Intracellular mycobacterial proteome

描述(申请人提供)：在进化过程中，结核分枝杆菌适应了与宿主巨噬细胞的同源相互作用，使其成为一种极其成功的细胞内病原体。人类潜伏性结核病的发病机制尚不清楚。令人担忧的是，至少在初始感染阶段，小鼠和人类的结核病病理不同。显然，人类结核病病理学研究的困难是阻碍人类对结核病进一步了解的主要障碍。同时，研究人和小鼠巨噬细胞内的分枝杆菌蛋白质组可能是了解结核分枝杆菌发病机制和病原体-宿主相互作用，特别是在系统水平上更容易获得和更有价值的手段。我们的研究兴趣归于结核分枝杆菌用于细胞内生长和持久性的中间代谢途径。本项目旨在利用高效液相色谱/线性离子陷阱-傅立叶变换质谱仪和定量蛋白质组学方法，研究结核分枝杆菌H37Rv在人和小鼠巨噬细胞内的蛋白质合成谱。我们将比较结核分枝杆菌H37Rv在人急性单核细胞白血病细胞系THP-1和C57BL/6来源的小鼠骨髓巨噬细胞系BMA3.1A7中生长的细胞内蛋白质组。基于蛋白质组图谱，我们将确定细胞内结核分枝杆菌H37Rv活跃的中间代谢途径，特别是由于缺乏-酮戊二酸脱氢酶而分裂的半个三羧酸(TCA)循环。当分枝杆菌在氧气和营养限制下生存时，分裂还原的TCA半周期和乙醛酸旁路变得突出。它们对于理解分枝杆菌在不同巨噬细胞内持续存在的生理学可能很重要。

DESCRIPTION (provided by applicant): Over the course of evolution, Mycobacterium tuberculosis has adapted a cognate interaction with host macrophages that allows it to become an extremely successful intracellular pathogen. The mechanism of latent tuberculosis in human remains unclear. It is a concern that tuberculosis pathology differs between murine and human at least at the primary infection stage. Difficulty in pathological study of tuberculosis in human subjects apparently is a major obstacle hindering further understanding of tuberculosis in man. Meanwhile, studying the intracellular mycobacterial proteome in human and murine macrophages may be a more accessible and worthwhile means to gain useful insight into the pathogenesis of M. tuberculosis and pathogen- host interaction, especially at a systems level. Our research interest vests in the intermediary metabolism pathway utilized by M. tuberculosis for intracellular growth and persistence. This project is to investigate the protein synthesis profile, the proteome, of M. tubeculosis H37Rv within human and murine macrophages using high resolution liquid-chromatography/linear ion trap-fourier transform mass spectrometry instrumentation and quantitative proteomics methods. We will compare the intracellular proteomes of M. tubeculosis H37Rv grown in the human acute monocytic leukemia cell line THP-1 versus in the C57BL/6-derived murine bone marrow macrophage cell line BMA3.1A7. Based on proteome profiles, we will identify the active intermediary metabolism pathway in intracellular M. tubeculosis H37Rv, particularly the half tricarboxylic acid (TCA) cycles split by the lack of ?-ketoglutarate dehydrogenase. The split reductive TCA half cycle along with the glyoxylate bypass come into prominence when mycobacteria strive for survival under oxygen and nutrient limitation. They may be important for understanding the physiology of intracellular persistence of mycobacteria within different macrophages.