Localization of Phosphatidylinositol (3,4,5)-Trisphosphate to Phagosomes in Entamoeba histolytica Achieved Using Glutathione S-Transferase- and Green Fluorescent Protein-Tagged Lipid Biosensors
Localization of Phosphatidylinositol (3,4,5)-Trisphosphate to Phagosomes in Entamoeba histolytica Achieved Using Glutathione S-Transferase- and Green Fluorescent Protein-Tagged Lipid Biosensors
复制标题
DOI：
10.1128/iai.00719-09
复制
发表时间：
2010-01-01
影响因子：
3.1
通讯作者：
Temesvari, Lesly A.
中科院分区：
文献类型：
--
作者：
Byekova, Yevgeniya A.;Powell, Rhonda R.;Temesvari, Lesly A.
溶组织内阿米巴是一种肠道原虫寄生虫，可引起阿米巴痢疾和肝脓肿。寄生虫的吞噬作用是一个关键的毒力过程，因为它是组织入侵和慢性感染建立的先决条件。虽然许多调节吞噬相关信号事件的蛋白质在溶组织埃希氏菌中的作用已经确定，但在这种寄生虫中，脂类在这一细胞过程中的功能仍然很大程度上未知。在其他系统中，磷脂酰肌醇(3，4，5)-三磷酸(PIP3)是磷脂酰肌醇3激酶(PI3-K)活性的主要产物，对吞噬作用是必不可少的。Pleckstrin同源(PH)结构域是与PIP3特异结合的蛋白质结构域。在这项研究中，我们利用谷胱甘肽S转移酶(GST)和绿色荧光蛋白(GFP)标记的PH结构域作为脂质生物传感器来表征PIP3在不同内吞过程中的时空分布。在接触红细胞的滋养体中，PIP3特异的生物传感器在延伸的伪足和吞噬体杯中积累，但在摄取液体标记葡聚糖的过程中不定位于胞吞细胞室。我们的结果表明，PIP3参与了溶组织埃希氏菌吞噬小体形成的早期阶段。此外，我们还证明了PIP3存在于溶组织埃希氏菌的质膜上，并且与哺乳动物细胞不同，这些水平不会被血清撤除所消除。最后，在滋养体中表达PH结构域抑制了红细胞的吞噬作用并增强了运动能力，为PI3-激酶信号在这些过程中的作用提供了遗传学证据。
Entamoeba histolytica is an intestinal protozoan parasite that causes amoebic dysentery and liver abscess. Phagocytosis by the parasite is a critical virulence process, since it is a prerequisite for tissue invasion and establishment of chronic infection. While the roles of many of the proteins that regulate phagocytosis-related signaling events in E. histolytica have been characterized, the functions of lipids in this cellular process remain largely unknown in this parasite. In other systems, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a major product of phosphoinositide 3 kinase (PI3-kinase) activity, is essential for phagocytosis. Pleckstrin homology (PH) domains are protein domains that specifically bind to PIP3. In this study, we utilized glutathione S-transferase (GST)- and green fluorescent protein (GFP)-labeled PH domains as lipid biosensors to characterize the spatiotemporal aspects of PIP3 distribution during various endocytic processes in E. histolytica. PIP3-specific biosensors accumulated at extending pseudopodia and in phagosomal cups in trophozoites exposed to erythrocytes but did not localize to pinocytic compartments during the uptake of a fluid-phase marker, dextran. Our results suggest that PIP3 is involved in the early stages of phagosome formation in E. histolytica. In addition, we demonstrated that PIP3 exists at high steady-state levels in the plasma membrane of E. histolytica and that these levels, unlike those in mammalian cells, are not abolished by serum withdrawal. Finally, expression of a PH domain in trophozoites inhibited erythrophagocytosis and enhanced motility, providing genetic evidence supporting the role of PI3-kinase signaling in these processes in E. histolytica.