The Legionella longbeachae Icm/Dot Substrate SidC Selectively Binds Phosphatidylinositol 4-Phosphate with Nanomolar Affinity and Promotes Pathogen Vacuole-Endoplasmic Reticulum Interactions
The Legionella longbeachae Icm/Dot Substrate SidC Selectively Binds Phosphatidylinositol 4-Phosphate with Nanomolar Affinity and Promotes Pathogen Vacuole-Endoplasmic Reticulum Interactions
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DOI：
10.1128/iai.01685-14
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发表时间：
2014-10-01
影响因子：
3.1
通讯作者：
Hilbi, Hubert
中科院分区：
文献类型：
--
作者：
Dolinsky, Stephanie;Haneburger, Ina;Hilbi, Hubert
军团菌属引起严重的肺炎军团病。环境细菌在自由生活的阿米巴原虫和人肺泡巨噬细胞中的细胞内复制，这些阿米巴原虫和人肺泡巨噬细胞位于被称为含军团菌空泡（LCV）的独特的内质网（ER）衍生区室中。LCV的形成需要细菌Icm/Dot IV型分泌系统（T4 SS），其将过多的不同“效应”蛋白易位到宿主细胞中，其中一些通过结合磷酸肌醇（PI）脂质而锚于病原体液泡。在这里，我们确定了无偏下拉分析在军团菌longbeachae裂解物的111 kDa SidC同源物作为主要的磷脂酰肌醇4-磷酸[PtdIns（4）P]结合蛋白。将PI结合结构域定位于20-kDa P4 C [ PtdIns（4）P binding of SidC]片段。等温滴定量热法表明L. longbeachae（SidC（Llo））以71 nM的Kd（解离常数）结合PtdIns（4）P，这比嗜肺军团菌的SidC直向同源物（SidC（Lpn））低3至4倍。用L. longbeachae、内源性SidC（Llo）或异位产生的SidC（Lpn）以Icm/Dot依赖的方式定位于PtdIns（4）P阳性LCV。安湖longbeachae Delta sidC缺失突变体在盘基网柄菌阿米巴LCV中的钙连接蛋白募集受到损害，并在卡氏棘阿米巴中被野生型细菌竞争。通过SidCLlo或其直系同源物SidCLpn和SdcA（Lpn）恢复钙连接蛋白募集。相反，钙连接蛋白募集恢复SidCLlo在L。嗜肺菌缺乏cDNAC和sdcA。总之，生物化学、遗传学和细胞生物学数据表明，SidCLlo是一个L。longbeachae效应子通过P4 C结构域以高亲和力结合LCV上的PtdIns（4）P，促进ER募集到LCV，从而在病原体-宿主相互作用中发挥作用。
Legionella spp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in free-living amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different "effector" proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays in Legionella longbeachae lysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4) P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [ PtdIns(4) P binding of SidC] fragment. Isothermal titration calorimetry revealed that SidC of L. longbeachae (SidC(Llo)) binds PtdIns(4) P with a K-d (dissociation constant) of 71 nM, which is 3 to 4 times lower than that of the SidC orthologue of Legionella pneumophila (SidC(Lpn)). Upon infection of RAW 264.7 macrophages with L. longbeachae, endogenous SidC(Llo) or ectopically produced SidC(Lpn) localized in an Icm/Dot-dependent manner to the PtdIns(4) P-positive LCVs. An L. longbeachae Delta sidC deletion mutant was impaired for calnexin recruitment to LCVs in Dictyostelium discoideum amoebae and out-competed by wild-type bacteria in Acanthamoeba castellanii. Calnexin recruitment was restored by SidCLlo or its orthologues SidCLpn and SdcA(Lpn). Conversely, calnexin recruitment was restored by SidCLlo in L. pneumophila lacking sidC and sdcA. Together, biochemical, genetic, and cell biological data indicate that SidCLlo is an L. longbeachae effector that binds through a P4C domain with high affinity to PtdIns(4) P on LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions.