Molecular characterization of autophagosomes from the yeast Saccharomyces cerevisiae

军团菌和布鲁氏菌属是引起军团病和布鲁氏菌病的严重病原体。在通过吞噬作用进入宿主细胞后，它们利用自噬机制被自噬体样细胞器吸收。为了避免溶酶体的分解，细菌然后阻止溶酶体与溶酶体融合，并最终触发其成熟为复制细胞器。含有细菌的自噬体在毒力中起着核心作用。因此，这种细胞器的分子表征是确定治疗这些感染的新靶点的一种方法。我们将从已经鉴定的酿酒酵母突变体中分离自噬体，这些突变体会积累自噬体，然后鉴定自噬体特异性膜蛋白和脂质。酵母自噬体标记物的同源性可以用来表征哺乳动物细胞中含细菌的细胞器。自噬体外膜蛋白可能参与了自噬体与溶酶体的融合。由于抑制这种融合对毒力至关重要，因此也计划对这些成分进行表征。自噬体生物发生缺陷的酵母突变体应该在一个新的自噬体前腔室中积累自噬体成分。因此，分析这些突变体中的自噬前体也是鉴定自噬体成分的计划工作的一部分。

Legionella and Brucella spp. are severe pathogens causing Legionnaires disease and brucelliosis. After entering host cells by phagocytosis, they exploit the autophagic machinery to be taken up in an autophagosome-like organelle. To escape lysosomal breakdown the bacteria then prevent its fusion with lysosomes and finally trigger its maturation to a replication organelle. The bacteria-containing autophagosome plays a central role in virulence. Thus molecular characterization of this organelle is a way to identify novel targets for the treatment of these infections. We will isolate autophagosomes from already identified Saccharomyces cerevisiae mutants, which accumulate autophagosomes, and then identify autophagosome specific membrane proteins and lipids. The homologes of the yeast autophagosomal markers then can be used to characterize the bacteria-containing organelles in mammalian cells. Proteins of the autophagosomal outer membrane probably are involved in fusion of autophagosomes with lysosomes. Since inhibition of this fusion is crucial for virulence, also characterization of these components is planned. Yeast mutants defective in autophagosome biogenesis should accumulate autophagosomal components at a novel preautophagosomal compartment. Analysis of the preautophagosome in these mutants, thus is also part of the planned work to identify autophagosomal constituents.