Heterotrimeric G-protein signaling is critical to pathogenic processes in Entamoeba histolytica

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.

Figure 1. E. histolytica G-protein subunits form a heterotrimer in a nucleotide-dependent manner.

Interactions between Gβ and Gγ subunits were detected with split-YFP protein complementation in COS-7 cells. (A) Human Gβ1 heterodimerized with human Gβ2, but not with E. histolytica Gγ subunits. (B) EhGβ1 interacts with EhGγ1 or EhGγ2 when co-expressed with EhGα1. (C) G-protein heterotrimer formation in the presence of excess GDP (“D”) or the non-hydrolyzable GTP analog, GTPγS (“T”), was examined with co-immunoprecipitation. EhGβ1 and EhGγ1 or EhGγ2 interacted selectively with EhGα1 in its GDP-bound, inactive state. Error bars represent standard error of the mean for three experiments. * represents statistically significant difference from zero, as determined by 95% confidence intervals excluding zero.

Figure 2. EhGα1 cycles between an active, GTP-bound state and an inactive, GDP-bound state.

(A) EhGα1 bound non-hydrolyzable GTPγS as determined by radionucleotide binding. The observed exchange rate, k_obs = 0.27 min⁻¹ ±0.06, indicates faster spontaneous GDP release than human Gα_i1 (k_obs = 0.06 min⁻¹ ±0.01). (B) EhGα1 hydrolyzed GTP[γ-³²P] at 0.21 min⁻¹ ±0.02, as determined by single turnover hydrolysis assays. No difference was observed for selenomethionine, lysine-methylated EhGα1 used for crystallization. (C) EhGα1 changes conformation upon binding the transition state mimetic aluminum tetrafluoride. Intrinsic EhGα1 fluorescence following excitation at 285 nm increases upon activation, reflecting burial of a conserved tryptophan on switch 2 (Trp-196). (D) EhGα1 adopts an active switch conformation upon addition of the non-hydrolyzable GTP analog GppNHp, as reflected by increased intrinsic tryptophan fluorescence. The kinetics of GppNHp-mediated activation are consistent with the kinetics of radiolabeled GTP analog binding (Fig. 1A). In contrast, addition of hydrolyzable GTP does not result in EhGα1 activation, indicating that nucleotide exchange, rather than GTP hydrolysis, is the rate-limiting step in the nucleotide cycle of EhGα1. (E, F) Two EhGα1 point mutants were profiled for effects on nucleotide cycle. The dominant negative S37C possessed negligible GTP binding. The constitutively active Q189L bound but did not hydrolyze GTP. Error bars in all panels represent standard error of the mean.

Figure 3. Evolutionary relationship of Gα subunits and identification of EhRGS-RhoGEF as a putative effector for activated EhGα1.

(A) Gα subunit protein sequences from E. histolytica, D. discoideum (D.d.), A. thaliana (A.t.), S. cerevisiae (S.c.), D. melanogaster (D.m), and H. sapiens (H.s.) were aligned and a bootstrapping consensus phylogram created using MEGA5 . Bootstrap values are indicated at each branch point. EhGα1 is distantly related to metazoan Gα subunits, specifically the adenylyl cyclase stimulatory Gα_s, adenylyl cyclase inhibitory Gα_i/o, phospholipase Cβ coupled Gα_q, and RGS-RhoGEF activating Gα_12/13 subfamilies. (B) Recombinant EhRGS-RhoGEF protein was immobilized on a surface plasmon resonance chip and EhGα1 protein flowed over in one of two nucleotide states. The EhRGS-RhoGEF biosensor bound EhGα1 selectively in the activated, GDP·AlF₄ ⁻-bound state (AMF).

Figure 4. Structure of EhGα1 reveals a conserved fold with unique features.

The crystal structure of EhGα1 was determined by single anomalous dispersion (SAD) using 2.6 Å resolution data (Table S1). (A) The EhGα1 Cα backbone is shown in green, bound to GDP in purple sticks. Conserved switch regions (SW 1–3) are dark blue. Trp-196 is solvent-exposed in the inactive state and buried when switch 2 adopts its activated conformation (e.g., Fig. 2C). Unique among Gα subunits, EhGα1 lacks an αB helix in the all-helical domain (red; labeled ‘αA-αC loop’) but possesses a unique short β-strand insert (β7) and a loop (orange) between the conserved α4 helix and β6 strand. Disordered regions in switch 3 (residues 222 and 223) and the β7- β6 loop (residues 302–310) are indicated by dashed lines. (B) Ser-37, conserved among Gα subunits, is an important ligand for Mg²⁺, a cofactor for GTP binding and hydrolysis. Mutation of Ser-37 to Cys is predicted to produce a dominant negative EhGα1 . Gln-189 is required for orienting the nucleophilic water during GTP hydrolysis; its mutation to Leu is predicted to cripple GTPase activity, yielding a constitutively active EhGα1.

