https://orcid.org/0000-0002-4314-6961
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Figures
Abstract
Amebiasis is a parasitic infection of the human intestines, primarily caused by Entamoeba histolytica. Its pathogenesis relies on the environmental sensing-induced cytoskeletal remodeling as the basic mechanism for motility and tissue invasion. We identified and characterized an atypical Venus Fly-Trap (VFT) receptor protein, EhVFT (CL6EHI_096680). While it shares homology with the ligand-binding domain of class C GPCRs, it is phylogenetically related to the Periplasmic Binding Protein (PBP) superfamily. This protein is uniquely lacking a transmembrane domain. Instead, the glycosylphosphatidylinositol (GPI) anchor is responsible for its cell membrane localization. Removal of the GPI signal led to unexpected mitosomal localization, highlighting the importance of GPI modification in subcellular targeting. Functional studies revealed that EhVFT knockdown reduced parasite motility and phagocytosis of mammalian cell following the reduction of expression of actin cytoskeleton-related genes, including myosin II, villidin, and gelsolin. Our findings suggest that EhVFT plays a role in regulating downstream signaling linked to Entamoeba motility and phagocytosis. This study provides novel insights into an atypical VFT protein in E. histolytica, an area previously understudied.
Author summary
Entamoeba histolytica is a human intestinal parasite that causes bloody diarrhea and often leads to fatal extraintestinal complications. This parasite can adhere to, consume, and invade intestinal cell layers in response to signals from its environment. In this study, we investigated a protein in the parasite E. histolytica, the Entamoeba histolytica Venus Fly Trap (EhVFT) domain receptor, which resembles a class C G-protein-coupled receptor in humans and Periplasmic Binding Protein (PBP) in bacteria known to sense signals and activate cellular responses. We found that EhVFT is located in the plasma membrane, facilitated by a lipid-associated anchoring mechanism known as the glycosylphosphatidylinositol (GPI) anchor. Removal of the predicted GPI-anchor signal caused the protein to be mistargeted to the mitosome, a degenerate and divergent mitochondrion-related organelle. It has also preserved the signal-sensing structure and the special structure commonly found in signal-sensing proteins. Knockdown of EhVFT expression significantly impaired the mobility and phagocytic activity of the parasite. Our findings suggest that EhVFT may be a crucial regulator of movement and invasion in E. histolytica, providing new insights into its biology and disease process.
Citation: Nugraha RYB, Jeelani G, Santos HJ, Nozaki T (2026) An atypical venus fly trap domain receptor regulates motility and phagocytosis in the protozoan parasite Entamoeba histolytica. PLoS Pathog 22(2): e1014019. https://doi.org/10.1371/journal.ppat.1014019
Editor: Katherine S. Ralston, University of California Davis, UNITED STATES OF AMERICA
Received: July 9, 2025; Accepted: February 20, 2026; Published: February 27, 2026
Copyright: © 2026 Nugraha et al. 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.
Data Availability: All data are found in the manuscript and Supporting information files.
Funding: This work was supported partly by Grants-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS) (JP21H02723 to T.N.), Fostering Joint International Research (B) from JSPS (JP21KK135 to T.N.), and Grant for Science and Technology Research Partnership for Sustainable Development (SATREPS) from Japan Agency for Medical Research and Development (AMED) and Japan International Cooperation Agency (JICA) (JP24jm0110022) to T.N., Grant for research on emerging and re-emerging infectious diseases from AMED (JP24fk0108680 to T.N.), Japan Society for the Promotion of Science (JSPS) KAKENHI (under grant number JP21K15427, JP24K10185 to G.J.) and support from the University of Tokyo Pandemic Preparedness, Infection, and Advanced Research Center (UTOPIA) and AMED (JP243fa627001) to T.N., and Indonesia Endowment Fund for Education (LPDP No. 20200822303634) to R.Y.B.N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The G-protein-coupled receptors (GPCRs) are the most prominent family of cell surface receptors, encoded by approximately 800 genes in human, and are characterized by seven transmembrane (7TM) helices [1,2]. These receptors respond to diverse extracellular stimuli, including photons, ions, amines, carbohydrates, lipids, and peptides [3]. Based on sequence homology, GPCRs are classified into: class A (rhodopsin-like), class B (secretin/B1 and adhesion/B2), class C (calcium-sending receptors, metabotropic glutamate receptors, GABA type B receptors, taste receptors, and several orphan receptors), and class F (smoothened and frizzled receptors) [4]. In mammals, GPCRs and their associated effectors are major regulators of actin cytoskeletal dynamics, which play as the key factor in regulating many cellular processes, such as gene expression [5], cell motility [6,7], exocytosis and endocytosis [8], cell polarity and adhesion [9,10], cell division and growth, cell morphology, as well as cell survival and death [11].
Canonical GPCR has 7TM helices and acts as a guanine-nucleotide exchange factor (GEF) for heterotrimeric G proteins, which are composed of Gα, Gβ, and Gγ subunits [3,12]. Ligand binding leads to GPCR conformational change that triggers GDP–GTP exchange on the Gα subunit and dissociation from the Gβγ dimer. This event activates or inhibits downstream effectors such as adenylyl cyclase (AC), phospholipase C (PLC), or ion channels, ultimately influencing intracellular signaling pathways including cAMP production, calcium mobilization, and kinase activation, which in turn regulate actin dynamics and gene expression [1,4,13].
Structurally, the extracellular domain of class C GPCRs typically has a bilobed structure, known as the Venus flytrap (VFT) domain, an architecture that is also conserved in Periplasmic Binding Proteins (PBPs), a class of bacterial proteins [14]. The extracellular domains of class C GPCRs contain large VFT modules that bind endogenous ligands like glutamate, GABA, calcium ions, and amino acids [15,16]. Ligand-induced closure of VFT domains brings the two lobes closer, triggering dimerization changes that propagate conformational signals through cysteine-rich domains and ultimately to the 7-transmembrane (7TM) heptahelical domain. This leads to G protein coupling and intracellular signal transduction [17,18]. PBPs are a crucial class of proteins found predominantly in Gram-negative bacteria, where they reside in the periplasmic space between the inner and outer membranes. These soluble proteins serve as initial receptors for substrates during active transport and chemotaxis processes [19]. PBPs bind diverse substrates, including sugars, amino acids, peptides, ions, and siderophores, with high affinity in a cleft between the lobes. PBPs interact directly with membrane-bound transport complexes composed of several proteins, including two hydrophobic membrane proteins and two ATPase subunits. In these ATP-binding cassette (ABC) transport systems, PBPs bind substrates in the periplasm and deliver them to the membrane components for translocation into the cytoplasm, energized by ATP hydrolysis [20,21]. Notably, this mechanism is conserved, and analogous transporters exist even in Gram-positive bacteria and mycoplasmas, where PBPs are replaced by membrane-anchored binding lipoproteins functioning similarly [22].
