Elemental imaging of M. tuberculosis during infection.

描述(申请人提供)：每年有超过180万人死于结核病的病原体结核分枝杆菌(M.tb)。目前的疫苗或化疗方案无法扭转这一趋势。我们的长期目标是更好地了解结核分枝杆菌的分子生物学，并发现这种成功的病原体使用的新的生存机制。宿主-病原体相互作用的一个重要方面是调节结核杆菌生存的微环境中金属元素的获取。这个项目的最终目标是确定金属反应基因在结核分枝杆菌整体基础生物学中的作用，并检查它们对分枝杆菌吞噬小体成熟的影响。到目前为止收集的初步数据为有关铜贩运系统的有力假设奠定了基础。此外，它还将我们的注意力引向了其他金属离子，它们可能与铜和铁同等重要，甚至更重要。幸运的是，我们开始了一项新的扫描电子显微镜(STEM)技术，用于在离子水平上对生物组织进行元素分析，可以分析同一样品中的多个离子(例如10种元素)。我们决定利用R21的资助机制来探索这项技术的优势，并将其应用于更好地了解结核分枝杆菌的分子发病机制。我们相信，我们的研究将为更详细地研究特定金属在结核分枝杆菌生存策略中的作用打开大门。在这个项目的第一个目标中，我们将利用分析电子显微镜的能力来描绘代表两种不同的小鼠结核病模型(BALB/c和SCID)的小鼠肺中的元素图谱。在第二个目标中，我们将描述分枝杆菌对关键离子在从早期到慢性结核病进展过程中显著改变其水平的反应。最后，在第三个目标中，我们将检查在金属离子采集系统中感染缺陷杆菌后分枝杆菌吞噬小体的成熟情况。总之，我们将充分利用高分辨率的微量分析系统(STEM)和DNA微阵列来剖析金属蛋白对金属动态平衡和结核分枝杆菌细胞内环境的贡献。我们相信，该项目的成果将勾勒出新的致病机制，并可能有助于设计更好的抗结核病药物/疫苗。公共卫生相关性：该项目与美国国立卫生研究院的使命非常相关。结核病对美国的健康人群以及免疫功能受损的个人造成了巨大的风险。

DESCRIPTION (provided by applicant): More than 1.8 million people die annually from infection with Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis. Current vaccines or chemotherapy regimens are not able to reverse this trend. Our long-term goal is to better understand the molecular biology of M.tb and to discover new mechanisms of survival employed by this successful pathogen. An important aspect of the host-pathogen interactions is the regulation of the acquisition of metal elements in the microenvironment where tuberculous bacilli live. The ultimate goal in this project is to identify the role of metal-responsive genes to the overall basic biology of M.tb and to examine their impact on mycobacterial phagosome maturation. Preliminary data gathered so far laid the foundation for strong hypotheses related to Cu trafficking systems. Additionally, it directed our attention to other metal ions that could be equally or even more important than Cu and Fe. Fortunately, we embarked on a new technology for Scanning Transmission Electron Microscopy (STEM) for elemental analysis of biological tissues on the ionic level that could analyze multiple ions (e.g. 10 elements) of the same sample. We decided to utilize the R21 mechanism of funding to explore the advantages of this technology and to apply it towards the better understanding of the molecular pathogenesis of M.tb. We believe that our studies will open the door for more detailed investigations of the role of specific metals in M.tb survival strategies. In the first aim of this project we will utilize the power of analytical electron microscopy to profile the elemental map in lungs of mice representing 2 different models of murine tuberculosis (BALB/c and SCID). In the second aim, we will profile the mycobacterial responses to key ions significantly changed their levels during progression from early to chronic tuberculosis. Finally, in the third aim, we will examine mycobacterial phagosome maturation following infection with bacilli defective in metal ion acquisition systems. Overall, we will take full advantage of the high resolution, micro- analytical system (STEM) and DNA microarrays to dissect the contribution of metalloproteins to metal homeostasis and to the intracellular environment of M. tb. We believe that the outcomes of this project will delineate novel mechanisms of pathogenesis and could help in designing better drugs/vaccines against tuberculosis. PUBLIC HEALTH RELEVANCE: This project is very relevant to the mission of the NIH. Tuberculosis causes a tremendous risk to healthy populations in the USA as well as immunocompromised individuals.