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Analysis of phosphatidylinositol mediated molecular mechanisms that related to phagocytosis process of the Entamoeba histolytica.

磷脂酰肌醇介导的溶组织内阿米巴吞噬过程相关分子机制分析。

基本信息

批准号：
20790323
负责人：
TSUKUI Kumiko
金额：
$ 2.75万
依托单位：
National Institute of Infectious Diseases
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2009
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-20790323/
关键词：
原虫感染症イノシトールリン脂質小胞輸送赤痢アメーバ貪食 RhoGEF Rac PtdIns4P

项目摘要

To understand the roles of phosphoinositides [PtdIns] in phagocytosis of parasitic eukaryotes, we examined the interaction of phosphatidylinositol-3-phosphate [PtdIns(3)P] and putative PtdIns-P-binding proteins during phagocytosis in the enteric protozoan parasite Entamoeba histolytica. It was previously shown that phagocytosis in E. histolytica is indispensable for virulence and is inhibited by PtdIns 3-kinase inhibitors. We demonstrated by time-lapse live imaging that during the initiation of phagocytosis, the PtdIns(3)P biomarker GFP-Hrs-FYVE, was translocated to the phagocytic cup, phagosome, and to tunnel-like structures connecting the plasma membrane and phagosomes. E. histolytica possesses 12 FYVE domain-containing proteins (EhFP1-12), 11 of which also contain the RhoGEF/DH domain. Among them EhFP4 was shown to be recruited to the tunnel-like structures and to the proximal region of the phagosome. We further demonstrated that EhFP4 physically interacted with 4 of 10 predominant Rho/Rac small GTPases. Phosphoinositide binding assay showed that EhFP4 unexpectedly bound to PtdIns(4)P via the carboxyl-terminal domain and that the FYVE domain modulates the binding specificity of EhFP4 to PtdIns-P. Expression of the FYVE domain from EhFP4 inhibited phagocytosis while enhancement was observed when mammalian Hrs-FYVE domain was expressed. Altogether, we demonstrated that PtdIns(3)P, PtdIns(4)P and EhFP4 coordinately regulate phagocytosis and phagosome maturation in this parasitic eukaryote.

为了了解磷脂酰肌醇[PtdIns]在寄生性真核生物吞噬作用中的作用，我们研究了肠道原生动物寄生虫溶组织内阿米巴（Entamoeba histolytica）在吞噬作用中磷脂酰肌醇-3-磷酸[PtdIns（3）P]和推定的PtdIns-P结合蛋白的相互作用。以前的研究表明，E.溶组织毒素是毒力不可缺少的，并被PtdIns 3-激酶抑制剂抑制。我们通过延时实时成像证明，在吞噬作用开始期间，PtdIns（3）P生物标志物GFP-Hrs-FYVE移位到吞噬杯、吞噬体以及连接质膜和吞噬体的隧道样结构。E. histolytica具有12个含FYVE结构域的蛋白质（EhFP 1 -12），其中11个还含有RhoGEF/DH结构域。其中EhFP 4被募集到吞噬体的隧道样结构和近端区域。我们进一步证明了EhFP 4与10种主要的Rho/Rac小GTP酶中的4种发生物理相互作用。磷脂酰肌醇结合实验表明，EhFP 4与PtdIns（4）P的结合是通过羧基端结构域实现的，而FYVE结构域调节EhFP 4与PtdIns（4）P的结合特异性。总之，我们证明了PtdIns（3）P、PtdIns（4）P和EhFP 4协调调节这种寄生性真核生物的吞噬作用和吞噬体成熟。