M. tuberculosis MazF toxins: target identification and stress adaptation

 描述(申请人提供)：结核分枝杆菌(Mtb)已经适应了从我们的免疫反应到抗菌疗法的广泛攻击，目的是根除这种微生物。然而，使结核分枝杆菌能够耐受这些压力、减缓复制或在潜在结核病感染(LTBI)时进入休眠状态的分子开关尚不清楚。关于大肠杆菌应激生存的分子基础的新研究一般指出毒素-抗毒素(TA)系统的主要作用，该系统是由编码两个小蛋白的相邻基因组成的操纵子，一个毒素及其同源抗毒素抑制TA蛋白-蛋白质复合体中的毒素活性。他们的表情一直是 与结核分枝杆菌应激存活和/或切换到LTBI的非复制持久状态特性有关。然而，几个瓶颈阻碍了对这种挑衅性联系进行严格测试的进展。这项提案招募了一个强大的团队来开发和应用一种新技术，5‘RNA-seq，以克服这些障碍，因为它们适用于Mtb的九个成员迷宫(抗毒素)-MazF(毒素)家族。我们的目标是将5‘RNA-SEQ技术应用于Mtb转录组中MazF-Mt靶标的全面检测。然后，我们将把它应用于在非压力条件下或在暴露于与结核病潜伏期相关的压力后生长的结核分枝杆菌培养物。最后，我们还将研究Mtb MazF毒素对rRNA的切割如何改变核糖体功能。这些方法应该能够准确地捕捉到MazF毒素在这些代谢状态下的靶向RNA，揭示RNA切割是如何改变翻译的，并识别在Mtb中触发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.