Molecular characterization of autophagosomes from the yeast Saccharomyces cerevisiae

军团菌和布鲁氏菌。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一个类似自噬小体的细胞器吸收。为了避免溶酶体的分解，细菌会阻止其与溶酶体的融合，最终触发其成熟为复制细胞器。含有细菌的自噬小体在毒力中起着核心作用。因此，这种细胞器的分子特征是确定治疗这些感染的新靶点的一种方法。我们将从已鉴定的积累自噬小体的酿酒酵母突变株中分离自噬小体，然后鉴定自噬小体特有的膜蛋白和脂类。酵母自噬标记的同源物可以用来表征哺乳动物细胞中含有细菌的细胞器。自噬外膜蛋白可能参与自噬小体与溶酶体的融合。由于抑制这种融合对毒力至关重要，因此还计划对这些成分进行表征。在自噬小体生物发生方面有缺陷的酵母突变株应该在新的自噬前隔室积累自噬小体成分。因此，分析这些突变体中的自噬前体体也是鉴定自噬体组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.