Brombacher Eva, Urwyler Simon, Ragaz Curdin, Weber Stefan S, Kami Keiichiro, Overduin Michael, Hilbi Hubert
Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
J Biol Chem. 2009 Feb 20;284(8):4846-56. doi: 10.1074/jbc.M807505200. Epub 2008 Dec 17.
The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.
军团病的病原体嗜肺军团菌，通过细胞内增殖/缺陷细胞器运输（Icm/Dot）IV型分泌系统和转运效应蛋白在吞噬细胞中形成复制性液泡，其中一些效应蛋白会破坏宿主的GTP和磷酸肌醇（PI）代谢。Icm/Dot底物SidC通过特异性结合磷脂酰肌醇4-磷酸（PtdIns(4)P）锚定在含军团菌液泡（LCV）的膜上。通过对新型嗜肺军团菌PI结合蛋白进行无偏差筛选，我们确定Rab1鸟嘌呤核苷酸交换因子（GEF）SidM/DrrA是主要的PtdIns(4)P结合蛋白。纯化的SidM特异性且直接地与PtdIns(4)P结合，而与SidM相互作用的Icm/Dot底物LidA优先结合PtdIns(3)P，但也结合PtdIns(4)P，并且嗜肺军团菌Arf1 GEF RalF不与任何PI结合。SidM的PtdIns(4)P结合结构域定位于12 kDa的C末端序列，称为“P4M”（SidM/DrrA的PtdIns4P结合）。分离出的P4M结构域主要是螺旋状的，并且在α-螺旋单体全长蛋白的背景下显示出更高的PtdIns(4)P结合活性。含有P4M的SidM构建体被Icm/Dot功能正常的嗜肺军团菌转运并定位于LCV膜，这表明SidM通过其P4M结构域锚定在LCV上的PtdIns(4)P上。嗜肺军团菌DeltasidM突变株在LCV上显示出显著更多的SidC，这表明SidM和SidC竞争液泡上有限量的PtdIns(4)P。最后，RNA干扰显示LCV上的PtdIns(4)P是由宿主磷脂酰肌醇4-激酶IIIβ特异性形成的。因此，嗜肺军团菌利用磷脂酰肌醇4-激酶IIIβ产生的PtdIns(4)P将效应蛋白SidC和SidM锚定在LCV上。
