M. tuberculosis MazF toxins: target identification and stress adaptation

 描述（由申请人提供）：结核分枝杆菌（Mtb）已经适应了广泛的攻击，从我们的免疫反应到抗菌治疗，旨在消除有机体的生存。然而，使结核分枝杆菌能够忍受这些压力，减缓复制或成为潜伏性结核感染（LTBI）的分子开关尚不清楚。新出现的研究大肠杆菌中的应激生存的分子基础一般指向毒素-抗毒素（TA）系统的主要作用，这是操纵子包含相邻的基因编码两个小的蛋白质，毒素和它的同源抗毒素，抑制毒素活性的TA蛋白质-蛋白质复合物。他们的表达方式 涉及Mtb应激存活和/或LTBI的非复制持续状态特征的转换。然而，一些瓶颈阻碍了对这种挑衅性关联进行严格测试的进展。该提案招募了一个强大的团队来开发和应用一种新技术，即5' RNA-seq，以克服这些障碍，因为它们适用于Mtb中的九个成员迷宫（抗毒素）- MazF（毒素）家族。我们的目标是应用5' RNA-seq技术全面检测Mtb转录组中的MazF-mt靶标。然后，我们将其应用于在非应激条件下或暴露于与TB潜伏期相关的应激后生长的Mtb培养物。最后，我们还将研究Mtb MazF毒素切割rRNA如何改变核糖体功能。这些方法应该导致在这些代谢状态下MazF毒素靶向的RNA的准确快照，揭示RNA切割如何改变翻译，并确定触发Mtb中毒素激活的环境信号。

 DESCRIPTION (provided by applicant): 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 infection (LTBI) are not known. Emerging studies on the molecular underpinnings of stress survival in Escherichia coli 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. Their expression has been implicated in Mtb stress survival and/or the switch to the non-replicating persistent state characteristic of LTBI. However, several bottlenecks have impeded progress toward rigorous testing of this provocative association. This proposal enlists a strong team to develop and apply a new technology, 5' RNA-seq, to overcome these obstacles as they apply to the nine member MazE (antitoxin) - MazF (toxin) family in Mtb. Our goal is to apply 5' RNA-seq technology toward comprehensive detection of MazF-mt targets in the Mtb transcriptome. We will then apply it to Mtb cultures grown under unstressed conditions or after exposure to stresses that are relevant to TB latency. Finally, we will also study how cleavage of rRNAs by Mtb MazF toxins modifies ribosome function. These approaches should lead to an accurate snapshot of RNAs targeted by MazF toxins under these metabolic states, reveal how RNA cleavage alters translation and identify the environmental signals that trigger toxin activation in Mtb.