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

摘要 结核病(TB)因其高发病率和高死亡率而成为全球关注的公共卫生问题。致使性 结核病的病原体结核分枝杆菌(Mtb)具有独特的富含霉菌酸的细胞膜，可诱导 宿主细胞中脂质的积累。在细胞内，它利用宿主脂类作为其感染的重要营养物质。 以及长期的细胞内存活。新的证据支持结核分枝杆菌分泌的一些脂肪酶起着至关重要的作用 在其细胞内持久性中所起的作用。然而，这些Mtb分泌的脂肪酶的确切功能和机制 它们与宿主胞浆蛋白相互作用的途径仍有待解开。此前，我们确定 Mtb Rv1075c属于类GDSL脂肪酶家族，具有脱酰基酶活性，能水解三乙酸甘油三酯和 三丁酸二酯。Rv1075c基因突变抑制结核分枝杆菌在巨噬细胞内的生长 减少受感染小鼠体内的细菌负荷。最近，我们观察到Rv1075c的脂肪酶活性增强 当巨噬细胞裂解液加入到酶反应中时，表明某些真核因子 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。我们已经鉴定了波形蛋白(Vim)是其中一种蛋白质。 与Rv1075c交互。据报道，脂肪细胞中的vim在脂滴周围形成支架，并 VIM是脂肪酶的功能伙伴，可促进脂肪分解。结合这些证据和我们的数据，我们 假设Mtb Rv1075c与VIM相互作用，VIM是巨噬细胞中脂滴的宿主，而 相互作用促进Rv1075c的脂解活性，使Mtb能够利用宿主脂类作为其能量来源 细胞内持久性。我们建议的研究目的是确定Rv1075c在心脏疾病中的作用机制。 巨噬细胞接触宿主脂滴的过程，并确定其与宿主VIM蛋白和 这种相互作用对结核分枝杆菌胞内生长的影响。我们将通过研究两个具体的例子来验证这个假设 目的：1)确定mtb rv1075c与真核细胞胞浆vim相互作用的机制；2)确定mtb rv1075c与真核细胞内vim的相互作用。 Rv1075c-Vim相互作用是否增强Rv1075c的脂肪酶和磷脂酶A的活性并促进 结核分枝杆菌在巨噬细胞内的存活。拟议的研究将揭示Rv1075c-vim的机制 相互作用并解决关于Rv1075c-vim相互作用如何影响细胞内结核分枝杆菌的基本问题 生存和成长。这些研究的结果将扩大我们对结核分枝杆菌利用宿主脂类的了解 并将有助于发现结核分枝杆菌新的致病机制。

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.