The Dot/Icm system of Legionella pneumophila is essential for virulence and delivers a large repertoire of effectors into infected host cells to create the Legionella containing vacuole. Since the secretion of effectors via the Dot/Icm system does not occur in the absence of host cells, we hypothesised that host factors actively participate in Dot/Icm effector translocation. Here we employed a high-throughput, genome-wide siRNA screen to systematically test the effect of silencing 18,120 human genes on translocation of the Dot/Icm effector, RalF, into HeLa cells. For the primary screen, we found that silencing of 119 genes led to increased translocation of RalF, while silencing of 321 genes resulted in decreased translocation. Following secondary screening, 70 genes were successfully validated as ‘high confidence’ targets. Gene set enrichment analysis of siRNAs leading to decreased RalF translocation, showed that ubiquitination was the most highly overrepresented category in the pathway analysis. We further showed that two host factors, the E2 ubiquitin-conjugating enzyme, UBE2E1, and the E3 ubiquitin ligase, CUL7, were important for supporting Dot/Icm translocation and L. pneumophila intracellular replication. In summary, we identified host ubiquitin pathways as important for the efficiency of Dot/Icm effector translocation by L. pneumophila, suggesting that host-derived ubiquitin-conjugating enzymes and ubiquitin ligases participate in the translocation of Legionella effector proteins and influence intracellular persistence and survival.

中文翻译：

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全基因组遗传筛选确定宿主泛素化对嗜肺军团菌 Dot/Icm 效应易位很重要

嗜肺军团菌的 Dot/Icm 系统对毒力至关重要，并将大量效应物传递到受感染的宿主细胞中以产生军团菌含有液泡。由于在没有宿主细胞的情况下不会通过 Dot/Icm 系统分泌效应物，我们假设宿主因子积极参与 Dot/Icm 效应物易位。在这里，我们采用高通量、全基因组 siRNA 筛选系统测试沉默 18,120 个人类基因对 Dot/Icm 效应器 RalF 易位到 HeLa 细胞中的影响。对于初级筛选，我们发现 119 个基因的沉默导致 RalF 的易位增加，而 321 个基因的沉默导致易位减少。经过二次筛选，70 个基因被成功验证为“高可信度”目标。导致 RalF 易位减少的 siRNA 的基因集富集分析表明，泛素化是通路分析中最过度代表的类别。L. pneumophila细胞内复制。总之，我们确定宿主泛素途径对L. pneumophila的 Dot/Icm 效应物易位的效率很重要，这表明宿主衍生的泛素结合酶和泛素连接酶参与了军团菌效应蛋白的易位并影响细胞内的持久性和存活。 .