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）因其高发病率和死亡率而成为世界范围内的公共卫生问题。 结核病的病原体结核分枝杆菌（Mtb）具有独特的富含分枝酸的细胞膜，可以诱导 在宿主细胞内积累脂质。在细胞内，它利用宿主脂质作为其感染的重要营养物质。 新出现的证据表明，结核分枝杆菌分泌的一些脂肪酶发挥着至关重要的作用。 然而，这些 Mtb 分泌的脂肪酶的确切功能和机制。 此前，我们确定了它们与宿主细胞质蛋白相互作用的通道仍有待阐明。 Mtb Rv1075c 属于 GDSL 样脂肪酶家族，具有脱酰酶活性并水解三醋精， rv1075c 的基因破坏突变会减弱 Mtb 在巨噬细胞和细胞内的生长。 最近，我们观察到 Rv1075c 的脂肪酶活性增强。 当巨噬细胞裂解物加入到酶促反应中时，表明一些真核因子 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。 筛选与 Rv1075c 相互作用的宿主蛋白，我们已确定波形蛋白 (VIM) 是其中一种蛋白。 据报道，脂肪细胞中的 VIM 与 Rv1075c 相互作用，在脂滴周围形成支架。 VIM 是脂肪酶的功能伙伴，可促进脂肪分解。将这些证据与我们的数据相结合，我们发现。 Mtb Rv1075c 与 VIM 相互作用，VIM 是巨噬细胞中宿主脂滴的支架，并且 相互作用促进了 Rv1075c 的脂解活性，使得 Mtb 可以利用宿主脂质作为其能量来源 我们提出的研究的目的是确定 Rv1075c 在细胞内的持久性机制。 访问巨噬细胞中宿主脂滴的过程并确定其与宿主 VIM 蛋白的相互作用 我们将通过两个具体的研究来检验这种相互作用对 Mtb 细胞内生长的影响。 目标： 1) 确定 Mtb Rv1075c 与真核细胞质 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 在巨噬细胞中的细胞内存活。拟议的研究将揭示 Rv1075c-VIM 的机制。 相互作用并解决有关 Rv1075c-VIM 相互作用如何影响 Mtb 细胞内的基本问题 这些研究的结果将扩大我们对结核分枝杆菌利用宿主脂质的认识。 感染期间，将导致发现 Mtb 发病机制的新机制。