Entamoeba histolytica is a protozoan parasite that causes human amebiasis, a disease with a broad clinical spectrum ranging from asymptomatic infection and intestinal dysentery to potentially fatal extraintestinal complications such as liver abscesses. Multiple virulence factors, such as D-galactose and N-acetyl-D-galactosamine (Gal/GalNAc) specific surface lectin [23], cysteine proteases [24], amoebapores [25], and extracellular vesicles [26], contribute to the pathogenicity of E. histolytica. Still, one key element involved in nearly all pathogenic processes is the actin cytoskeleton [27]. Due to the absence of extranuclear microtubules, actin microfilaments are the sole structural component responsible for critical cellular functions, including motility, adhesion, endocytosis, invasion, and defense against host immune responses [28].
Although the importance of heterotrimeric G-proteins in Entamoeba pathogenesis is well recognized [29], relatively little is known about the GPCRs or PBP domain-containing proteins in Entamoeba. The first indication of GPCR activity came from observations that histamine enhances parasite virulence, an effect reversed by Histamine 2 (H2) receptor antagonists, suggesting the presence of H2-like receptors on the parasite surface [30,31]. Epinephrine has also been shown to initiate encystation in E. histolytica through increased intracellular cAMP levels, and similar effects are observed with G-protein modulators such as forskolin, pertussis toxin, and cholera toxin, implying the presence of β1-adrenergic-like receptors and conserved G-protein signaling mechanisms [32]. Another study identifies a putative GPCR, EhGPCR-1, as a binding partner of the Rab GTPase EhRabB and reports that it participates in phagocytosis [33]. However, subsequent domain analysis using the NCBI Conserved Domain Database reveals that EhGPCR-1 contains a Wnt-binding domain but lacks canonical GPCR features [34,35]. Only one study has systematically identified potential GPCRs in E. histolytica, which are important for cAMP signaling, highlighting four candidates: EHI_196900 (containing GPHR_N and ABA_GPCR domains), EHI_153230 (rhodopsin superfamily), EHI_105070 (Gpa2_C domain), and EHI_096680 (Atrial Natriuretic Factor (ANF) receptor ligand-binding domain) [13]. Among these candidates, EHI_096680 presents a unique structural paradox. Although it contains a Venus Flytrap (VFT) domain characteristic of Class C GPCRs, it lacks the canonical 7-transmembrane (7TM) helices required for G-protein coupling [13]. Database analysis suggests this protein belongs to the Periplasmic Binding Protein (PBP) superfamily, the evolutionary ancestors of VFT domains.
In this study, we present the structural, molecular, and functional characterization of EHI_096680, hereafter referred to as the Entamoeba histolytica Venus Fly Trap domain receptor (EhVFT). EhVFT is a transmembrane domain-lacking, PBP superfamily protein in E. histolytica. EhVFT is conserved among Entamoeba species and, interestingly, still localizes to the plasma membrane via an N-terminal signal peptide and a C-terminal GPI anchor. Structural analysis reveals similarity to the bacterial PBPs and VFT module of class C GPCR proteins. Functional studies using gene silencing and ectopic expression strategies reveal that EhVFT modulates actin cytoskeleton dynamics, thereby influencing parasite motility, morphology, and phagocytosis—but not trogocytosis—of mammalian cell which collectively contribute to parasite virulence. Our findings suggest that a novel, non-canonical signaling mechanism in E. histolytica potentially represents an evolutionary adaptation in protozoan signal transduction.
Results
EhVFT shares structural features with class C GPCR extracellular domains but lacks transmembrane regions
To investigate the presence of class C GPCR homologs in Entamoeba spp., a BLAST search was performed using several queries that included the ANF receptor domain, such as the GABA type B receptor (GABA-B-R, Uniprot ID Q9UBS5), the calcium-sensing receptor (CaSR, Uniprot ID P41180), and the metabotropic glutamate receptor (mGluR, Uniprot ID P23385). This analysis identified a single gene, EHI_096680 (UniProt ID C4LWI0), also referred to as CL6EHI_096680 (UniProt ID A0A175JHL1), as the top hit with the lowest E-value in E. histolytica and other Entamoeba species. Meanwhile, BLAST analysis using bacterial PBPs (E. coli LIVBP, PDB ID 2LIV; Atu2422, PDB ID 3IP9) as queries against the Entamoeba database returned no results. The CL6EHI_096680 gene identifier was selected for subsequent analyses based on sequencing results (S1a and S1b Fig). Although the percentage identity between Entamoeba VFTs and metazoan GPCRs was relatively low (~20%), sequence identity among Entamoeba species ranged from approximately 60% to 80% (S1c and S1d Fig), indicating strong conservation within the genus.
To investigate the structural and evolutionary characteristics of CL6EHI_096680 (hereafter referred to as EhVFT), we performed a comprehensive phylogenetic analysis. Initially, EhVFT homolog proteins were retrieved through BLASTp searches against the NCBI database. However, the majority of sequences exhibited low similarity to EhVFT, with E-values greater than 1e–10. Therefore, as a different approach, homolog proteins were searched based on the 3D structure similarity using Foldseek. The phylogeny revealed that six Entamoeba VFT sequences formed a monophyletic group with a bootstrap proportion (BP) of 0.66, consistent with the clade’s evolutionary distinctiveness. The closest outgroups to the Entamoeba VFT monophyly included periplasmic binding protein 1-like (PBP1) sequences from diverse bacteria, as well as human ANF receptors, and inotropic glutamate NMDA receptors from humans and rats. The detection of numerous bacterial PBPs suggests that lateral gene transfer may have contributed to the evolution of EhVFT. Interestingly, E. nutalli VFT exhibited greater similarity with the human GABA type B receptor, calcium-sensing receptor, and metabotropic glutamate receptor than other Entamoeba VFTs, which formed distinct clades. Overall, the phylogenetic analysis highlights EhVFT as part of a distinct VFT lineage with affinities to bacterial PBPs and metazoan class C GPCR VFTs, indicating a complex evolutionary history shaped by both divergence and potential gene transfer (Fig 1a).
(A) Phylogenetic tree constructed using maximum likelihood method showing the evolutionary relationship of the Entamoeba VFT protein (highlighted in blue) with representative VFT family members: bacterial PBPs (dark gray) and mammalian VFT (red) including calcium-sensing receptors (CaSR), metabotropic and inotropic glutamate receptors (mGluR and iGluR), and GABA type B receptors (GABA-B-R). Colored branches represent taxonomically distinct groups. Bootstrap values are indicated in each clade branch with 0 – 1 range support. (B) Dot plot of the correlation between transmembrane (TM) conservation (x-axis) and length of amino acid (aa) sequence (y-axis) across the selected homologs based on pairwise alignment scores. The E. histolytica protein (in red dots) lacked the transmembrane domain among the homologous proteins. (C) Predicted tertiary structures of the hypothetical E. histolytica protein, generated by AlphaFold3, reveal conserved bilobed domains resembling Venus Fly-Trap-like structures commonly found in bacterial PBPs (Atu2422 PDB ID 3IP9, Agrobacterium tumefaciens PBP) and class C GPCR extracellular domains (HsCaSR PDB ID 7DD6, Homo sapiens Calcium Sensing Receptor). SP, signal peptide; LB, lobe; C-ter, C-terminal; CRD, cysteine-rich domain; TM, transmembrane.
