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

为了了解磷酸肌苷[PtdIns]在寄生真核生物吞噬中的作用，我们研究了肠原生动物溶组织内阿米巴寄生虫吞噬过程中磷脂酰肌醇-3-磷酸[PtdIns(3)P]和推测的PtdIns-P结合蛋白的相互作用。以前的研究表明，溶组织芽胞杆菌的吞噬作用对毒力是必不可少的，并且被PtdIns 3-激酶抑制剂所抑制。我们通过延时实时成像证明，在吞噬开始时，PtdIns(3)P生物标志物GFP-Hrs-FYVE被转移到吞噬杯、吞噬体以及连接质膜和吞噬体的隧道状结构中。E. histolytica含有12个含FYVE结构域的蛋白（EhFP1-12），其中11个也含有RhoGEF/DH结构域。其中EhFP4被证明被招募到隧道状结构和吞噬体的近端区域。我们进一步证明EhFP4与10个主要Rho/Rac小gtpase中的4个物理相互作用。磷酸肌肽结合实验表明，EhFP4通过羧基末端结构域与PtdIns(4)P结合，FYVE结构域调节了EhFP4与PtdIns-P的结合特异性。从EhFP4中表达FYVE结构域可抑制吞噬，而在哺乳动物中表达Hrs-FYVE结构域可增强吞噬。总之，我们证明了PtdIns(3)P、PtdIns(4)P和EhFP4在这种寄生真核生物中协同调节吞噬作用和吞噬体成熟。

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.