Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts.
Cell Microbiol. 2020 Apr;22(4):e13151. doi: 10.1111/cmi.13151.
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.
嗜肺军团菌需要 Dot/Icm 易位系统在宿主细胞的空泡腔内进行复制。在宿主细胞内居留期间，缺乏易位底物 SdhA 的菌株会形成一个可渗透的空泡，使细菌暴露于宿主细胞质中。在原代巨噬细胞中，由于细菌暴露于细胞质中，突变体在细胞内生长缺陷，会引发细胞焦亡死亡反应。为了了解 SdhA 在细胞内生长过程中如何维持空泡完整性，我们针对宿主膜转运基因进行了高通量 RNAi 筛选，以鉴定在缺乏 SdhA 的情况下拮抗空泡完整性的因素。耗尽参与内吞摄取和再循环的宿主蛋白会导致细胞内生长增强，而围绕 ΔsdhA 突变体的可渗透空泡数量减少。在这些过程中涉及到三种不同的 Rab GTPases：Rab11b、Rab8b 和 Rab5 同工型，它们的耗尽会导致 sdhA 突变体周围的空泡完整性增强。受这些 Rab 调节的蛋白质负责干扰适当的空泡膜维持，因为耗尽下游效应物 EEA1、Rab11FIP1 或 VAMP3 可挽救 sdhA 突变体的空泡完整性和细胞内生长。为了测试与这些效应物相关的特定囊泡成分可能会破坏复制空泡的模型，与野生型（WT）空泡相比，EEA1 和 Rab11FIP1 在 sdhA 突变体空泡周围的密度增加。Rab5 同工型或 Rab11b 的耗尽减少了这种异常再分布。这些发现与 SdhA 干扰内吞和再循环膜转运事件一致，这些事件会在感染过程中破坏空泡完整性。