Sequence length variation among the homologs prompted us to assess their membrane-spanning properties. Surprisingly, transmembrane domain prediction [36] revealed that approximately 292 out of 402 of the Foldseek hits lacked transmembrane regions, including the Entamoeba homologs (Fig 1b, shown in red dots). Furthermore, structural modeling of EhVFT by AlphaFold3 [37] indicated a strong resemblance to bacterial PBP (Agrobacterium tumefaciens Atu2422) [38] and the extracellular VFT module characteristic of class C GPCRs, as exemplified by the human CaSR VFT domain [39]. The VFT structure typically consists of two globular lobes connected by a hinge, with the inter-lobal region serving as the primary ligand-binding site (Fig 1c) [14,40]. However, in line with the previous finding, this 3D model also showed the absence of the canonical 7TM helical domain in EhVFT. Intriguingly, the 7TM helical domain was replaced by a hydrophobic amino acid stretch at the C-terminal region (S1e Fig) [41]. This raised the possibility of this amino acid sequence being either a single spanning transmembrane domain or a glycosylphosphatidylinositol (GPI) signal sequence [42]. To further predict the modification that happened in this C-terminal sequence, NetGPI 1.1 was used [43], and the apparent free energy of membrane insertion (Gapp) was calculated [42]. The ΔGapp scores were between 0–1 kcal/mol, suggesting the potential for glycosylphosphatidylinositol (GPI) anchoring. Similarly, important residues for GPI modification, called omega site, were also predicted to certain degrees of likelihood by NetGPI 1.1 (S1f Fig) [42–45]. Despite the absence of the 7TM domain, EhVFT was still predicted to have an N-terminal signal peptide (SP), as indicated by SignalP 6.0 with a score of 0.9991 (S1g Fig) [46].
To gain further insight into residue-level conservation, multiple sequence alignment (MSA) was performed comparing Entamoeba VFTs (CL6EHI_096680) with some reference proteins: mammalian class C GPCR and bacterial PBP (Fig 2). The secondary structure conservation, α-helix (red) and β-strand (blue), was first observed. The MSA suggested that the middle part of the sequence was aligned well, specifically starting from α-helix III until β-strand I. This finding aligns with a previous study [14], which identified the presence of amino acid binding residues in that specific region. Those residues, which were also previously identified as critical for amino acid binding, were also conserved in Entamoeba proteins (highlighted in yellow). They fulfilled the 8-residue motif: [S]-x(22)-[TS]-x(13,14)-[R]-x(4)-[D]-x(2)-[Q]-x(24,25)-[Y]-[GA]-x(74,84)-[ED]19 which is commonly found in the VFT module of the periplasmic binding protein-like 1 (PBP1) and class C GPCR [14].
EhVFT (CL6EHI_096680) was aligned against representatives of class C GPCRs (HsCaSR, PDB ID 5K5S; RnmGLUR1, PDB ID 1EWK; RnmGLUR3, PDB ID 2E4X) and bacterial periplasmic binding protein/PBP (EcoLIVBP, PDB ID 2LIV; Atu2422, PDB ID 3IP9). Conserved amino acid binding sites were highlighted in yellow. Secondary structures were also aligned and showed in the same letter colors: ⍺-helix in red (roman number ordering) and β-sheet in blue (alphabet ordering). HsCaSR: Homo sapiens Calcium Sensing Receptor Venus Fly Trap Module; RnmGLUR1: Rattus novergicus metabotropic glutamate receptor 1; RnmGLUR3: Rattus novergicus metabotropic glutamate receptor 3; EcoLIVBP: E. coli PBP Leucine/Isoleucine/Valine Binding Protein; and Atu2422: Agrobacterium tumefaciens PBP.
EhVFT is a cell membrane-localized protein with an extracellular topology
To investigate the subcellular localization of EhVFT, we generated engineered strains using the pEhExHA expression vector containing the EhVFT gene, as one is schematically illustrated in Fig 3a. By considering all the domain predictions in the previous section, we carefully inserted a Hemagglutinin (HA) tag just before the predicted GPI-anchor signal (omega site). This engineered strain was hereafter referred to as EhVFT-HA-GPI. The primers used for plasmid construction are listed in S1 Table. Immunofluorescence analysis of stable transformants revealed that HA-tagged EhVFT localized predominantly to the plasma membrane and internal vesicular membranes, exhibiting a distribution pattern similar to that of the intermediate lectin subunit (Igl) (Fig 3b and c). In the absence of permeabilizing agents, HA signals were concentrated along the outer surface of the cell membrane, indicating that EhVFT is positioned on the plasma membrane and oriented toward the extracellular environment (Fig 3b and d).
(A) Schematic representation of the constructs used to study the epitope-tagged, ectopically expressed EhVFT protein: SP, ANF receptor domain, and GPI-anchor signal; the experimental construct includes an HA tag inserted between the ANF receptor domain and GPI-anchor. (B) Immunofluorescence images showing expression of HA-tagged EhVFT and HA mock in trophozoites. Cells were stained with rabbit anti-HA (green) and mouse anti-Igl (red) antibodies. Merged images show areas of co-localization (yellow). White arrows indicate representative regions used for fluorescence intensity profiling. White arrowheads showed green intensity aggregation inside the vesicle. DIC and merged channels are provided for structural reference. Signals were compared with and without 0.2% saponin as a permeabilizing agent. Observation was done for around 40 images from two independent trials. Scale bars: 5 µm. (C) Line-scan fluorescence intensity profiles from merged images of saponin positive and (D) negative samples, plotting HA (green) and IgI (red) signals across the selected regions (white arrows). Peaks indicate localization at the plasma membrane (PM), vesicular membranes (VM), and vesicles (Ves). SP, Signal Peptide; ANF, Atrial Natriuretic Factor; GPI, Glycosylphosphatidylinositol; HA, hemaglutinin; Igl, intermediate subunit of galactose (Gal)- and N-acetyl-D-galactosamine (GalNAc)-inhibitable lectin; and DIC, Differential Interference Contrast.
To validate this finding independently of the ectopic expression system, a polyclonal rabbit anti-EhVFT antibody was generated against the recombinant EhVFT protein. Immunofluorescence staining using this antibody confirmed membrane localization of both the endogenous and epitope-tagged, ectopically expressed forms of EhVFT (S2a–c Fig). Notably, the specificity of the antibody signal was higher in non-permeabilized cells, further supporting the extracellular orientation of the protein. In summary, EhVFT (CL6EHI_096680) is a membrane-localized protein with an extracellular-facing topology, consistent with its predicted role in ligand recognition and signal transduction.
