Mycobacterium tuberculosis carbon metabolism in vivo

结核分枝杆菌体内碳代谢

基本信息

批准号：
7446585
负责人：
John D. McKinney
金额：
$ 27万
依托单位：
ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-15 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7446585
关键词：
3-nitropropionate Aerosols Amination Attenuated Bacteria Candidiasis Carbon Catabolism Cavia Cell Death Cells Chromosomes Chronic Coenzyme A Communicable Diseases Enzymes Event Exposure to Fatty Acids Genes Glucose Glycine Glyoxylates Goals Growth Human In Vitro Infection Infusion procedures Invaded Invasive Isocitrate Lyase Knock-out Label Laboratories Malate Synthase Metabolic Metabolic Pathway Metabolism Microbe Microfluidics Mus Mycobacterium tuberculosis NMR Spectroscopy Other Biochemical Pathway Oxidoreductase Pathway interactions Pharmacologic Substance Phenotype Prokaryotic Cells Propionates Reaction Reporting Research Research Personnel Role Suppressor Mutations Testing Therapeutic Time Tissues Toxic effect Tracer Translations Tuberculosis Vertebrates Video Microscopy Virulence antimicrobial drug attenuation drug development fatty acid metabolism feeding fungus glyoxylate in vivo inhibitor/antagonist interest killings macrophage microorganism mutant novel oxidation paralogous gene pathogen prevent response size small molecule tool tuberculosis drugs

项目摘要

DESCRIPTION (provided by applicant): Invasive bacteria must acquire and assimilate carbon substrates from host tissues in order to replicate and persist in vivo. Metabolic pathways that are essential for bacterial survival during infection are potentially interesting targets for antimicrobial drug development. The glyoxylate cycle, which is required for metabolism of fatty acid substrates, is widespread among prokaryotes but absent in vertebrates. Previously we reported that isocitrate lyase 1 (ICL1) and 2 (ICL2), which catalyze the first unique reaction in the glyoxylate cycle, are jointly required for Mycobacterium tuberculosis (MTB) growth, survival, and virulence in vivo. Recently we discovered that fatty acids are markedly toxic towards ICL-deficient bacteria, even when metabolizable substrates such as glucose are present. These observations suggest that fatty acids are catabolized by MTB during infection and the glyoxylate cycle is required to prevent the accumulation of a toxic metabolic intermediate(s). We propose three Specific Aims to test this hypothesis. In AIM 1 we will delete the MTB glcB gene encoding malate synthase, which catalyzes the second unique reaction in the glyoxylate cycle. We predict that growth, survival, and virulence of the glcB mutant will be attenuated in ex vivo-infected macrophages and in aerosol-infected mice and guinea pigs. We predict that the glcB mutant will be unable to grow on fatty acids in vitro and will be killed by them. In AIM 2 we will delete the MTB fadB1, fadB2, and fadB3 genes encoding paralogs of hydroxyacyl-CoA dehydrogenase, which catalyzes the penultimate step in the fatty acid beta-oxidation cycle. We predict that growth, survival, and virulence of the fadB1-3 triple-knockout mutant will be attenuated in macrophages, mice, and guinea pigs. We predict that the fadB1-3 mutant will be unable to grow on fatty acid substrates in vitro but will not be killed by them, due to the mutant's inability to degrade fatty acids. In AIM 3 we will focus on the mechanism of fatty acid toxicity towards ICL-deficient bacteria. We will identify suppressor mutations that allow ICL-deficient bacteria to grow in the presence of fatty acids. We will use 13C nuclear magnetic resonance spectroscopy (13C-NMR) to profile metabolite pools in wild-type versus ICL-deficient bacteria fed 13C-labeled fatty acids. Our goal is to identify a potentially toxic metabolite(s) derived from fatty acid catabolism that accumulates in the absence of ICL. Lastly, we will use microfluidics and video microscopy to analyze the real-time single-cell response of ICL-deficient bacteria after infusion of fatty acids. The studies proposed in this application will elucidate the role of MTB fatty acid metabolism during infection, and could potentially identify novel targets for drug development.

描述（由申请人提供）：入侵细菌必须从宿主组织获取并同化碳底物，以便在体内复制和持续存在。感染期间细菌生存所必需的代谢途径是抗菌药物开发的潜在有趣目标。脂肪酸底物代谢所需的乙醛酸循环在原核生物中广泛存在，但在脊椎动物中不存在。之前我们报道过异柠檬酸裂解酶 1 (ICL1) 和 2 (ICL2) 催化乙醛酸循环中的第一个独特反应，是结核分枝杆菌 (MTB) 体内生长、存活和毒力共同需要的。最近我们发现，即使存在葡萄糖等可代谢底物，脂肪酸对 ICL 缺陷的细菌也具有明显的毒性。这些观察结果表明，脂肪酸在感染期间被 MTB 分解代谢，并且需要乙醛酸循环来防止有毒代谢中间体的积累。我们提出三个具体目标来检验这一假设。在 AIM 1 中，我们将删除编码苹果酸合酶的 MTB glcB 基因，该基因催化乙醛酸循环中的第二个独特反应。我们预测，glcB 突变体的生长、存活和毒力在离体感染的巨噬细胞以及气溶胶感染的小鼠和豚鼠中都会减弱。我们预测glcB突变体将无法在体外依靠脂肪酸生长并会被脂肪酸杀死。在 AIM 2 中，我们将删除编码羟酰辅酶 A 脱氢酶旁系同源物的 MTB fadB1、fadB2 和 fadB3 基因，该基因催化脂肪酸 β-氧化循环中的倒数第二步。我们预测 fadB1-3 三重敲除突变体在巨噬细胞、小鼠和豚鼠中的生长、存活和毒力将减弱。我们预测 fadB1-3 突变体将无法在体外在脂肪酸底物上生长，但不会被脂肪酸底物杀死，因为该突变体无法降解脂肪酸。在 AIM 3 中，我们将重点研究脂肪酸对 ICL 缺陷细菌的毒性机制。我们将鉴定出抑制突变，使 ICL 缺陷的细菌能够在脂肪酸存在的情况下生长。我们将使用 13C 核磁共振波谱 (13C-NMR) 来分析喂食 13C 标记脂肪酸的野生型细菌与 ICL 缺陷型细菌的代谢库。我们的目标是确定一种源自脂肪酸分解代谢的潜在有毒代谢物，该代谢物在没有 ICL 的情况下会积累。最后，我们将使用微流体和视频显微镜来分析 ICL 缺陷细菌在注入脂肪酸后的实时单细胞反应。本申请中提出的研究将阐明 MTB 脂肪酸代谢在感染过程中的作用，并有可能确定药物开发的新靶点。