RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics.
RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics.
复制标题
DOI：
10.1371/journal.ppat.0020034
复制
发表时间：
2006-04
期刊：
影响因子：
6.7
通讯作者：
Isberg RR
中科院分区：
文献类型：
--
作者：
Dorer MS;Kirton D;Bader JS;Isberg RR
嗜肺军团菌将多种细菌效应蛋白易位到宿主细胞中，以指导细菌复制泡的形成。新出现的共识是，这种隔室的形成涉及到内质网（ER）和高尔基体之间的膜材料的招募。为了研究该模型，使用靶向方法在果蝇细胞中使用RNA干扰来敲低参与膜运输的蛋白质的表达。令人惊讶的是，很少有单一敲除ER-高尔基体转运蛋白降低L。嗜肺复制通过分析成对的双链RNA，确定了共同引起细胞内复制缺陷的组合，这与膜交通从多个来源漏斗进入复制泡的模型一致。特别是，同时耗尽的中间室和高尔基体栓系因子转运蛋白颗粒与ER SNARE蛋白Sec 22一起降低复制效率，表明在分泌系统中的不同位点引入病变降低复制效率。与需要多次同时命中的分泌交通中的敲除相反，ER相关降解的单个胞质组分（包括Cdc 48/p97和相关辅因子）的敲除足以抑制细胞内复制。在哺乳动物细胞中，Cdc 48/p97复合物的需求是保守的，其中复制空泡显示出强烈的泛素化蛋白，Cdc 48/p97的优选底物的招聘。这种复合物促进了泛素化蛋白和细菌效应子从复制液泡中的移位，这与维持高水平复制需要监视液泡表面的模型一致。这一工作表明L. pneumophila具有进入分泌系统中多个位点的能力，并为Cdc 48/p97复合物在促进病原体的细胞内复制和维持复制空泡中的作用提供了第一个证据。 嗜肺军团菌是引起军团菌性肺炎的病原菌。巨噬细胞吞噬细菌并试图杀死它。军团菌避免这种杀伤，而是在巨噬细胞内生长，利用宿主细胞成分创造一个生长环境。细菌通过将细菌蛋白质（称为效应子）注入宿主细胞来指导其复制生态位的形成。这些效应器劫持宿主功能。在这项研究中，作者确定了细菌劫持的一些宿主途径。作者使用了来自果蝇的巨噬细胞样细胞，因为这些细胞中的蛋白质功能可以使用一种称为RNA干扰的技术来破坏，这种技术破坏了编码蛋白质的RNA信息，导致这些蛋白质的定向丢失。基于先前关于军团菌生物学的知识，选择候选蛋白质进行破坏。这份报告强调了两个观察结果，有助于我们了解军团菌的生物学。令人惊讶的是，一些宿主成分的缺失可以被容忍，因为其他宿主成分可以取代它们。这一规则的一个例外是军团菌复制液泡外部的蛋白质复合物，它可能有助于细菌将其蛋白质运送到宿主细胞中的适当部位。
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. Legionella pneumophila is a pathogenic bacterium that causes Legionnaires pneumonia. Immune cells, called macrophages, engulf the bacterium and attempt to kill it. Legionella avoids this killing and instead grows inside the macrophage, creating a growth niche using host cell components. The bacterium directs the formation of its replication niche by injecting bacterial proteins, called effectors, into the host cell. These effectors hijack host functions. In this study, the authors identify some of the host pathways that the bacterium hijacks. The authors used macrophage-like cells derived from fruit flies because protein function can be disrupted in these cells using a technique called RNA interference, which destroys the RNA messages that encode for proteins, resulting in directed loss of these proteins. Candidate proteins were chosen to disrupt based upon previous knowledge about the biology of Legionella. This report highlights two observations that contribute to our understanding of the biology of Legionella. Surprisingly, the absence of some host components could be tolerated because other host components could take their place. One exception to this rule was a protein complex on the outside of the Legionella replication vacuole that may help the bacterium deliver its proteins to appropriate sites in the host cell.
登录
查看更多内容
影响因子：
3.4
作者：
Cheng, LW;Portnoy, DA
通讯作者：
Portnoy, DA
影响因子：
3.4
作者：
Amer, AO;Swanson, MS
通讯作者：
Swanson, MS
影响因子：
56.9
作者：
Nagai, H;Kagan, JC;Roy, CR
通讯作者：
Roy, CR
影响因子：
11.8
作者：
Boutros, M;Agaisse, H;Perrimon, N
通讯作者：
Perrimon, N
影响因子：
3.5
作者：
Brunger, AT;DeLaBarre, B
通讯作者：
DeLaBarre, B