Pyrazinamide and Trans-Translation in Mycobacterium tuberculosis

描述(申请人提供)：吡津胺(PZA)是一种关键的一线结核病(TB)药物，在缩短化疗持续时间方面发挥着独特的作用。尽管PZA在结核病治疗中很重要，但它在结核分枝杆菌(Mtb)中的靶点仍然难以捉摸。我们最近发现了PZA的一个新靶点是核糖体蛋白S1或RpsA，这是一种参与翻译和核糖体拯救过程的重要蛋白质，称为反式翻译，参与管理停滞的核糖体以及应激过程中受损的蛋白质和RNA。我们发现，RpsA的过表达增加了对PZA的耐药性，而耐PZA的结核分枝杆菌临床分离株DHM444没有常见的PncA突变机制，RpsA C端第438位氨基酸的丙氨酸缺失突变是PZA耐药的新机制。我们在生化上证实了PZA的活性成分吡津酸(POA)与野生型结核分枝杆菌RpsA结合，而不是突变型RpsA，并且抑制了反式翻译，而不是规范翻译。这些发现证实了RpsA是PZA的靶点。然而，目前尚不清楚C端区RpsA的改变如何影响POA和tmRNA的结合和PZA的易感性，以及反式翻译tmRNA(SsrA)和SmpB的成分缺陷是否会改变PZA的易感性。我们假设，反式翻译途径对于结核分枝杆菌持久生存是重要的，该途径的缺陷会导致对PZA和各种压力的易感性增加，并降低持久性。为了解决这些假说，我们将结合遗传学、生化、结构生物学和动物研究来阐明反式翻译在PZA易感性和持久生物学中的作用。我们将创造在RpsA的C末端以及在反译(SsrA和SmpB)方面有缺陷的突变体，并在持续分析中评估它们对PZA和不同胁迫条件的敏感性变化。我们还将在小鼠结核病感染模型中评估它们的毒力和持久性表型，以及它们对结核病化疗和PZA的反应。此外，我们将确定新发现的PZA耐药基因rpsA突变的临床分离株对PZA耐药的频率。最后，我们将使用结构生物学的方法来确定RpsA C末端的变化如何影响与POA的结合，并有助于PZA抗性。我们的研究结果将为PZA的作用和耐药机制以及反译在持久生存中的作用提供新的见解，并验证反译成分作为持久药物靶点的有效性。这些研究将为设计新的、更有效的药物提供有用的信息，这些药物以持久者为目标，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.