Functional phosphoinositide lipidomics of Legionella-containing vacuoles

军团菌属是在自由生活的阿米巴原虫和肺中的巨噬细胞内生长的环境细菌，从而引起称为“军团病”的严重肺炎。细菌在宿主细胞中的不同的膜结合区室中复制，“含军团菌的空泡”（LCV）。LCV的形成是一个复杂而稳健的过程，其涉及细菌IV型分泌系统和超过100种不同的分泌“效应”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。该项目的目的包括使用生物化学，遗传学和细胞微生物方法对宿主磷脂酰肌醇（PI）脂质和与军团菌属LCV形成有关的其他脂质进行详细的分子分析。为此，我们将纯化完整的LCV并通过质谱法确定PI、脂质组和蛋白质组。为了从功能上表征PI在LCV形成中的作用，我们将（i）建立并比较嗜肺军团菌和长滩军团菌的纯化完整LCV的PI和脂质目录，（ii）实时分析LCV上PI和PI代谢酶的动力学，（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.