Leishmania major promastigote entry of the of the autophagic compartment and amastigote escape from the parasitophorous vacuole

在最初的三年里，我们将(i)利用最先进的高压冷冻和电子断层扫描技术，观察巨噬细胞中含有寄生液泡的利什曼原虫（利什曼原虫），重点研究巨噬细胞与自噬的相互作用，以及巨噬细胞从寄生液泡中逃逸；（ii）我们将尝试降低巨噬细胞系和原代巨噬细胞的自噬和溶酶体成熟；（iii）我们将建立一种新的相关电子显微镜检测方法。在第二个3年资助期内(i)，我们的目标是解剖导致promastigote进入自噬途径和amastigoite逃逸的分子基础。（ii）我们将把相关的电子显微技术应用于我们质谱筛选的新目标蛋白。此外，我们将测试已知阻断吞噬溶酶体成熟的L. donovani和已知诱导大型寄生液泡的L. amazonensis。（三）此外，我们将重点研究人类中性粒细胞中的寄生液泡，这些液泡只能被promastigotes感染，树突状细胞只能被amastigotes感染。

During the first three years we will (i) visualize the leishmania major containing parasitophorous vacuoles in human macrophages using state of the art high pressure freezing and electron tomography focusing on promastigote interactions with autophagy and amastigote escape from the parasitophorous vacuoles (ii) we will try to down regulate autophagy and lysosomal maturation in macrophage cell lines and primary macrophages (iii) we will establish a new assay for correlative electron microscopy. During the second 3-year funding period (i) we aim to dissect the molecular basis leading to promastigote entry of the autophagic pathway and amastigote escape. (ii) We will apply the correlative electron microcopy technique on new target proteins coming from our mass spectrometry screen. In addition we will test L. donovani known to block phagolysosomal maturation and L. amazonensis known to induce large parasitophorous vacuoles. (iii) Moreover we will focus on the parasitophorous vacuoles in human neutrophils that can only be infected with promastigotes and dendritic cells that can only be infected with amastigotes.