Genome exploration through toxin-mediated ribosome stalling

项目摘要 大约90%感染结核分枝杆菌(Mtb)的人发展为无症状 潜伏感染，非传染性。虽然有些人可能会根除这种感染，但那些做到了的人 不包括可从潜伏性结核病转变为具有传染性的活动性结核病的大量人群。 在免疫受损的人中，包括感染艾滋病毒的人，重新激活的可能性尤其大。这个 分子开关，使结核分枝杆菌能够减缓或停止复制，进入休眠状态，并建立潜在的结核病感染 都有很差的特征。对这些开关的透彻了解对于开发诊断至关重要 以便能够预测再激活风险和2)更短、更有效的潜伏结核病感染治疗方案。 毒素-抗毒素(TA)系统与潜在结核病感染的建立密切相关，因为它们的毒素 成分通常下调结核分枝杆菌细胞的生长，并在与此相关的压力下被激活 州政府。然而，TA系统的非凡冗余决定了个人的贡献 每种毒素都使用传统的遗传和分子生物学方法进行挑战。我们建议使用 一组强大的基因组规模工具，用于跟踪转录本、核糖体和蛋白质的命运 激活一组裂解tRNA的毒素以了解应激的分子机制 生死存亡。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征 识别新的ORF，并将其作为改进的结核分枝杆菌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.