿主。成年斑马鱼患干酪性肉芽肿性结核病类似于活动性人类结核病。MM还在斑马鱼幼体中产生肉芽肿疾病，由于其光学透明度，可以实时监测宿主与病原体的相互作用。将胚胎模型与我们在海洋分枝杆菌中开发的各种基于荧光的工具结合使用，我们已经能够实时监测和调节感染、传播和肉芽肿形成的基本阶段。在这里，我们将在斑马鱼中测试一系列海洋分枝杆菌突变体，以探索分枝杆菌抵抗甚至篡夺宿主免疫持续存在的分子机制。我们将开发其他技术来评估感染期间细胞内海洋分枝杆菌的亚细胞定位，并使用pH敏感的GFP报告程序来确定吞噬体酸化与细菌内酸化的相关性。最后，我们将继续对Rd1毒力基因座的研究。具体地说，我们将探索Rd1分泌的决定簇诱导上皮细胞产生宿主基质金属蛋白酶-9的身份和分子机制，从而促进肉芽肿的形成和细菌的扩张。从我们提议的实验中获得的信息将进一步加深我们对结核病的基本知识。 公共卫生相关性：结核病是由致病的分枝杆菌引起的，通常以免疫系统无法根除的长期感染为特征。该应用程序使用一个简单且遗传上容易处理的斑马鱼幼体模型来探索脊椎动物免疫系统与感染开始时的分枝杆菌之间发生的最早的相互作用。通过了解这些早期的相互作用，我们希望确定治疗干预的关键步骤。

DESCRIPTION (provided by applicant): Tuberculosis, caused by Mycobacterium tuberculosis, is one of the greatest infectious causes of disease and mortality worldwide. The problem has been exacerbated in recent years by the emergence of drug resistant strains and the concurrent HIV epidemic. There is no reliable vaccine against tuberculosis and there is increasing evidence that natural infection and disease cannot prevent subsequent reinfection. Thus host immunity is only partially effective in preventing and eliminating infection. To enhance our understanding of the basic interplay between pathogenic mycobacteria and host immunity, we will use a closely related pathogenic species Mycobacterium marinum. M. marinum causes a systemic tuberculosis-like disease in ectotherms, and a peripheral granulomatous disease in humans called fish tank granuloma. M. marinum is a powerful model for the study of tuberculosis owing to its genetic and pathogenic similarities to M. tuberculosis, its relatively rapid growth, and the existence of multiple natural hosts that serve as animal models. Adult zebrafish develop caseating granulomatous tuberculosis similar to active human tuberculosis. Mm also produces granulomatous disease in zebrafish larvae, which allow live monitoring of host-pathogen interactions due to their optical transparency. Using the embryo model in conjunction with a variety of fluorescence-based tools we developed in M. marinum, we have been able to monitor and modulate the essential stages of infection, dissemination and granuloma formation in real-time. Here, we will test a series of M. marinum mutants in the zebrafish to explore the molecular mechanisms by which mycobacteria resist and even usurp host immunity to persist. We will develop additional techniques to assess the subcellular localization of intracellular M. marinum during infection, and use a pH sensitive GFP reporter to determine correlate phagosomal acidification to intrabacterial acidification. Finally, we will continue our studies of the RD1 virulence locus. Specifically, we will pursue the identity of, and the molecular mechanism by which, RD1-secreted determinants induce the production of host matrix metalloproteinase-9 by epithelial cells, thus facilitating granuloma formation and bacterial expansion. The information obtained from our proposed experiments will further our fundamental knowledge about tuberculosis. PUBLIC HEALTH RELEVANCE: Tuberculosis is caused by pathogenic mycobacteria, and is often characterized by a long-term infection that the immune system is unable to eradicate. This application uses a simple and genetically tractable zebrafish larval model to explore the earliest interactions that occur between the immune system of Vertebrate Animals: and mycobacteria at the onset of infection. By understanding these early interactions we hope to identify key steps for therapeutic intervention.