Function of a novel Mycobacterium tuberculosis lipase and its interaction with host proteins

新型结核分枝杆菌脂肪酶的功能及其与宿主蛋白的相互作用

• 批准号: 10686799
• 负责人: Ying Kong
• 金额: $ 7.7万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-19 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10686799
• 关键词: Address; Adipocytes; Affect; Amino Acids; Antibodies; Attenuated; Bacteria; Binding; Biochemical Reaction; Biological Assay; Carbohydrates; Cell Culture Techniques; Cell Nucleus; Cells; Chimeric Proteins; Complement; Data; Docking; Energy-Generating Resources; Enzyme-Linked Immunosorbent Assay; Enzymes; Family; Fatty Acids; Foamy Macrophage; Genes; Growth; Human; Hybrids; Immunoprecipitation; Individual; Infection; Knowledge; Lipase; Lipids; Lipolysis; Lung; Macrophage; Microscopic; Modeling; Morbidity - disease rate; Mus; Mutation; Mycobacterium tuberculosis; Mycolic Acid; Nonesterified Fatty Acids; Nutrient; Pathogenesis; Pathway interactions; Phagosomes; Phospholipases A; Plasmids; Play; Process; Proteins; Public Health; Reaction; Reporting; Research; Role; Site-Directed Mutagenesis; System; Testing; Thin Layer Chromatography; Time; Triglycerides; Tuberculosis; Vimentin; Yeasts; cell envelope; cell type; inhibitor; liquid chromatography mass spectrometry; mortality; mutant; mycobacterial; novel; pathogen; scaffold; tributyrin

ABSTRACT Tuberculosis (TB) is a worldwide public health concern because of its high morbidity and mortality. The causative pathogen of TB, Mycobacterium tuberculosis (Mtb), has a unique mycolic acid-rich cell envelope, and can induce accumulation of lipids in the host cells. Inside cells, it exploits host lipids as important nutrients for its infection and long-term intracellular survival. Emerging evidences support that some lipases secreted by Mtb play vital roles in its intracellular persistence. However, the exact functions of these Mtb-secreted lipases and mechanisms that channel their interactions with host cytosolic proteins remain to be unraveled. Previously, we determined that Mtb Rv1075c belongs to a GDSL-like lipase family with a deacylase activity and hydrolyzes triacetin and tributyrin. The gene-disrupting mutation of rv1075c attenuates Mtb’s intracellular growth in macrophages and reduces bacterial load in the infected mice. Recently, we observed that Rv1075c’s lipase activity was enhanced when macrophage lysate was added into the enzymatic reaction, indicating that some eukaryotic factors contribute to boosting Rv1075c’s lipase activity. Subsequently, we performed an ultimate yeast 2-hybrid to screen for host proteins interacting with Rv1075c. We have identified that vimentin (VIM) is one of the proteins interacting with Rv1075c. It has been reported that VIM in adipocytes forms a scaffold around lipid droplets and VIM is a functional partner of lipase to facilitate lipolysis. Combining these evidences with our data, we hypothesize that Mtb Rv1075c interacts with VIM that scaffolds host lipid droplets in macrophages, and the interaction facilitates Rv1075c’s activity in lipolysis so that Mtb can utilize host lipids as energy source for its intracellular persistence. The objective of our proposed studies is to identify mechanism of Rv1075c in the process of accessing host lipid droplets in macrophages and determine its interaction with host VIM protein and the impact of this interaction on Mtb intracellular growth. We will test this hypothesis by pursuing two specific aims: 1) Identify the mechanism by which Mtb Rv1075c interacts with the eukaryotic cytosolic VIM; 2) Determine whether the Rv1075c-VIM interaction enhances Rv1075c’s lipase and phospholipase A activity and facilitates Mtb intracellular survival in macrophages. The proposed research will uncover mechanism of the Rv1075c-VIM interaction and address fundamental questions about how the Rv1075c-VIM interaction affects Mtb intracellular survival and growth. The results from these studies will expand our knowledge of Mtb’s utilization of host lipids during infection and will lead to discovery of new mechanisms of Mtb pathogenesis.

抽象的 结核病（TB）由于其发病率高和死亡而成为全球公共卫生的关注。因果关系 结核病的病原体，结核分枝杆菌（MTB）具有独特的霉菌酸细胞包膜，可以诱导 脂质在宿主细胞中的积累。在细胞内部，它利用宿主脂质作为其感染的重要营养素 和长期细胞内存活。新兴的证据支持MTB分泌的一些脂肪酶发挥至关重要 在其细胞内持久性中的作用。但是，这些MTB分泌的脂肪酶和机制的确切功能 该引导他们与宿主胞质蛋白的相互作用仍有待解散。以前，我们确定 MTB RV1075C属于具有脱酰基酶活性的GDSL样脂肪酶家族，并水解三乙酸和 Trigutyrin。 RV1075C的基因破裂突变减弱了MTB在巨噬细胞中的细胞内生长 减少受感染小鼠的细菌负荷。最近，我们观察到RV1075C的脂肪酶活性得到了增强 当将巨噬细胞裂解物添加到酶促反应中时，表明某些真核生物因子 有助于提高RV1075C的脂肪酶活性。随后，我们进行了最终的酵母2杂交 屏幕宿主蛋白与RV1075C相互作用。我们已经确定波形蛋白（VIM）是蛋白质之一 与RV1075C相互作用。据报道，脂肪细胞中的VIM在脂质液滴周围形成脚手架， VIM是脂肪酶的功能伙伴，可促进脂肪解。将这些证据与我们的数据相结合，我们 假设MTB RV1075C与VIM相互作用，脚手架寄生在巨噬细胞中的脂质液滴，并且 相互作用促进RV1075C在脂解中的活性，因此MTB可以利用宿主脂质作为其能源 细胞内持久性。我们提出的研究的目的是确定RV1075C的机制 访问巨噬细胞中宿主脂质液滴的过程，并确定其与宿主VIM蛋白的相互作用 这种相互作用对MTB细胞内生长的影响。我们将通过追求两个特定的特定来检验这一假设 目的：1）确定MTB RV1075C与真核胞质VIM相互作用的机制； 2）确定 RV1075C-VIM相互作用是否增强RV1075C的脂肪酶和磷脂酶A活性并促进 MTB巨噬细胞中细胞内存活。拟议的研究将发现RV1075C-VIM的机制 互动和解决有关RV1075C-VIM相互作用如何影响MTB细胞内的基本问题 生存和成长。这些研究的结果将扩大我们对MTB对宿主脂质的利用的了解 在感染过程中，将导致发现MTB发病机理的新机制。