Genome exploration through toxin-mediated ribosome stalling

项目摘要 大约90%感染结核分枝杆菌（Mtb）的个体发展为无症状的结核病。 潜伏感染，即非传染性。虽然有些人可能会根除这种感染， 不包括可从潜伏性结核病转化为具有传染性的活动性结核病的大量人群。 在免疫受损的个体中，包括感染艾滋病毒的个体，重新激活尤其可能。的 使结核分枝杆菌能够减缓或停止复制、进入休眠状态并建立潜伏性结核感染的分子开关 特征不明显。对这些开关的透彻理解对于开发1）诊断是至关重要的 能够预测再活化风险和2）更短、更有效的潜伏性TB感染治疗方案。 毒素-抗毒素（TA）系统与潜伏性TB感染的建立密切相关，因为它们的毒素 组分通常下调Mtb细胞生长，并且响应于与此相关的应激而被激活。 状态然而，TA系统的异常冗余使得确定TA系统的个体贡献成为可能。 每种毒素都使用常规的遗传和分子生物学方法进行挑战。我们建议使用 强大的基因组规模的工具，以跟踪转录本，核糖体和蛋白质的命运，以应对 激活一组tRNA裂解毒素，以了解压力背后的分子机制 生存然后，我们利用我们的发现，这些毒素的密码子特异性核糖体停滞特性 鉴定新的ORF，并将其作为改进Mtb基因组注释的可靠工具。

Project Summary Approximately 90% of individuals infected with Mycobacterium tuberculosis (Mtb) develop an asymptomatic latent infection, which is non-infectious. Although some individuals may eradicate this infection, those who do not comprise a large reservoir of persons who can convert from latent to active TB, which is infectious. Reactivation is especially likely in immune-compromised individuals, including those infected with HIV. The molecular switches that enable Mtb to slow or stop replication, become dormant and establish latent TB infection are poorly characterized. A thorough understanding of these switches is critical for development of 1) diagnostics to enable prediction of reactivation risk and 2) shorter, more effective treatment regimens for latent TB infection. Toxin-antitoxin (TA) systems are strongly implicated in establishment of latent TB infection because their toxin components typically downregulate Mtb cell growth and are activated in response to stresses relevant to this state. Yet, the extraordinary redundancy of TA systems make determination of the individual contributions of each toxin challenging using conventional genetic and molecular biological approaches. We propose to use a powerful battery of genome-scale tools to track the fate of transcripts, ribosomes and proteins in response to activation of a subset of tRNA-cleaving toxins to understand the molecular mechanisms that underlie stress survival. We then exploit our finding that the codon-specific ribosome-stalling characteristic of these toxins identifies novel ORFs and apply this as a reliable tool for improved Mtb genome annotation.