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.
Entamoeba histolytica causes an estimated 50 million intestinal infections and 100,000 deaths per year worldwide. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, constituting a signaling pathway which, when perturbed, is seen to regulate multiple cellular processes required for pathogenesis. Like mammalian counterparts, EhGα1 forms a heterotrimer with EhGβγ that is dependent on guanine nucleotide exchange and hydrolysis. Despite engaging a classical G-protein effector, EhRGS-RhoGEF, EhGα1 diverges from mammalian Gα subunits and cannot be classified within mammalian Gα subfamilies, as highlighted by distinct structural features in our crystal structure of EhGα1 in the inactive conformation. To identify roles of G-protein signaling in pathogenesis-related cellular processes, we engineered trophozoites for inducible expression of EhGα1 or a dominant negative mutant, finding that G-protein signaling perturbation affects host cell attachment and the related process of contact-dependent killing, as well as trophozoite migration and Matrigel transmigration. A transcriptomic comparison of our engineered strains revealed differential expression of known virulence-associated genes, including amoebapores and cytotoxic cysteine proteases. The expression data suggested, and biochemical experiments confirmed, that cysteine protease secretion is altered upon G-protein overexpression, identifying a mechanism by which pathogenesis-related trophozoite behaviors are perturbed. In summary, E. histolytica encodes a vital heterotrimeric G-protein signaling pathway that is likely amenable to pharmacologic manipulation.