EhVFT exhibits canonical features of a plasma membrane GPI-anchored protein
One plausible explanation for the membrane localization of a protein lacking a transmembrane domain is the presence of specific post-translational modifications (PTMs), such as glycosylphosphatidylinositol (GPI) anchoring, as predicted in the S1f and g Fig. To assess the functional significance of the predicted N-terminal signal peptide (SP) and C-terminal GPI-anchor signal, we generated truncated versions of EhVFT (Fig 4a). Interestingly, a stable transformant expressing EhVFT with a truncated C-terminal GPI-anchor signal (EhVFT-HA-ΔGPI; lacking the final 26 amino acids) exhibited punctate anti-HA signal pattern based on IFA (Fig 4b). Moreover, the anti-HA signals strongly co-localized with adenosine 5’-phosphosulfate kinase (APSK), a well-established marker of mitosomes in E. histolytica (Fig 4b) [47] with an average overlap coefficient of ~0.85, comparable to an unrelated control mitosomal protein, Entamoeba Transmembrane Mitosomal Protein of 30 kDa (ETMP30) (Fig 4c, unpaired t-test, p = 0.4153) [48]. In contrast, removal of both the N-terminal signal peptide (amino acids 1–19) and the C-terminal GPI-anchor signal (construct HA-EhVFT-ΔSPΔGPI) resulted in diffuse cytoplasmic localization, consistent with the distribution of cysteine synthase 1 (CS1) [49], a known cytoplasmic marker (Fig 4b, bottom panel). The colocalization of HA-EhVFT-ΔSPΔGPI with CS1 was quantitatively confirmed by an average overlap coefficient of ~0.65 in Fig 4d. This observation further supports the important role of the N-terminal sequence in directing the proper protein localization, possibly as a functional signal peptide.
(A) Schematic diagrams of the constructs used: two truncated versions lacking of GPI sequence (EhVFT-HA-ΔGPI) and lacking of both SP and GPI sequences (HA-EhVFT-ΔSPΔGPI). An HA-tag fused directly to the C- and N-terminus of the ANF receptor domain, respectively. (B) Immunofluorescence images showing expression of HA-tagged truncated EhVFT in trophozoites. EhVFT-HA-ΔGPI strain was stained with mouse anti-HA (green) and rabbit anti-APSK (red) antibodies. HA-tagged ETMP30 (HA-ETMP30) ectopic expression strain and HA mock were used as control. Merged images showed overlapping regions in yellow. DIC and merged images are provided for structural reference. Observation was done for around 40 images from two independent trials. Scale bars: 5 µm. (C) Co-localization quantification using dot plots that representing overlap coefficient between green and red signals from EhVFT-HA-ΔGPI and HA-ETMP30 (N = 20 images). Meanwhile, HA-EhVFT-ΔSPΔGPI strain (B, lowest panel) was stained with mouse anti-HA (green) and rabbit anti-CS1 (red) antibodies. Observation was done for around 20 images. (D) Dot plots representing overlap coefficient between green (HA) and red (CS1) signals from HA-EhVFT-ΔSPΔGPI (N = 20). (E) Western blot analysis confirming the expression of EhVFT constructs in E. histolytica. Upper panel: anti-HA blot showing bands corresponding to the expected size of epitope-tagged protein. Lower panel: CS1 expression as loading control. FL, full length (EhVFT-HA-GPI); APSK, Adenosine 5’-Phosphosulfate (APS) Kinase; ETMP30, Entamoeba Transmembrane Mitosomal Protein of 30 kDa; and CS1, Cysteine Synthase 1.
Western blot analysis (Fig 4e) revealed that the full-length EhVFT-HA-GPI construct appeared as a single, diffuse band at approximately 49 kDa. In contrast, the EhVFT-HA-ΔGPI construct produced multiple lower-molecular-weight bands, likely reflecting partial degradation or altered PTMs due to the absence of the C-terminal GPI-anchor signal. The double-truncated construct (HA-EhVFT-ΔSPΔGPI) appeared as the lowest molecular weight band (~43 kDa), as expected based on its reduced sequence length. In summary, EhVFT contains an N-terminal signal peptide and a putative C-terminal GPI-anchor signal, supporting its classification as a canonical GPI-anchored membrane protein.
To explore the binding partners, activation, and trafficking mechanism of EhVFT, co-immunoprecipitation (co-IP) experiments were conducted by comparing the two ectopic expression strains, EhVFT-HA-GPI and EhVFT-HA-ΔGPI, along with HA mock as a control. A total of 2336 proteins were identified after mass spectrometry analysis of EhVFT-HA-GPI anti-HA co-IP (n = 2, as shown in S2 Table). Among them, 24 proteins were exclusively pulled down in the EhVFT-HA-GPI (Quantitative Value (QV) of HA mock = 0, Table 1 and S2 Table). Additionally, 91 proteins were identified as enriched during the pull-down experiments (Fold Change ≥ 2, Tables 1, S2). The majority of the pulled-down protein was EhVFT (EHI_096680), the bait protein. Several actin cytoskeleton-associated proteins were identified among the exclusive and enriched hits, suggesting a role for the bait protein in cytoskeletal remodeling. These included: Fimbrin/Plastin containing a calponin homology (CH) domain (EHI_193940; mean QV 0.91; exclusive hit); the focal adhesion complex protein Paxillin/LIM zinc finger domain-containing protein (EHI_022960; mean QV 13.3 vs 2.82 in mock); an autophagy-related VASt domain-containing protein (EHI_024350; 5.34 vs 0.91), the Arp2/3 complex subunit 4 (EHI_030820; 6.35 vs 1.36), a Pleckstrin homology (PH) domain-containing protein (EHI_117920; 4.8 vs 1.36), and an AIG1 family protein (EHI_136950; 2.24 vs 0.45). Interestingly, components of the GPI biosynthesis and anchoring machinery were also detected as exclusive hits, including PIG-X (EHI_178650; mean QV 2.24), PIG-S (EHI_005120; mean QV 2.61), and PIG-K (EHI_092280; mean QV 1.33), suggesting a potential interaction between the bait protein and the GPI transamidase complex. In addition, several vesicle trafficking-related proteins were identified. These included the GTP-binding protein EhSar1, which initiates COPII vesicle formation at the endoplasmic reticulum (EHI_031410 and EHI_075040; mean QV: 15.9 and 15.47 vs 7) [50], the Adaptor Protein-1 (AP-1) complex subunit mu-2/Clathrin (EHI_124460; 2.61 vs 0.45 and EHI_089880; mean QV 1.28) which were important for golgi – plasma membrane cargo transportation [51]; the SNARE complex protein EhSyntaxinD1 (EHI_117250; mean QV 0.91); and a Rab-GTPase-TBC domain-containing protein implicated in vesicle fusion (EHI_009510; 9.5 vs 3.62) [52] (Table 1, S2). All suggest the bait protein may traffic from the ER, Golgi, and plasma membrane. Moreover, all the proteins above were absent in the ΔGPI co-IP proteome list, suggesting that those proteins specifically interacted with the full-length strain (S3 Table).
