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 ΔsdhA 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.

中文翻译：

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内吞和再循环运输途径的成分干扰含军团菌的液泡的完整性。

嗜肺军团菌需要Dot / Icm易位系统才能在宿主细胞内的液泡隔室中复制。缺少易位底物SdhA的菌株在驻留于宿主细胞期间会形成可渗透的液泡，从而使细菌暴露于宿主细胞质。在原代巨噬细胞中，突变体对于细胞内生长是有缺陷的，由于细菌暴露于胞质溶胶而引起了焦细胞死亡。为了了解SdhA如何在细胞内生长期间保持液泡完整性，我们针对宿主膜运输基因进行了高通量RNAi筛选，以鉴定在不存在SdhA的情况下拮抗液泡完整性的因子。参与内吞摄取和再循环的宿主蛋白的消耗导致细胞内生长增强和ΔsdhA突变体周围的可渗透液泡水平降低。感兴趣的是涉及这些过程的三种不同的Rab GTPases：Rab11b，Rab8b和Rab5同工型，它们在耗尽时会导致sdhA突变体周围的液泡完整性增强。由这些Rab调控的蛋白质负责干扰适当的液泡膜维持，因为下游效应子EEA1，Rab11FIP1或VAMP3的耗竭挽救了液泡完整性和sdhA突变体的细胞内生长。为了测试与这些效应子相关的特定囊泡成分可能起到破坏复制液泡稳定性的模型，与野生型（WT）液泡相比，EEA1和Rab11FIP1的sdhA突变液泡密度更高。Rab5亚型或Rab11b的消耗减少了这种异常的重新分布。