Figure 5. Heterotrimeric G-protein signaling increases trophozoite migration across porous membranes and Matrigel layers.

(A) Trophozoites were stably transfected to express wildtype EhGα1 or dominant negative EhGα1^S37C under tetracycline control. (B) EhGα1^wt-expressing trophozoites showed greater migration across a porous membrane in the absence of stimuli (serum-free) while amoebae expressing EhGα1^S37C showed lower migration toward both serum-free and serum-containing nutritive media. Migration of HM-1:IMSS trophozoites was not significantly different from the non-induced EhGα1^wt and EhGα1^S37C strains. Tetracycline treatment was 5 µg/mL over 24 hours. (C) Trophozoites expressing EhGα1^wt were better able to migrate through a Matrigel layer than uninduced controls. Conversely, EhGα1^S37C expression greatly reduced Matrigel transmigration. Parent strain HM-1:IMSS trophozoites were unaffected by tetracycline treatment and were indistinguishable from non-induced EhGα1^wt and EhGα1^S37C. Error bars represent standard error of the mean. * represents statistical significance by an unpaired, two-tailed Student's t-test (p<0.05) for four independent experiments.

Figure 6. Heterotrimeric G-protein signaling positively regulates E. histolytica attachment to host cells as well as host cell killing.

(A) Trophozoites attach to CHO cell monolayers, primarily through a galactose-inhibitable lectin. Overexpression of EhGα1^wt enhanced monolayer attachment, while expression of EhGα1^S37C reduced attachment. Parent strain HM-1:IMSS trophozoites were unaffected by tetracycline treatment and were indistinguishable from non-induced EhGα1^wt and EhGα1^S37C. Attached trophozoites quantities were obtained by multiplying detached cell concentrations by a dilution factor. * indicates a statistically significant difference (p<0.05) between quadruplicate experiments. Error bars represent standard error of the mean. * indicates statistical significance by an unpaired, two-tailed Student's t-test (p<0.05) for four independent experiments. (B) Amoebae overexpressing EhGα1^wt or EhGα1^S37C displayed enhanced or reduced abilities to kill Jurkat (human T-lymphocyte) cells, respectively, as measured by LDH release in a membrane integrity assay. Cell killing by HM-1:IMSS trophozoites was not altered by tetracycline treatment. 0.5% Triton X-100 was added to Jurkat cells to define 100% host cell lysis. Tetracycline treatment was 5 µg/mL over 24 hours. Error bars represent standard error of the mean. * indicates statistical significance by an unpaired, two-tailed Student's t-test (p<0.05) for three independent experiments, with four technical replicates each.

Figure 7. Heterotrimeric G-protein signaling alters E. histolytica transcription to modulate cysteine protease secretion.

(A) 96 genes or 394 genes were differentially transcribed upon overexpression of EhGα1^wt or EhGα1^S37C, respectively, when compared to uninduced controls as determined by RNA-seq. 21 transcripts were oppositely regulated in trophozoites expressing EhGα1^wt vs EhGα1^S37C. (B) Differentially transcribed genes were categorized by putative function based on prior studies, homology to genes of known function, or predicted protein domains of known function. “Virulence/encystation” category includes genes known to modulate E. histolytica pathogenesis, such as cysteine proteases . (C) Both intracellular and secreted cysteine protease activities were assessed with an azo-collagen assay. EhGα1^wt overexpression enhanced cysteine protease secretion, while EhGα1^S37C expression resulted in less extracellular (E), despite higher intracellular (I), cysteine protease activity, suggesting that transcriptional responses downstream of heterotrimeric G-protein signaling modulate E. histolytica pathogenic processes in part by regulating cysteine protease secretion. Tetracycline treatment in all experiments was 5 µg/mL over 24 hours. * = statistical significance by an unpaired, two-tailed Student's t-test (p<0.05) for four independent experiments.