Determination of the mode of action of the antibiotic pyrazinamide

项目摘要/摘要： 结核病(TB)是由结核分枝杆菌(Mtb)引起的，是由 单一细菌。2020年，全球约有1000万人患上结核病，其中约150万人 人们死于结核病。结核病是可以治愈的，但治疗是有毒和漫长的(至少6个月)。在毒品的情况下 耐药结核病的治疗持续时间更长(9至24个月)，患者存活的机会也降低了。 在用于治疗结核病的4种一线抗结核药物中，吡嗪酰胺(PZA)因其对结核病的不良反应而臭名昭著 MTB体外培养。PZA最初是在感染结核杆菌的动物身上进行的筛查中发现的，这解释了为什么 PZA在体外的低活性并没有阻止它被发现为一种主要的抗结核病药物。穷人的主要后果 PZA的体外活性是缺乏对PZA作用模式的了解，从而导致PZA的能力降低 与耐PZA结核病作斗争。我们最近建立了一个体外模型，在该模型中，PZA显示出高水平的 对结核分枝杆菌的杀菌活性。在这个应用中，我们将验证基因/通路的参与 通过构建选定的突变体并测量它们的基因筛查来确定改变PZA易感性的基因 对PZA的敏感性。此外，我们还将分离自发的抗PZA克隆，以鉴定Bona 在我们的体外模型中，确定PZA靶点并测量对PZA的耐药性频率。PZA敏感性 随后将在结核感染的巨噬细胞模型中验证所产生的突变体的特征。除 确定PZA易感性的遗传决定因素，我们将描述PZA如何影响结核分枝杆菌 新陈代谢。使用我们的体外模型，我们将使用代谢组学来分析结核分枝杆菌的代谢组，我们将 测量PZA对结核杆菌呼吸链功能的影响。对PZA敏感性发生改变的突变株将是 作为野生型Mtb的补充，用于阐明PZA的作用机制。我们预计这个项目将 增进我们对PZA作用机制的了解，将有助于抗击耐PZA结核病。我们 相信这项研究也将为使用与感染更密切相关的体外条件铺平道路 环境，以改进抗击结核病和其他传染病的药物的发现和研究。

Project summary/Abstract: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and is the leading cause of death due to a single bacterium. In 2020, around 10 million people developed TB worldwide and approximately 1.5 million people died from TB. TB is curable but the treatment is toxic and long (minimum of 6 month). In the case of drug resistant TB, the treatment lasts even longer (9 to 24 months) and the chances of patient survival are reduced. Among the 4 first-line anti-TB drugs used to treat TB, pyrazinamide (PZA) is notorious for its poor activity against Mtb in vitro. PZA was originally identified in a screen performed in Mtb-infected animals which explains why the poor activity of PZA in vitro did not stop its discovery as a major anti-TB drug. The main consequence of the poor activity of PZA in vitro is the lack of understanding of PZA mode of action which results in a reduced capacity to fight against PZA-resistant TB. We recently established an in vitro model in which PZA displays a high bactericidal activity against Mtb. In this application, we will validate the involvement of the genes/pathways identified in a genetic screen to alter PZA susceptibility by constructing selected mutants and measuring their susceptibility to PZA. Moreover, we will also isolate spontaneous PZA-resistant clones in order to identify bona fide PZA targets and measure the frequency of resistance to PZA in our in vitro model. The PZA susceptibility profile of the mutants generated will then be validated in a macrophage model of TB infection. In addition to the identification of genetic determinants for PZA susceptibility, we will characterize how PZA impacts Mtb metabolism. Using our in vitro model, we will analyze the metabolome of Mtb using metabolomics and we will measure the impact of PZA on Mtb respiratory chain functions. Mutants with altered susceptibility to PZA will be used in addition to wild-type Mtb to elucidate the mechanism of action of PZA. We anticipate that this project will advance our knowledge on the mechanism of action of PZA and will help the fight against PZA-resistant TB. We believe this study will also pave the way for the use of in vitro conditions more closely related to the infection settings in order to improve the discovery and the study of drugs to fight against TB and other infectious diseases.