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，在缩短化疗持续时间方面发挥独特作用。尽管PZA在结核病治疗中的重要性，但其在结核分枝杆菌（Mtb）中的靶点仍然难以捉摸。我们最近发现了一个新的靶点PZA作为核糖体蛋白S1或RpsA，一个重要的蛋白质参与翻译和核糖体拯救过程，称为反式翻译，参与管理停滞的核糖体和受损的蛋白质和RNA在压力。我们发现RpsA过表达增加了PZA抗性，并且PZA抗性M.结核病临床分离株DHM 444在RpsA基因C端第438位氨基酸处发生丙氨酸缺失突变，这是一种新的PZA耐药机制。我们从生物化学上证实了PZA的活性成分吡嗪酸（POA）与野生型M。结核病RpsA，但不是突变体RpsA和抑制反式翻译，而不是典型的翻译。这些发现验证了RpsA作为PZA的靶点。然而，目前尚不清楚RpsA在C-末端区域的改变如何影响POA和tmRNA的结合以及PZA敏感性，以及反式翻译tmRNA（SsrA）和SmpB的组分的缺陷是否改变PZA敏感性。我们推测反式翻译途径对M.结核病持续生存，并且该途径中的缺陷导致对PZA和各种应激的易感性增加和持续性降低。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式翻译在PZA易感性和持久生物学中的作用。我们将创建在RpsA的C-末端和反式翻译（SsrA和SmpB）缺陷的突变体，并评估它们在持久试验中对PZA和不同胁迫条件的敏感性的改变。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，我们将确定新鉴定的PZA耐药基因rpsA突变的PZA耐药临床分离株的频率。最后，我们将使用结构生物学方法来确定RpsA的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作用和耐药性机制以及反式翻译在持续生存中的作用提供新的见解，并验证反式翻译的组分作为持续药物靶标。这些研究将为设计新的和更强大的药物提供有用的信息，这些药物针对持续存在的结核病，以缩短结核病治疗的时间。

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.