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      Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in Entamoeba histolytica

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          Abstract

          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.

          Author Summary

          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.

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          Most cited references65

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          Heterotrimeric G protein activation by G-protein-coupled receptors.

          Heterotrimeric G proteins have a crucial role as molecular switches in signal transduction pathways mediated by G-protein-coupled receptors. Extracellular stimuli activate these receptors, which then catalyse GTP-GDP exchange on the G protein alpha-subunit. The complex series of interactions and conformational changes that connect agonist binding to G protein activation raise various interesting questions about the structure, biomechanics, kinetics and specificity of signal transduction across the plasma membrane.
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            Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.

            Protein interactions are a fundamental mechanism for the generation of biological regulatory specificity. The study of protein interactions in living cells is of particular significance because the interactions that occur in a particular cell depend on the full complement of proteins present in the cell and the external stimuli that influence the cell. Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragments. Numerous protein interactions have been visualized using the BiFC assay in many different cell types and organisms. The BiFC assay is technically straightforward and can be performed using standard molecular biology and cell culture reagents and a regular fluorescence microscope or flow cytometer.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                November 2012
                November 2012
                15 November 2012
                : 8
                : 11
                : e1003040
                Affiliations
                [1 ]Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
                [2 ]Department of Biochemistry & Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
                [3 ]R. L. Juliano Structural Bioinformatics Core, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
                [4 ]Department of Physiology & Pharmacology, West Virginia University School of Medicine, Robert C. Byrd Health Sciences Center, Morgantown, West Virginia, United States of America
                University of Virginia Health System, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: DEB AJK FSW DPS. Performed the experiments: DEB AJK REM PMG MM BRST. Analyzed the data: DEB AJK REM MM BRST DPS. Contributed reagents/materials/analysis tools: MM. Wrote the paper: DEB AJK DPS.

                [¤]

                Current address: Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana, United States of America.

                Article
                PPATHOGENS-D-12-01439
                10.1371/journal.ppat.1003040
                3499586
                23166501
                a432ec3d-0070-49cc-bff6-fdda5cacf826
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 15 June 2012
                : 3 October 2012
                Page count
                Pages: 17
                Funding
                This work was supported by NIH grant GM082892 (to DPS) and individual F30 NRSA fellowships MH074266 to AJK and DK091978 to DEB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Proteins
                Protein Structure
                Genetics
                Microbiology
                Parasitology

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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