Pyrazinamide and Trans-Translation in Mycobacterium tuberculosis

吡嗪酰胺和结核分枝杆菌的反式翻译

• 批准号: 8705383
• 负责人: YING ZHANG
• 金额: $ 42.49万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-17 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8705383
• 关键词: Address; Affect; Alanine; Amino Acids; Animals; Binding; Binding Sites; Biochemical; Biological Assay; Biology; C-terminal; Clinical; Complex; Crystallography; Defect; Deletion Mutation; Drug Targeting; Frequencies; Genes; Genetic; Infection; Maps; Multidrug-Resistant Tuberculosis; Mutation; Mycobacterium tuberculosis; Outcome; Outcome Study; Pathway interactions; Pharmaceutical Preparations; Phenotype; Play; Predisposition; Process; Proteins; Pyrazinamide; Pyrazinamide resistance; RNA; Resistance; Ribosomes; Role; Site; Site-Directed Mutagenesis; Stress; Structure; Testing; Translation Process; Translations; Tuberculosis; Virulence; attenuation; base; chemotherapy; design; improved; insight; killings; macrophage; mouse model; mutant; overexpression; pyrazinoic acid; response; ribosomal protein S1; structural biology; tmRNA; tool; tuberculosis drugs; tuberculosis treatment

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.

描述（申请人提供）：吡嗪酰胺（PZA）是一种重要的一线结核病（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，但结核分枝杆菌 (Mtb) 中 PZA 的作用靶点仍然难以捉摸。我们最近确定了 PZA 的一个新靶标为核糖体蛋白 S1 或 RpsA，这是一种参与翻译和核糖体拯救过程（称为反式翻译）的重要蛋白质，参与应激期间停滞核糖体以及受损蛋白质和 RNA 的管理。我们发现RpsA过表达会增加PZA耐药性，并且不具有pncA突变共同机制的PZA耐药结核分枝杆菌临床分离株DHM444在RpsA C末端第438个氨基酸处存在丙氨酸缺失突变，这是PZA耐药的新机制。我们通过生化证实，PZA 的活性成分吡嗪酸 (POA) 与野生型结核分枝杆菌 RpsA 结合，但不与突变型 RpsA 结合，并且抑制反式翻译而不是经典翻译。这些发现证实 RpsA 是 PZA 的靶点。然而，目前尚不清楚C端区域RpsA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成分的缺陷是否会改变PZA敏感性。我们假设反式翻译途径对于结核分枝杆菌持续存活很重要，并且该途径的缺陷导致对 PZA 和各种应激的易感性增加以及持续性降低。为了解决这些假设，我们将结合遗传、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。我们将创建 RpsA C 末端和反式翻译（SsrA 和 SmpB）有缺陷的突变体，并评估它们对 PZA 敏感性的改变以及持久测定中的不同应激条件。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 抗性基因 rpsA 发生突变的 PZA 抗性临床分离株的频率。最后，我们将使用结构生物学方法来确定 RpsA C 末端的变化如何影响与 POA 的结合并导致 PZA 耐药。我们的研究结果将为 PZA 作用和耐药机制以及反式翻译在持久性存活中的作用提供新的见解，并验证反式翻译的成分作为持久性药物靶点。这些研究将为设计新的、更强大的药物提供有用的信息，这些药物针对持续者，以缩短结核病治疗的持续时间。