Regulation of RecA Intein splicing in M. tuberculosis

项目描述（由申请人提供）：该项目的长期目标是了解结核分枝杆菌（Mtb）在感染过程中调节其对宿主环境反应的手段，从而开发出更好的干预策略和生物标志物来对抗结核（TB）疾病。内含物是一种可移动的蛋白质元素，可以插入使它们所处的蛋白质失活。这些内含物的自催化切除导致其宿主蛋白的功能激活，为快速的翻译后基因调控提供了理想的机制。在三种Mtb蛋白（DnaB， RecA和SufB）中发现了内部蛋白，它们都在哺乳动物感染期间暴露于DNA损伤条件后的DNA复制和恢复过程中发挥核心作用。蛋白在生物化学水平上得到了广泛的研究，并被用作蛋白质工程的工具。然而，尽管最近的研究表明，FDA批准的抗癌药物顺铂可以防止内嵌蛋白剪接，但对内嵌蛋白在结核分枝杆菌中的生物学作用知之甚少。这个飞行员

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.