It is noteworthy that several proteins annotated as mitochondrial heat shock protein 70 (mtHsp70) were identified in the co-IP results of the full-length EhVFT, and were predicted to be involved in protein folding processes (S2 Table). These included EHI_013760 (Mean QV 3.68, exclusive hit), EHI_166050 (mean QV 5.02 vs 0.45), EHI_127700 (4.54 vs 0.45), EHI_007150 (4.54 vs 0.45), EHI_175600 (3.20 vs 0.45), EHI_070450 (3.20 vs 0.45), EHI_101120 (3.10 vs 0.45), EHI_071800 (2.72 vs 0.45), and EHI_100810 (2.24 vs 0.45). However, only one mtHsp70, EHI_007150 (3.04 vs 0.74), was detected in the ΔGPI co-IP proteome (S3 Table). Given that the ectopically expressed ΔGPI protein localised to the mitosome, this finding suggests that the interaction between full-length EhVFT and mtHsp70 proteins may occur outside the mitosome, possibly in the ER.
EhVFT regulates the motility and phagocytosis via actin cytoskeleton organization
To investigate the physiological function of EhVFT, we silenced its expression and examined the resulting phenotypes. Transcriptional gene silencing was achieved by transfecting trophozoites with the psAP2-Gunma vector containing the first 416 bp of the EhVFT coding region. Three independent biological clones of the EhVFT-silenced strain (EhVFTgs), alongside empty vector transformants serving as controls, were established. Gene silencing was validated by quantitative real-time PCR (qRT-PCR) (Fig 5a and b). The EhVFTgs exhibited slightly defective growth compared to the psAP2 mock control (S3a and b Fig). As a membrane protein that can receive external stimuli, many genes may be affected by the absence of EhVFT. To identify genes regulated by EhVFT, RNA-seq analysis was performed. As expected, RNA-seq results demonstrated a substantial decrease in EhVFT transcript abundance (Log Fold Change (LogFC) –8.72) in EhVFTgs, indicating successful gene silencing. In contrast, EhVFT transcript levels in psAP2 mock were comparable to that of a house-keeping gene, RNA polymerase II (EHI_056690) (S3c Fig). RNA-seq revealed that 73 gene transcripts were downregulated upon EhVFT silencing (Fig 5c). Notably, 17 of these genes were involved in actin cytoskeleton organization, including myosin II (EHI_110180, –1.38), actin (EHI_131230, –1.12), Calponin homology (CH) domain-containing proteins (EHI_050660, –1.87; EHI_154330, –1.28; EHI_189500, –1.27), autophagy protein Atg8 (EHI_130660, –1.51), gelsolin (EHI_009570, –1.48), villidin (EHI_021260, –1.29; EHI_150430, –1.18), coronin (EHI_122800, –1.24), and a small GTPase (EHI_059670, –1.29). Additionally, 18 genes related to transcription and translation, four cell surface proteins, 12 proteins with other predicted functions, and 22 hypothetical proteins were also downregulated (Tables 2; S4). Gene ontology (GO) analysis revealed that most of the downregulated genes were significantly associated with actin cytoskeleton-related processes, including actin filament severing, binding, and organization (Fig 5d). Unexpectedly, only five genes were upregulated, likely as compensatory responses, including tRNA intron endonuclease (EHI_042160, 6.50), 60S ribosomal protein L40 (EHI_119590, 2.98), and three proteins of unknown function (EHI_193640, 6.05; EHI_028980, 1.10; and EHI_112070, 1.10) (S4 Table). Altogether, these results suggest that EhVFT plays a regulatory role in modulating the actin cytoskeleton regulatory network in Entamoeba.
(A) Semi-quantitative RT-PCR showing the knockdown efficiency of EhVFT expression in silenced (EhVFTgs) versus control (psAP2 mock) trophozoites. Three independent clones were established for each strain (#1 – 3). EhRNA polymerase II (EHI_056690) was used as an internal control. (B) Quantitative RT-PCR (qRT-PCR) results measuring the EhVFT expression level in EhVFTgs versus psAP2 mock control. By using EhRNA polymerase II expression level serving as the internal normalization control, double delta Ct value (2–ΔΔCt) was calculated. (C) Volcano plot illustrating differential gene expression between EhVFTgs and psAP2 mock control trophozoites. Each dot represents a single gene; significantly downregulated genes (blue; LogFoldChange (LogFC)<-1, q-value<0.05) and upregulated genes (red; LogFC > 1, q-value<0.05) are highlighted. (D) Gene Ontology (GO) enrichment analysis of downregulated genes in EhVFTgs cells. X-axis is Fold Enrichment, dot size indicates the number of associated genes, and color scale represents –log10(FDR, False Discovery Rate).
Since the actin cytoskeleton components are crucial for Entamoeba pathogenicity [28], the pathogenicity properties, such as migration/motility, adhesion, and endocytosis activities, were further evaluated in the EhVFTgs. In a transwell migration assay using serum as a chemoattractant, EhVFTgs exhibited significantly impaired migration after three hours of incubation, regardless of serum starvation (Fig 6a, 2-way ANOVA, no serum starvation adj.p = 0.0021, serum starvation adj.p = 0.0117). Moreover, detailed live-cell motility tracking stained by PlasMem Bright Red revealed that psAP2 mock cells had an average total displacement of 50.6 ± 19.3 µm (mean ± SD) and an average net displacement of 33 ± 18.1 µm over 150 sequential image captures (~161.2 seconds). At the same time, EhVFTgs migrated only about half these distances (Fig 6b and c, 24.5 ± 17.7 µm and 15.3 ± 13.3 µm respectively, unpaired t-test p < 0.0001). Average migration speed was similarly reduced with 0.31 ± 0.12 µm/sec in mock and 0.15 ± 0.11 µm/sec in EhVFTgs (Fig 6d, unpaired t-test p < 0.0001). Although motion linearity remained unchanged (Fig 6e, 64 ± 22 vs 60 ± 25%, unpaired t-test p = 0.2949), the search radius, a metric reflecting motility spread, was significantly reduced in EhVFTgs (Fig 6f, 34.6 ± 17.3 vs 16.2 ± 13.3 µm, unpaired t-test p < 0.0001). Morphologically, EhVFTgs cells were more rounded (Fig 6g, 49.8 ± 8.3 vs 61.8 ± 12.7%, unpaired t-test p < 0.0001) and exhibited smaller cell areas than controls (Fig 6h, 822.1 ± 191.8 vs 739.3 ± 173 µm2, unpaired t-test p = 0.0013), consistent with disrupted actin dynamics. To assess the impact on phagocytosis, heat-killed Jurkat cells were used as baits. EhVFTgs showed reduced phagocytosis efficiency, which plateaued after one hour (Fig 6i, 2-way ANOVA adj.p < 0.0001 at 15, 30, 45, and 60 minutes of co-incubation). Conversely, ectopic expression of full-length EhVFT enhanced mammalian phagocytic activity within the same period (Fig 6j, 2-way ANOVA adj.p < 0.0001 at 15, 30, 45, and 60 minutes of co-incubation). In addition to phagocytosis, we also examined trogocytosis, the nibbling of live mammalian host cells by E. histolytica. When live Jurkat cells were used as baits in the trogocytosis assay, no significant difference in activity was observed between EhVFTgs and control strains after two hours (S3d Fig, 2-way ANOVA adj.p > 0.1234 at 60, 90, and 120 minutes of co-incubation). In summary, EhVFT plays a key role in modulating Entamoeba virulence by regulating migration, motility, cell morphology, and phagocytosis, but not trogocytosis in mammalian cells. These effects align with transcriptomic data indicating that EhVFT influences the organization of the actin cytoskeleton.
