Dorer Marion S, Kirton Donald, Bader Joel S, Isberg Ralph R
Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts, USA.
PLoS Pathog. 2006 Apr;2(4):e34. doi: 10.1371/journal.ppat.0020034. Epub 2006 Apr 28.
Legionella pneumophila translocates multiple bacterial effector proteins into host cells to direct formation of a replication vacuole for the bacterium. The emerging consensus is that formation of this compartment involves recruitment of membrane material that traffics between the endoplasmic reticulum (ER) and Golgi. To investigate this model, a targeted approach was used to knock down expression of proteins involved in membrane trafficking, using RNA interference in Drosophila cells. Surprisingly, few single knockdowns of ER-Golgi transport proteins decreased L. pneumophila replication. By analyzing double-stranded RNAs in pairs, combinations were identified that together caused defects in intracellular replication, consistent with the model that membrane traffic funnels into the replication vacuole from multiple sources. In particular, simultaneous depletion of the intermediate compartment and Golgi-tethering factor transport protein particle together with the ER SNARE protein Sec22 reduced replication efficiency, indicating that introduction of lesions at distinct sites in the secretory system reduces replication efficiency. In contrast to knockdowns in secretory traffic, which required multiple simultaneous hits, knockdown of single cytosolic components of ER-associated degradation, including Cdc48/p97 and associated cofactors, was sufficient to inhibit intracellular replication. The requirement for the Cdc48/p97 complex was conserved in mammalian cells, in which replication vacuoles showed intense recruitment of ubiquitinated proteins, the preferred substrates of Cdc48/p97. This complex promoted dislocation of both ubiquitinated proteins and bacterial effectors from the replication vacuole, consistent with the model that maintenance of high-level replication requires surveillance of the vacuole surface. This work demonstrates that L. pneumophila has the ability to gain access to multiple sites in the secretory system and provides the first evidence for a role of the Cdc48/p97 complex in promoting intracellular replication of pathogens and maintenance of replication vacuoles.
嗜肺军团菌将多种细菌效应蛋白转运到宿主细胞中，以指导为该细菌形成复制泡。目前逐渐形成的共识是，这个区室的形成涉及内质网（ER）和高尔基体之间运输的膜材料的募集。为了研究这个模型，采用了一种靶向方法，利用果蝇细胞中的RNA干扰来敲低参与膜运输的蛋白质的表达。令人惊讶的是，很少有内质网-高尔基体转运蛋白的单基因敲低会降低嗜肺军团菌的复制。通过成对分析双链RNA，鉴定出了共同导致细胞内复制缺陷的组合，这与膜运输从多个来源汇入复制泡的模型一致。特别是，中间区室和高尔基体拴系因子转运蛋白颗粒与内质网SNARE蛋白Sec22的同时缺失降低了复制效率，表明在分泌系统的不同位点引入损伤会降低复制效率。与需要多个同时敲低的分泌运输敲低不同，内质网相关降解的单个胞质成分（包括Cdc48/p97和相关辅因子）的敲低足以抑制细胞内复制。Cdc48/p97复合物的需求在哺乳动物细胞中是保守的，在哺乳动物细胞中，复制泡显示出泛素化蛋白的强烈募集，泛素化蛋白是Cdc48/p97的首选底物。这个复合物促进了泛素化蛋白和细菌效应蛋白从复制泡的错位，这与高水平复制需要对泡表面进行监测的模型一致。这项工作表明嗜肺军团菌有能力进入分泌系统的多个位点，并为Cdc48/p97复合物在促进病原体细胞内复制和维持复制泡方面的作用提供了首个证据。
