Pyrazinamide and Trans-Translation in Mycobacterium tuberculosis

描述（由申请人提供）：吡嗪酰胺（PZA）是一种关键的一线结核病（TB）药物，在缩短化疗时间方面发挥着独特的作用。尽管PZA在结核病治疗中具有重要意义，但PZA在结核分枝杆菌（Mtb）中的靶点仍然难以捉摸。我们最近发现了PZA的一个新靶点，即核糖体蛋白S1或RpsA，这是一种重要的蛋白质，参与翻译和核糖体拯救过程，称为反式翻译，涉及在应激过程中管理停滞的核糖体和受损的蛋白质和RNA。我们发现，RpsA过表达增加了PZA的耐药性，并且没有pncA突变的PZA耐药结核分枝杆菌临床分离株DHM444在RpsA c端第438个氨基酸上存在丙氨酸缺失突变，这是PZA耐药的新机制。我们从生化角度证实了PZA的活性成分吡嗪酸（pyrazinoic acid， POA）与野生型结核分枝杆菌RpsA结合，而不是突变型RpsA，并且抑制反翻译而不是规范翻译。这些发现证实了RpsA是PZA的靶点。然而，c端区RpsA的改变如何影响POA与tmRNA的结合和PZA的易感性，以及反翻译tmRNA （SsrA）和SmpB成分的缺陷是否会改变PZA的易感性，目前尚不清楚。我们假设，反翻译途径对结核分枝杆菌的持久性生存很重要，而该途径的缺陷导致PZA易感性增加和各种应激降低持久性。为了解决这些假设，我们将结合遗传学、生物化学、结构生物学和动物研究来阐明反翻译在PZA易感性和持久性生物学中的作用。我们将创建在RpsA的c端和反翻译中有缺陷的突变体（SsrA和SmpB），并在持久性试验中评估它们对PZA和不同应激条件的敏感性变化。我们还将评估它们在结核感染小鼠模型中的毒力和持久性表型以及它们对结核化疗和PZA的反应。此外，我们将确定PZA耐药临床分离株中新发现的PZA耐药基因rpsA突变的频率。最后，我们将使用结构生物学方法来确定RpsA的c端变化如何影响与POA的结合并促进PZA抗性。我们的研究结果将为PZA的作用和耐药机制以及反翻译在持久性生存中的作用提供新的见解，并验证反翻译成分作为持久性药物靶点。这些研究将为设计新的和更有效的药物提供有用的信息，以缩短结核病治疗的持续时间。

DESCRIPTION (provided by applicant): Pyrazinamide (PZA) is a critical first-line tuberculosis (TB) drug that plays a unique role in shortening the duration of chemotherapy. Despite its importance in TB treatment, the target of PZA in Mycobacterium tuberculosis (Mtb) has remained elusive. We recently identified a new target of PZA as the ribosomal protein S1 or RpsA, a vital protein involved in both translation and ribosome rescuing process called trans-translation involved in management of stalled ribosomes and damaged proteins and RNA during stress. We found that RpsA overexpression conferred increased PZA resistance and that a PZA-resistant M. tuberculosis clinical isolate DHM444 without the common mechanism of pncA mutations harbored an alanine deletion mutation at 438th amino acid in the C-terminus of RpsA as a new mechanism of PZA resistance. We confirmed biochemically that the active component of PZA, pyrazinoic acid (POA), bound to wild type M. tuberculosis RpsA but not the mutant RpsA and inhibited trans-translation rather than canonical translation. These findings validate RpsA as a target of PZA. However, it is not clear how alterations in RpsA in C-terminal region affect the binding of POA and tmRNA and PZA susceptibility and whether defect in components of trans-translation tmRNA (SsrA) and SmpB alters PZA susceptibility. We hypothesize that trans-translation pathway is important for M. tuberculosis persister survival and that defect in this pathway leads to increased susceptibility to PZA and various stresses and reduced persistence. To address these hypotheses, we will use a combined genetic, biochemical, structural biology and animal studies to elucidate the role of trans-translation in PZA susceptibility and persister biology. We will create mutants defective in the C-terminus of the RpsA and also in trans-translation (SsrA and SmpB) and assess their alterations in sensitivity to PZA and diverse stress conditions in persister assays. We will also evaluate their virulence and persistence phenotypes in mouse model of TB infection and their response to TB chemotherapy and PZA. In addition, we will determine the frequency of PZA- resistant clinical isolates with mutations in the newly identified PZA resistance gene rpsA. Finally we will use a structural biology approach to determine how changes in the C-terminus of RpsA affect the binding to POA and contribute to PZA resistance. The outcome of our studies will provide new insight into the mechanisms of PZA action and resistance and the role of trans-translation in persister survival and validate components of trans-translation as persister drug targets. These studies will provide useful information for design of new and more powerful drugs that target persisters for shortening the duration of TB treatment.