(A) Bar graph showing percentage of migrated trophozoites of the EhVFTgs, blue) compared to psAP2 mock control (red). The assay was done with (right) and without (left) prior serum starvation. Experiments were done in three technical replicates from two independent experiments. (B–H) Dot plots showing quantitative motility analysis based on time-lapse confocal microscopy and ICY software analysis, showing significant reductions in motility parameters—including displacement, trajectory length, and speed—in EhVFTgs relative to control. Data represented mean ± SD from N = 106 cells for psAP2 mock and N = 102 cells for EhVFTgs in two independent experiments. Significant differences were stated after unpaired t-test analysis. (I) Flow cytometry-based phagocytosis assay revealing impaired uptake of heat-killed Jurkat cells by EhVFTgs trophozoites (red) in comparison to control (blue). (J) Ectopically expressed form of EhVFT-HA-GPI enhanced phagocytic activity, as shown by increased internalization of heat-killed Jurkat cells compared to mock control. Data represented mean ± SD from at least three technical replicates and three independent experiments. Significant differences were stated after 2-way ANOVA analysis. P value: < 0.1234 (ns, not significant); < 0.0332 (*); < 0.0021 (**); < 0.0002 (***); and 0.0001 (****).
Discussion
During the plasmid construction process, we observed a discrepancy in intron sequence recognition between the AmoebaDB database and the mRNA sequencing results (S1a Fig). In AmoebaDB, EHI_096680 intron length was 47 bp (nt 840 – 886, GenBank: DS571163.1) [53], whereas mRNA sequencing results showed 119 bp intron (nt 840 – 958) matching another database ID CL6EHI_096680 (NCBI GenBank: BDEQ01000001.1). This difference may result from the use of different sequencing protocols to generate these databases or, perhaps, intrinsic factors of Entamoeba histolytica itself. Both genome sequences were generated using the whole-genome shotgun method with the Illumina system. However, the coverage of genome assembly for EHI_096680 was only 12.5x, whereas for CL6EHI_096680, it was 30x. Intrinsic factors should be considered as an essential aspect. Genomic DNA for EHI_096680 was prepared from E. histolytica strain HM-1: IMSS (ATCC number 30459) [53], while CL6EHI_096680 was from E. histolytica HM-1: IMSS clone 6 (CL6), which had been generated by single cell cloning of the strain by Louis S. Diamond [54,55] and continuously maintained in Japan until 2001 (NCBI BioProject accession PRJDB4673) [56]. Cultivation conditions, as well as in vitro and in vivo environments, also affect the gene expression and intracellular adaptation [57]. Additionally, approximately one-fourth of E. histolytica genes are predicted to contain introns, with 6% of these genes containing multiple introns. Moreover, its chromosomes do not condense, and its ploidy remains uncertain [53]. These unique characteristics of E. histolytica may lead to mis-annotation and misrecognition by some bioinformatic tools that are otherwise well-established for mammalian genomes. Nevertheless, the intron length found in CL6EHI_096680 is consistent with findings at the amino acid level in other species, particularly E. invadens and E. moshkovskii (S1b Fig). Therefore, based on the sequencing results, the CL6EHI_096680 version was used for further analysis.
Based on the InterPro domain prediction, the EhVFT is predicted to have several domains. First, it has a periplasmic binding protein-like 1 (PBP1, IPR028082) characterized mainly by two globular subdomains, each with a central β-sheet flanked by two or three helices [58]. Second is a class C GPCR GABA-B receptor domain (IPR002455). The last is an ANF receptor domain (IPR001828) that belongs to the extracellular ligand-binding domain of a wide range of receptors, including bacterial amino acid-binding proteins [13,59]. ANF is a hormone secreted by the heart atrium that functions to increase glomerular filtration and inhibit sodium reabsorption in the kidney. The extracellular domain of the ANF receptor consists of two lobes, each comprising a central β-sheet flanked by α-helices. It also tends to spontaneously form dimers [60]. Other class C GPCRs such as CaSR, GABA-B-R, and mGluR are also known to have a similar extracellular domain characteristic to that found in the ANF receptor. Similarly, as shown in the MSA and structural analysis (Fig 1c and Fig 2), EhVFT was demonstrated to retain the characteristics of the bacterial PBP and the VFT domain of class C GPCRs. The VFT commonly preserves the region located between ⍺-helix III and β-sheet I as the region for ligand binding, typically an amino acid. Amino acid recognition by PBPs and class C GPCR is encoded by an 8-residue motif: [S]-x(22)-[TS]-x(13,14)-[R]-x(4)-[D]-x(2)-[Q]-x(24,25)-[Y]-[GA]-x(74,84)-[ED]19 which is also conserved in the VFT [14,40].
Consistent with the presence of an ANF receptor domain, EhVFT clustered with class C GPCR proteins, bacterial PBPs, and human ANF receptors based on the phylogenetic analysis (Fig 1a). Interestingly, although EhVFT also contains a predicted GABA-B-R domain, the phylogenetic analysis revealed a more coherent and distinctive pattern than initially anticipated. Rather than clustering with canonical GABA-B receptors, EhVFT and other Entamoeba VFT sequences formed a distinct monophyletic clade, whose closest neighbors were bacterial PBP sequences, followed by class C GPCR VFTs including ionotropic glutamate receptors (GluRs), metabotropic glutamate receptors (mGluRs), and calcium-sensing receptors (CaSRs). This pattern suggests that lateral gene transfer may have contributed to the acquisition of EhVFT. Furthermore, multiple sequence analysis (MSA) (Fig 2) revealed that the predicted secondary structure and the amino acid-binding residues of EhVFT were well conserved and aligned closely with those of related proteins.
