Role of Mycobacterium tuberculosis protease in pathogenesis and host response

描述（由申请人提供）：结核分枝杆菌是一种非常成功的人类病原体，已进化为在宿主体内生存。我们的长期目标是阐明M。结核病干扰巨噬细胞的杀微生物功能并逃避宿主免疫应答。了解M.结核病与宿主的相互作用对设计治疗干预措施和疫苗战略很重要。我们已经鉴定了Rv 2224 c，一种预测的蛋白酶，作为巨噬细胞和体内存活的关键。我们推测Rv 2224 c是一个重要的毒力因子，它通过蛋白水解切割M。结核病基质和调节宿主免疫应答。我们的初步研究表明，Rv 2224 c输出到分枝杆菌的细胞被膜和分泌到细胞外。蛋白酶中被破坏的突变体在巨噬细胞和体内的生长受到损害。用蛋白酶突变体感染的小鼠显示出显著减少的肺病理学，并且比野生型感染的小鼠存活更长时间。在这个提议中，将研究蛋白酶在宿主-病原体相互作用中的作用。具体目标是：1. Rv 2224 c及其潜在底物在M.结核我们将研究Rv 2224 c在分枝杆菌中的定位，并对Rv 2224 c进行结构-功能分析。我们将通过研究候选生理底物来研究蛋白酶的生物活性和特异性。2.在巨噬细胞和体内研究蛋白酶与宿主免疫应答的相互作用。我们将研究对Rv 2224 c突变体的免疫应答的性质，特别是确定T和B细胞在免疫中的作用，并在巨噬细胞和体内进行蛋白酶与宿主相互作用的结构-功能分析。3.剖析蛋白酶对巨噬细胞功能的调节。我们将询问感染突变体是否会改变巨噬细胞的反应，并测试Rv 2224 c：：tn突变体是否对IFN γ介导的免疫反应超敏感。最后，我们将研究Rv 2224是否直接与宿主细胞组分相互作用， 互动研究

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is a highly successful human pathogen that has evolved to survive within the host. Our long-term goals are to delineate the mechanisms by which M. tuberculosis interferes with the microbicidal functions of macrophages and evades host immune responses. Understanding the molecular mechanisms underlying M. tuberculosis- host interactions is important for designing therapeutic interventions and vaccine strategies. We have identified Rv2224c, a predicted protease, as being critical for survival in macrophages and in vivo. We hypothesize that Rv2224c is an important virulence factor that proteolytically cleaves M. tuberculosis substrates and modulates the host immune response. Our preliminary studies indicate that Rv2224c is exported to the cell envelope of mycobacteria and secreted extracellularly. A mutant disrupted in the protease is impaired for growth in macrophages and in vivo. Mice infected with a protease mutant show markedly reduced lung pathology and survive longer than wild type-infected mice. In this proposal will study the role of the protease in host-pathogen interactions. The specific aims are: 1. Molecular and biochemical characterization of Rv2224c and its potential substrate(s) in M. tuberculosis. We will study the localization of Rv2224c in mycobacteria and perform structure-function analysis of Rv2224c. We will investigate the biological activity and specificity of the protease by studying a candidate physiologic substrate. 2. Study the interaction of the protease with the host immune response in macrophages and in vivo. We will examine the nature of the immune response to Rv2224c mutant, specifically determine the role of T and B cells in immunity and perform structure-function analysis of protease interaction with the host in macrophages and in vivo. 3. Dissect the modulation of macrophage function by the protease. We will ask whether infection with the mutant elicits altered macrophage responses and test whether the Rv2224c::tn mutant is hyper-susceptible to IFNy-mediated immune responses. Finally, we will study whether Rv2224 directly interacts with host cell components using protein-protein interaction studies.