Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole
Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole
复制标题
DOI：
10.1111/cmi.13151
复制
发表时间：
2020-04-01
影响因子：
3.4
通讯作者：
Isberg, Ralph R.
中科院分区：
文献类型：
--
作者：
Anand, Ila S.;Choi, Wonyoung;Isberg, Ralph R.
嗜肺军团菌需要Dot/ICM易位系统在宿主细胞内的空泡室中复制。缺乏转位底物SdhA的菌株在寄主细胞中停留时形成一个可渗透的空泡，使细菌暴露在寄主细胞质中。在原代巨噬细胞中，突变体不利于细胞内生长，由于细菌暴露在胞浆中，导致焦链细胞死亡反应增强。为了了解SdhA如何在细胞内生长过程中保持液泡完整性，我们进行了针对宿主膜运输基因的高通量RNAi筛选，以确定在缺乏SdhA的情况下对抗液泡完整性的因素。耗尽参与内吞和再循环的宿主蛋白导致细胞内生长增强，并在Delta sdhA突变体周围形成较低水平的可渗透空泡。有趣的是参与这些过程的三种不同的Rab GTP酶：Rab11b、Rab8b和Rab5亚型，当耗尽时，导致sdhA突变体周围的液泡完整性增强。受这些RAB调控的蛋白质负责干扰适当的液泡膜维持，因为下游效应器EEA1、Rab11FIP1或VAMP3的耗尽拯救了sdhA突变体的液泡完整性和细胞内生长。为了测试与这些效应器相关的特定囊泡成分可以破坏复制空泡稳定的模型，EEA1和Rab11FIP1显示与野生型(WT)空泡相比，sdhA突变空泡的密度增加。Rab5亚型或Rab11b的缺失减少了这种异常的重新分布。这些发现与SdhA干扰内吞和循环膜运输事件是一致的，这些事件在感染期间破坏了液泡的完整性。
Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the Delta sdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.