Transcriptome and proteome remodeling by Mycobacterium tuberculosis MazF toxins

项目摘要 结核分枝杆菌(Mtb)已经适应了从我们的免疫反应中存活下来的各种攻击。 到抗菌疗法--旨在根除这种微生物。然而，分子开关能够使 结核分枝杆菌可以承受这些压力，减缓复制或作为一种潜在的结核病(TB)感染而进入休眠状态 都是未知的。关于应激生存的分子基础的新兴研究通常指向一个主要的 毒素-抗毒素(TA)系统的作用，TA系统是由编码两个小蛋白的相邻基因组成的操纵子， 一种毒素及其同源抗毒素，可抑制TA蛋白-蛋白质复合体中的毒素活性。然而，有几个 瓶颈阻碍了对这种挑衅性联系进行严格测试的进展。这份提案提出了 一个强大的多学科团队，拥有拟议工作的所有核心组件的专业知识-RNA-seq，助教 系统、MTB生物学/生理学和生物信息学/计算生物学。基因组规模的方法 在PI的实验室开发的5‘RNA-SEQ将用于克服这些障碍，因为它们适用于 Mtb中的11个成员迷宫(抗毒素)-MazF(毒素)家族。5‘RNA-SEQ将促进全面检测 Mtb转录组中MazF靶标在非应激条件下或在暴露于 与潜伏的结核病感染有关。最后，将研究MazF毒素对Mtb蛋白质组的影响。 总的来说，这些方法将识别在结核分枝杆菌中触发毒素激活的环境信号，提供 在这些代谢状态下被MazF毒素靶向的RNA的准确快照，并揭示了如何 毒素介导的RNA裂解改变了结核分枝杆菌的生理。这些目标与“优先事项1：改进”非常吻合 NIAID结核病研究战略计划五个组成部分中的“结核病基础知识” 于2018年9月发布。

Project Summary Mycobacterium tuberculosis (Mtb) has adapted to survive a wide range of assaults—from our immune response to antimicrobial therapeutics—intended to eradicate the organism. However, the molecular switches that enable Mtb to endure these stresses, to slow replication or to become dormant as a latent tuberculosis (TB) infection are not known. Emerging studies on the molecular underpinnings of stress survival generally point to a major role for toxin-antitoxin (TA) systems, which are operons comprising adjacent genes encoding two small proteins, a toxin and its cognate antitoxin that inhibits toxin activity in the TA protein-protein complex. However, several bottlenecks have impeded progress toward rigorous testing of this provocative association. This proposal enlists a strong multidisciplinary team with expertise in all core components of the proposed work—RNA-seq, TA systems, Mtb biology/physiology and bioinformatics/computational biology. The genome-scale approach developed in the PI’s laboratory, 5’ RNA-seq, will be used to overcome these obstacles as they apply to the eleven-member MazE (antitoxin) – MazF (toxin) family in Mtb. 5’ RNA-seq will facilitate comprehensive detection of MazF targets in the Mtb transcriptome under unstressed conditions or after exposure to stresses that are relevant to latent TB infection. Finally, the impact of MazF toxins on the Mtb proteome will be investigated. Collectively, these approaches will identify the environmental signals that trigger toxin activation in Mtb, provide an accurate snapshot of RNAs targeted by MazF toxins under these metabolic states, and reveal clues to how toxin-mediated RNA cleavage alters Mtb physiology. These goals align well with “Priority 1: Improve Fundamental Knowledge of TB” of the five components of the NIAID Strategic Plan for Tuberculosis Research released in September 2018.