Genetic Basis of Mycobacterial Invasion

描述（由申请人提供）：结核病，由结核分枝杆菌引起，是世界上最大的疾病和死亡传染病之一。近年来，由于出现耐药菌株和同时流行艾滋病毒，这一问题更加严重。没有可靠的结核病疫苗，越来越多的证据表明，自然感染和疾病不能预防随后的再感染。因此，宿主免疫仅在预防和消除感染方面部分有效。为了加强我们对致病性分枝杆菌和宿主免疫之间基本相互作用的理解，我们将使用一个密切相关的致病性物种海洋分枝杆菌。海洋分枝杆菌在恒温动物中引起全身性结核样疾病，在人类中引起称为鱼缸肉芽肿的外周肉芽肿疾病。由于海洋分枝杆菌与结核分枝杆菌在遗传和致病性上的相似性，其生长速度相对较快，并且存在多种天然宿主作为动物模型，因此海洋分枝杆菌是研究结核病的有力模型。成年斑马鱼发展成干酪样肉芽肿结核，类似于活动性人类结核。Mm也在斑马鱼幼虫中产生肉芽肿病，由于其光学透明性，可以实时监测宿主-病原体相互作用。利用胚胎模型结合我们在海洋分枝杆菌中开发的各种基于荧光的工具，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。我们将开发额外的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报告器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毒力位点的研究。具体来说，我们将探讨rd1分泌的决定因子诱导上皮细胞产生宿主基质金属蛋白酶-9的身份及其分子机制，从而促进肉芽肿的形成和细菌扩张。从我们提出的实验中获得的信息将进一步加深我们对结核病的基本认识。

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.