Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia.
Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.
Cell Microbiol. 2021 Oct;23(10):e13368. doi: 10.1111/cmi.13368. Epub 2021 Jun 8.
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.
嗜肺军团菌的 Dot/Icm 系统对于毒力至关重要，它将大量效应蛋白输送到感染的宿主细胞中，形成含有军团菌的空泡。由于 Dot/Icm 系统的效应蛋白分泌在没有宿主细胞的情况下不会发生，我们假设宿主因素积极参与 Dot/Icm 效应蛋白的易位。在这里，我们采用了高通量、全基因组 siRNA 筛选方法，系统地测试了沉默 18120 个人类基因对 Dot/Icm 效应蛋白 RalF 易位到 HeLa 细胞中的影响。在初级筛选中，我们发现沉默 119 个基因会导致 RalF 的易位增加，而沉默 321 个基因则会导致易位减少。在二次筛选后，有 70 个基因被成功验证为“高可信度”靶点。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显著的过度代表类别。我们进一步表明，两种宿主因子，E2 泛素连接酶 UBE2E1 和 E3 泛素连接酶 CUL7，对于支持 Dot/Icm 易位和 L. pneumophila 细胞内复制是重要的。总之，我们确定了宿主泛素途径对于 L. pneumophila Dot/Icm 效应蛋白易位的效率很重要，这表明宿主来源的泛素连接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。
