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性的公共卫生问题。致病 结核病的病原体，结核分枝杆菌（Mtb），具有独特的富含分枝菌酸的细胞包膜，并且可以诱导 脂质在宿主细胞中的积累。在细胞内，它利用宿主脂质作为其感染的重要营养物质 和长期的细胞内存活。新出现的证据支持结核杆菌分泌的一些脂肪酶起着重要作用 在其细胞内的持久性的作用。然而，这些结核分枝杆菌分泌的脂肪酶的确切功能和机制， 但其与宿主胞质蛋白相互作用的通道仍有待解开。此前，我们确定 Mtb Rv 1075 c属于具有脱酰基酶活性的GDSL样脂肪酶家族，并水解三醋精， 三丁酸甘油酯rv 1075 c的基因破坏突变减弱了Mtb在巨噬细胞中的细胞内生长， 减少感染小鼠体内的细菌负荷。最近，我们观察到Rv 1075 c的脂肪酶活性增强 当将巨噬细胞裂解物加入酶促反应中时， 有助于提高Rv 1075 c的脂肪酶活性。随后，我们进行了最终的酵母双杂交， 筛选与Rv 1075 c相互作用的宿主蛋白。我们已经确定，波形蛋白（Vim）是一种蛋白质， 与Rv 1075 c相互作用。据报道，脂肪细胞中的Vim在脂滴周围形成支架， Vim是脂肪酶的功能伴侣，以促进脂肪分解。结合这些证据和我们的数据，我们 假设Mtb Rv 1075 c与Vim相互作用，VIM是巨噬细胞中脂滴的宿主， 相互作用促进Rv 1075 c在脂解中的活性，使得Mtb可以利用宿主脂质作为其能量来源。 胞内持久性我们提出的研究的目的是确定Rv 1075 c在 进入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， 这种相互作用对结核分枝杆菌细胞内生长的影响。我们将通过两个具体的实验来验证这个假设。 目的：1）鉴定结核分枝杆菌Rv 1075 c与真核细胞质Vim相互作用的机制; 2）确定 Rv 1075 c-VIM相互作用是否增强Rv 1075 c的脂肪酶和磷脂酶A活性， 巨噬细胞中结核分枝杆菌的细胞内存活。这项研究将揭示Rv 1075 c-VIM的作用机制 相互作用，并解决有关Rv 1075 c-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利用宿主脂质的了解 并将导致发现新的Mtb发病机制。

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.