Determination of the mode of action of the antibiotic pyrazinamide

项目概要/摘要： 结核病（TB）是由结核分枝杆菌（Mtb）引起的，是由于结核病引起的死亡的主要原因。 单一细菌2020年，全球约有1000万人患结核病， 人们死于肺结核。结核病是可以治愈的，但治疗是有毒的和长期的（至少6个月）。就毒品而言， 耐药结核病，治疗持续时间更长（9至24个月），患者生存的机会减少。 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活性而臭名昭著。 体外结核分枝杆菌。PZA最初是在Mtb感染动物中进行的筛选中确定的，这解释了为什么 PZA体外活性差并没有阻止其作为主要抗结核药物的发现。穷人的主要后果 PZA在体外的活性是缺乏对PZA作用模式的理解，这导致降低的能力， 对抗抗PZA结核病。我们最近建立了一个体外模型，其中PZA显示出高的 对结核分枝杆菌的杀菌活性在本申请中，我们将验证基因/通路的参与 通过构建选择的突变体并测量它们在遗传筛选中被鉴定为改变PZA易感性。 对PZA的敏感性此外，我们还将分离自发的PZA抗性克隆，以鉴定Bona 真正的PZA靶点，并在我们的体外模型中测量对PZA的耐药频率。PZA易感性 然后将在TB感染的巨噬细胞模型中验证所产生的突变体的谱。除了有 鉴定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分析结核分枝杆菌的代谢组， 测量PZA对Mtb呼吸链功能的影响。对PZA敏感性改变的突变体将被 除野生型Mtb外，还使用PZA来阐明PZA的作用机制。我们预计，该项目将 这将进一步加深我们对PZA作用机制的了解，并将有助于对抗PZA耐药结核病。我们 我相信这项研究也将为在体外条件下使用更密切相关的感染铺平道路 在这些环境中，为了改进药物的发现和研究，以对抗结核病和其他传染病。

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.