Functional phosphoinositide lipidomics of Legionella-containing vacuoles

军团菌属。是一种环境细菌，生长在自由生活的阿米巴和肺内巨噬细胞内，从而导致一种被称为“军团病”的严重肺炎。细菌在宿主细胞中复制，在不同的膜结合的隔间，即“含军团菌的空泡”(LCV)。LCV的形成是一个复杂而强大的过程，它涉及到一个IV型细菌分泌系统和100多种不同分泌的“效应器”蛋白。这些蛋白质被注入宿主细胞，在那里它们调节信号转导和膜动力学。本项目的目标包括使用生化、遗传学和细胞微生物方法对宿主磷脂酰肌醇(PI)脂类和其他与军团菌LCV形成有关的脂类进行详细的分子分析。为此，我们将提纯完整的LCV，并用质谱仪测定PI、脂体和蛋白质组。为了从功能上表征PI在LCV形成中的作用，我们将(I)建立和比较嗜肺军团菌和长军团菌纯化的完整LCV的PI和脂质目录，(Ii)实时分析LCV上PI和PI代谢酶的动态，(Iii)表征与LCV形成有关的PI激酶和PI磷酸酶，以及(Iv)研究PI在LCV形成中的功能作用。

Legionella spp. are environmental bacteria that grow within free-living amoebae and within macrophages in the lung, thereby causing a severe pneumonia termed “Legionnaires’ disease”. The bacteria replicate in host cells in distinct membrane-bound compartments, the “Legionella-containing vacuoles” (LCVs). Formation of LCVs is a complex and robust process, which involves a bacterial type IV secretion system and more than 100 different secreted “effector” proteins. These proteins are injected into host cells, where they modulate signal transduction and membrane dynamics. The aims of this project comprise a detailed molecular analysis using biochemical, genetic and cellular microbial methods of host phosphoinositide (PI) lipids and other lipids implicated in LCV formation by Legionella spp. To this end, we will purify intact LCVs and determine the PIs, the lipidome and the proteome by mass spectrometry. To functionally characterize the role of PIs in the formation of LCVs, we will (i) establish and compare PI and lipid catalogs of purified intact LCVs of Legionella pneumophila and Legionella longbeachae, (ii) analyze the dynamics of PIs and PImetabolizing enzymes on LCVs in real-time, (iii) characterize PI kinases and PI phosphatases implicated in LCV formation, and (iv) study the functional role of PIs for LCV formation.