Host pathways important for Coxiella burnetii infection revealed by genome-wide RNA interference screening.
Host pathways important for Coxiella burnetii infection revealed by genome-wide RNA interference screening.
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DOI：
10.1128/mbio.00606-12
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发表时间：
2013-01-29
期刊：
影响因子：
6.4
通讯作者：
Roy CR
中科院分区：
文献类型：
--
作者：
McDonough JA;Newton HJ;Klum S;Swiss R;Agaisse H;Roy CR
贝氏柯克斯体是一种在溶酶体样空泡内复制的细胞内病原体。Dot/Icm型IVB分泌系统被贝氏梭菌用于将效应蛋白易位到宿主细胞质中，其可能调节宿主因子功能。为了鉴定贝氏隐孢子虫细胞内生长所需的宿主决定簇，使用通过小干扰RNA（siRNA）的基因沉默进行全基因组筛选。在siRNA转染的HeLa细胞中通过免疫荧光显微镜测量贝氏隐孢子虫的复制。新鉴定的宿主因子包括逆转录酶复合物的组分，其介导内吞途径和高尔基体之间的货物循环。降低逆转录聚合物运载物-衔接子VPS 26-VPS 29-VPS 35复合物或逆转录聚合物相关的分选连结蛋白的水平消除了贝氏梭菌复制。当沉默时，几个基因导致贝氏梭菌感染细胞内的空泡扩大或空泡数量增加。编码syntaxin-17的STX 17基因的沉默导致含有贝氏隐球菌的液泡的同型融合中的显著缺陷，表明syntaxin-17在调节该过程中的作用。最后，沉默贝氏隐孢子虫复制所需的宿主基因与Dot/Icm效应子易位中的缺陷相关，而沉默影响液泡形态但不影响复制的基因不影响Dot/Icm易位。这些数据表明，贝氏梭菌液泡成熟对于产生允许Dot/Icm功能的生态位是重要的。因此，全基因组筛选揭示了参与贝氏梭菌感染期间发生的顺序事件的宿主决定因素，所述顺序事件由细菌摄取、液泡运输和酸化、Dot/Icm系统的激活、液泡的同型融合和细胞内复制定义。人类Q热是由贝氏柯克斯体引起的。感染贝氏梭菌的特征是其独特的能力，即在细胞内的大空泡区室中复制，类似于溶酶体的苛刻酸性环境。其发病机制的核心是通过Dot/Icm型IVB分泌系统将细菌效应蛋白递送到宿主细胞胞质溶胶中。这些蛋白质可以与宿主因子相互作用并操纵宿主因子，从而导致细菌在其中生长的液泡的产生和维持。在人类细胞中使用高通量全基因组筛选，我们确定了对贝氏隐孢子虫感染的几个方面很重要的宿主因素，包括促进空泡扩张的空泡运输和膜融合事件。此外，我们表明，成熟的贝氏梭菌液泡是必要的，创造一个环境允许的点/Icm交付的细菌效应蛋白进入宿主胞质溶胶。
Coxiella burnetii is an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used by C. burnetii to translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required for C. burnetii intracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication of C. burnetii was measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogated C. burnetii replication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles within C. burnetii-infected cells. Silencing of the STX17 gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containing C. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed for C. burnetii replication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate that C. burnetii vacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur during C. burnetii infection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication. Q fever in humans is caused by the bacterium Coxiella burnetii. Infection with C. burnetii is marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets of C. burnetii infection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of the C. burnetii vacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol.