Role of Mycobacterium tuberculosis protease in pathogenesis and host response

结核分枝杆菌是一种非常成功的人类病原体，它已经进化成在 主持人。我们的长期目标是阐明结核分枝杆菌干扰人类健康的机制 巨噬细胞的杀菌功能和逃避宿主免疫反应。理解分子 结核分枝杆菌与R\OSI相互作用的机制对设计治疗干预措施很重要 和疫苗策略。我们已经确定Rv2224c是一种预测的蛋白水解酶，对于在 巨噬细胞和活体内。我们假设Rv2224c是一种重要的毒力因子，在蛋白降解方面 裂解结核分枝杆菌底物，调节宿主免疫反应。我们的初步研究 说明Rv2224c输出到分枝杆菌的细胞膜，并以细胞外的方式分泌。变种人 被破坏的蛋白水解酶会损害巨噬细胞和体内的生长。感染了一种蛋白酶的小鼠 与野生型感染的小鼠相比，突变小鼠的肺部病理明显减轻，存活时间更长。在这 提案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. 酵母Rv2224c及其潜在底物(S)的分子和生化特性 肺结核。我们将研究Rv2224c在分枝杆菌中的定位并进行结构功能 Rv2224c.我们将通过研究一种新的方法来研究该酶的生物学活性和特异性。 候选生理底物。2.研究蛋白酶与宿主免疫的相互作用 巨噬细胞和体内的反应。我们将研究对Rv2224c的免疫反应的性质 突变体，特异性地确定T和B细胞在免疫中的作用并进行结构-功能分析 巨噬细胞内和体内的蛋白酶与宿主的相互作用。3.剖析巨噬细胞的调控机制 通过蛋白水解酶发挥作用。我们将询问感染突变体是否会引起巨噬细胞的改变 反应并测试Rv2224c：：Tn突变体是否对IFNy介导的免疫超敏 回应。最后，我们将研究Rv2224是否通过蛋白质直接与宿主细胞成分相互作用 互动研究。

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-}r\osi 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 wildype-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 proteinprotein interaction studies.