Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in <em>Entamoeba histolytica</em>

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.异三聚体G蛋白信号通路（Heterotrimeric G-protein signaling pathways）是机体生理活动的核心组成部分，其中多条通路可通过药理学手段进行干预调控。本研究在阿米巴结肠炎的致病菌——溶组织内阿米巴（Entamoeba histolytica）中，成功鉴定出功能性异三聚体G蛋白亚基。溶组织内阿米巴的Gα亚基EhGα1具备经典的核苷酸循环特性，且可通过典型的核苷酸状态选择性结合模式，与EhGβγ二聚体以及候选效应蛋白EhRGS-RhoGEF发生相互作用。与之迥异的是，EhGα1的晶体结构揭示了其独特的分子特征，其分类地位并不属于常规的哺乳动物Gα亚家族。以诱导型方式过表达野生型EhGα1的溶组织内阿米巴滋养体，其杀伤宿主细胞的能力显著增强，该表型可能完全或部分源于宿主细胞黏附效率的提升。过表达EhGα1的滋养体同时表现出更强的跨基质胶（Matrigel）屏障迁移能力，这一效应可能由基础迁移状态的改变所介导。而诱导表达显性负性EhGα1突变体，则会呈现出完全相反的表型。转录组学研究结果显示，异三聚体G蛋白表达失衡对致病相关滋养体行为的调控，涵盖了毒力因子的转录调控以及半胱氨酸蛋白酶运输过程的改变。综上，本研究表明溶组织内阿米巴拥有一条趋异的异三聚体G蛋白信号轴，该信号轴可精准调控与该病原体致病过程相关的多项关键细胞进程。