Similar to EhVFT, the Dictyostelium discoideum GPCR, GrlE, also harbors the exact three domains. It is also closely related to both the GABA-B receptor and the mGluR. Hence, it can bind both GABA and glutamate competitively, which is essential during different developmental stages. This phenomenon contradicts the high specificity characteristic of mammalian GPCRs toward their ligands [61,62]. Likewise, EhVFT may also exhibit less specificity, indicating that it has multiple effects following activation by different ligands under various stages and conditions.
The membrane expression of EhVFT was as expected (Fig 3). Although the carboxyl-terminal transmembrane domain was absent, that part was replaced by a GPI-anchor signal. The GPI-anchor signal is composed of a carboxyl-terminal hydrophobic domain separated from the GPI-anchor modification site (the ω-site) by a short stretch of hydrophilic amino acids. To differentiate it from the actual single-spanning transmembrane domain, the biological hydrophobicity scale prediction of the carboxyl-terminal sequence, based on the apparent free energy of membrane insertion (Gapp), can be used. In the case of EhVFT, this value also fell within a narrow range of ~0 kcal/mol, indicating efficient anchoring (Fig 2 and S1f and S1g). In some cases, although a hydrophobic amino acid stretch is present, some natural GPI signals may be embedded in the ER membrane, while others may not. The GPI transamidase can only recognize substrates that are not too tightly embedded in the ER membrane, which is in line with the ΔGapp score prediction [42]. Interestingly, a membrane-associated proteome study doesn’t identify EhVFT (CL6EHI_096680 or EHI_096680), despite its membrane localization demonstrated in this study. By cross-checking other proteins in the list, it is likely that the identification can’t cover the entire membrane proteome, as another GPI-anchored protein (GPI-AP), Igl2 (EHI_183000), is also not included in the list [63].
Some GPI-related machinery proteins were also detected from the co-IP results (Table 1 and S2). Those included PIG-X, PIG-S, and PIG-K. The biosynthesis of GPI-AP in mammals, as reviewed before [64], involves over 20 intra-ER-golgi catalytic steps starting from GPI synthesis, GPI addition onto the proproteins by GPI transamidase (GPI-T), and GPI-AP maturation. In comparison to human GPI-AP biosynthesis enzymes, Entamoeba has also preserved nearly all of the GPI-AP biosynthesis enzymes listed in a previous study [23], including the ER transmembrane pentameric protein, GPI-T, which is composed of five subunits: GAAP1, PIG-K, PIG-S, PIG-T, and PIG-U [65]. This suggests the importance of this system in the Entamoeba’s survival and pathogenesis. For instance, the silencing of a partially characterized GlcNAc-phosphatidylinositol deacetylase (PIG-L), one of the enzymes involved in GPI biosynthesis, causes suppression of cell growth, inhibition of endocytosis, and adhesion [23]. Notably, Entamoeba GPI anchor is typically a Phosphatidylinositol Phospholipase C (PI-PLC)- and PI-PLD-resistant type of GPI anchor due to the presence of an acyl moiety esterified to the inositol ring of the GPI anchor [66].
In our study, the EhVFT protein was found to localize to the plasma membrane when the GPI-anchor signal was present (Fig 3), supporting its putative role in sensing extracellular signals. However, removal of the GPI-anchor signal resulted in a striking change in localization, with the protein misdirected to the mitosome, a mitochondrion-related organelle (Fig 4). This shift underscores the critical role of the GPI-anchor in directing EhVFT to the correct subcellular compartment. The mislocalization upon GPI removal likely disrupts its signal-sensing function, underscoring the essential role of precise membrane targeting in the activity of the VFT proteins in E. histolytica. Despite the mitosome main function is in the sulfate activation pathway [67–70], the discovery of the truncated EhVFT in the mitosome suggests that this organelle may play novel roles in the unfolded protein response or protein degradation pathway. The ΔGPI co-IP proteome revealed that the unfolded protein response and Endoplasmic Reticulum-associated Degradation (ERAD) pathway were the majority of proteins, both in the exclusive and enriched hits. Additionally, many mitosome proteins were also detected in the list, which is consistent with the immunofluorescence results (Fig 4 and S3 Table). These included the mitosome importer machineries: Tom60 (EHI_053160, 7.1 fold), Tom40 (EHI_104420, 1.5 fold), and mtHsp70 (EHI_007150, 4.1 fold) [71,72]. Other typical mitosome proteins detected were MBOMP30 (EHI_178630, 2.1 fold) [73], inorganic pyrophosphatase (IPP, EHI_124880, 2.7 fold), and APSK (EHI_179080, 1.3 fold) [68]. Recent studies reveal such a degradation pathway involving the import of cytosolic misfolded proteins into the mitochondria to resolve cytosolic proteostasis [74,75]. However, no such protein degradation machinery has been detected in the previous mitosome proteome [67]. Although this finding still can’t be clearly explained, the importance of the carboxyl-terminal GPI-anchor signal for membrane localization has been demonstrated. Although more reliable experiments, such as chemical or enzymatic cleavage, as well as radiolabeling of the GPI anchor, are needed, the involvement of GPI anchor transamidase complex protein (PIG-S and PIG-K) and ER-Golgi-Plasma membrane trafficking machineries (EhSar1, AP-1, Syntaxin, etc), which are absent in the ΔGPI ectopic expression strain co-IP proteome (Table 1, S2), suggests that GPI modification is authentic.
Although the knockdown of EhVFT was not lethal to the parasite (S3 Fig), its virulence factors, such as motility and phagocytosis, were significantly affected (Fig 6). Migration and motility were significantly reduced in both velocity and directionality. It is interesting to note that, despite the phagocytosis capability being partially reduced, the trogocytosis capability against mammalian cells was unaffected (S3d Fig). This distinction suggests that phagocytosis and trogocytosis, while both involving membrane dynamics, rely on different underlying mechanisms that are differentially impacted by the absence of EhVFT. This phenomenon may be explained as follows: (1) Many key genes, which are upregulated in the cell capable of bead phagocytosis [76], were downregulated in the EhVFTgs (Table 2). Among those are: CRIB domain containing protein (EHI_124620), gelsolin (EHI_009570), villidin (EHI_021260, EHI_150430), myosin II heavy chain (EHI_110180), coronin (EHI_122800), calponin homology (CH) protein (EHI_189500, EHI_007640), glutamic acid-rich protein GLE1 (EHI_082590), and fibrinogen-binding protein (EHI_098440); (2) Myosin II, as a central component of the cytoskeleton, is known to bind F-actin and is essential for contractility and intercellular motility between intestinal epithelial cell monolayers and liver invasion [77]. The uroid is a rear superstructure necessary as a stepping point for forward motility and pseudopod formation. In the presence of concanavalin A, myosin II is three times more concentrated in the uroid, and chemical inhibition of myosin heavy chain phosphorylation, which inhibits capping, prevents uroid formation [78,79]. Also, the myosin II null phenotype showed abnormal movement, failure of uroid formation, and failure of concanavalin A capping [80]; (3) The fact that EhVFT is a GPI-AP may contribute to the importance of the lipid raft structure during the phagocytosis process. Lipid raft has been shown to have a significant role in phagocytosis in human macrophages [81]. E. histolytica is also shown to have raft-like plasma membrane domains where the GPI-AP is expressed. All of the Gal/GalNAc lectin subunits were concentrated in this fraction, which is in line with the raft-dependent function of endocytosis and adhesion [82–84]; and (4) in line with the RNA-seq results, the co-IP also showed involvement of deeper actin cytoskeleton components (Table 1, S2): Arp2/3 complex (subunit 4, EHI_030820), a member of the “phagocytosis complex” [85], was enriched 3.1 fold; fimbrin/plastin (EHI_193940) was in the exclusive hit list; and paxillin (EHI_022960) was enriched 3.7 fold. Although both are actin-dependent processes, phagocytosis involves the uptake of larger particles, and therefore requires a more complex and extensively organized actin cytoskeleton, along with associated regulatory proteins, many of which were found to be downregulated upon EhVFT silencing. In contrast, trogocytosis involves the transfer of membrane fragments and depends on a more limited set of cytoskeletal components [85]. This study was limited to observing phagocytosis in mammalian cells, as bead and bacterial phagocytosis weren’t observed.
