Regulation of RecA Intein splicing in M. tuberculosis

描述(由申请人提供)：该项目的长期目标是了解结核分枝杆菌(Mtb)在感染期间调节其对宿主环境的反应的方法，以便能够开发针对结核病(TB)疾病的改进干预策略和生物标志物。内含子是一种可移动的蛋白质元件，可以插入方式使它们所居住的蛋白质失活。这些内含子的自动催化切除导致其宿主蛋白的功能激活，为快速翻译后基因调控提供了理想的机制。在三种Mtb蛋白(Dna B、RecA和SufB)中发现内含子，所有这些蛋白都在DNA复制和恢复过程中发挥核心作用，这些蛋白在哺乳动物感染过程中遇到DNA损伤。内含子已经在生化水平上得到了广泛的研究，并已被开发为蛋白质工程的工具。然而，尽管最近的研究表明，阻止内含素与FDA批准的抗癌药物顺铂剪接会抑制结核分枝杆菌的生长，但对内含素在结核分枝杆菌中的生物学作用知之甚少。这位飞行员 该项目将测试内含子剪接受宿主相关环境条件调节的假设，并产生一个敏感的基于荧光的功能增益报告系统，适用于在感染过程中监测细菌内的Mtb内含子剪接。这项研究计划的完成将提供关键的概念和技术基础，这也将是未来研究所需要的，以解决在宿主感染期间内含子在结核分枝杆菌中提供一种快速的翻译后调节形式的可能性。这项工作具有特别高的影响，因为内含子剪接有可能建立结核分枝杆菌基因调控的新范例，可能控制宿主内的结核分枝杆菌复制，以及内含子剪接作为可能的药物靶点的展示作用。

DESCRIPTION (provided by applicant): The long term goal of this project is to understand the means by which M. tuberculosis (Mtb) regulates its response to the host environment during infection, so that improved intervention strategies and biomarkers can be developed against tuberculosis (TB) disease. Inteins are mobile protein elements that insertionally inactivate the proteins they inhabit. Autocatalytic excision of these inteins results in functional activation of their host proteins, providing an ideal mechanism for rapid post-translational gene regulation. Inteins are found in three Mtb proteins (DnaB, RecA and SufB), all of which have central roles in the process of DNA replication and recovery following exposure to the DNA damaging conditions encountered during mammalian infection. Inteins have been extensively studied at the biochemical level and have been exploited as tools for protein engineering. However, little is known about the biological roles of inteins in Mtb despite recent studies showing that prevention of intein splicing with the FDA approved anti-cancer drug cisplatin inhibits Mtb growth. This pilot project will test the hypothesis that intein splicing is modulated by host-associated environmental conditions and generate a sensitive fluorescence-based gain-of-function reporter system suitable for monitoring Mtb intein splicing within bacteria during infection. Completion of this research plan will provide critical conceptual and technical foundations that will also be needed for future studies addressing the possibility that inteins provide a rapid form of post-translational regulation in Mtb during host infection. This work is of particularly high impact because of the potential for intein splicing to establish new paradigms for gene regulation in Mtb that may control Mtb replication within the host, as well as the demonstrated role of intein splicing as a possible drug target.