Although many protein binding partners were successfully pulled down in the co-IP experiments, no tightly bound proteins with high QV were identified (Table 1, S2). Even though resembling the class C GPCR in VFT structure, EhVFT could not pull down any heterotrimeric G-protein complex members, probably due to the absence of the transmembrane domain. As a limitation of the study, the activation mechanism and the subsequent intracellular cascade reactions following EhVFT activation remain unclear. However, some proteins may act as mediators of EhVFT activation signal. One such protein, the Leucine-rich repeat containing protein (EHI_164460, Table 1), has been implicated in phagosome formation. Its localization appears to be phagosome specific in an Atg8-dependent manner [86]. Interestingly, based on our BLASTp and domain analysis, this protein is predicted to be cytoplasmic and lacks a transmembrane domain. It contains multiple functional domains, including an RNA1 domain, Ran GTPase-activating protein (RanGAP) which is implicated in cytoplasmic–nuclear cargo transportation, and protein phosphatase 1 regulatory subunit 42 (PPP1R42) domain, known to contribute to centrosome separation. In addition, several cell membrane-associated proteins were detected: galactose-inhibitable lectin, putative (Lgl, EHI_058330) and cysteine protease binding family protein 3 (CPBF3, EHI_161650) (Table 1). These findings raise the possibility that EhVFT may interact with Lgl or CPBF3, although further experimental evidence is required to confirm such interactions.
Nevertheless, some hypotheses regarding the EhVFT activation cascade can be proposed, which refers to the mammalian system. Although the GPI-anchored receptor (GPI-AR) does not have any cytoplasmic domain, in the case of CD59, for instance, signal transduction can be achieved by a different mechanism utilizing the lipid raft microdomain. The binding of a GPI-AR ligand induces stable GPI-AR cluster signalling rafts and a trans-bilayer raft phase by recruiting inner-leaflet molecules which have saturated alkyl chains, such as H-Ras [87], Lyn [87,88], and Gαi2 [87,88]. A previous CD59 immunoprecipitation experiment also successfully pulled down Gi1, Gi2, and Gi3, indicating their role as effector proteins of GPI-AR [89]. Then the clustering of CD59 and involvement of its effector, Gαi2, can activate Lyn to immobilize the cluster via F-actin [88]. Additionally, it also induces the IP3-Ca2+ response via PLCγ2 activation [90]. In the cluster, they interact via both protein-protein and lipid-lipid (raft) interactions. The residency time of these inner leaflet molecules is often limited. However, one molecule may come after another, allowing effective interactions beneath the cluster raft and transducing the important signal [87,88]. Altogether, these observations underscore an important limitation of the current study and emphasize the need for future investigations using complementary biochemical and structural approaches to identify the signaling partners of EhVFT and elucidate its precise mode of action.
Another limitation of this study is the inability to identify a preferred ligand for EhVFT. Although the receptor retains an amino acid-binding motif between α-helix III and β-sheet I, our attempts to detect ligand interactions using isothermal titration calorimetry (ITC) with CaCl₂ (CaSR ligands), γ-aminobutyric acid (GABA; GABAbR ligand), and Na-L-glutamate (mGluR ligand) were unsuccessful (S4 Fig). Despite extensive optimization, no reproducible heat changes indicative of binding was observed. This technical limitation precludes identification of the external stimulus that activates this non-canonical receptor and further underscores the necessity of employing alternative biophysical or structural approaches in future studies.
In summary, this study presents a comprehensive structural, cellular, and functional characterization of EhVFT (CL6EHI_096680). The structural composition of EhVFT provides significant insight into the evolution of signaling receptors in protozoa. While EhVFT shares homology with the extracellular portion of Class C GPCRs, the absence of a 7TM domain prevents it from functioning as a canonical GPCR. Instead, EhVFT is likely a true VFT-related protein derived from the Periplasmic Binding Protein (PBP) superfamily. PBPs are bacterial proteins involved in nutrient sensing and transport that are considered the evolutionary ancestors of eukaryotic VFT domains. Since E. histolytica is an ancient lineage, the presence of EhVFT likely reflects an evolutionary intermediate acquired through ancient horizontal gene transfer, confirming the pathway that eventually led to the fusion of VFT domains with 7TM helices in higher eukaryotes. This study identified EhVFT not as a classical GPCR, but as a membrane-tethered VFT-domain receptor distinct from the canonical G-protein signalling machinery. EhVFT functions through a mechanism distinct from heterotrimeric G-protein coupling, consistent with our co-immunoprecipitation data which found no interaction with G-protein subunits. Our findings provide novel insights into the signalling mechanisms governing parasite motility and mammalian phagocytosis. It also highlights the diversity of protozoan signalling, in which ancient PBP-like domains have been adapted for environmental sensing and virulence regulation, independent of 7TM architectures. This work lays important groundwork for future studies on receptor-mediated signal transduction and its role in the parasite’s pathogenesis.
Materials and methods
Organism, cultivation, and chemicals
Trophozoites of Entamoeba histolytica strains HM-1:IMSS clone 6 [54] and G3 [91] were axenically cultured in 6 ml screw-capped Pyrex glass tubes containing Diamond’s BI-S-33 (BIS) medium at 35.5°C [92,93]. All trophozoites used for the experiment were prepared during the logarithmic phase at 24 hours of cultivation. Jurkat cell (T cell leukemia) was maintained at 37°C with RPMI medium (Invitrogen-Gibco, New York, USA) supplemented with 10% fetal bovine serum on a 25 cm3 flask (IWAKI, Shizuoka, Japan). The anti-HA 16B12 mouse monoclonal antibody was obtained from Biolegend (San Diego, USA). The anti-HA rabbit polyclonal antibody was obtained from MBL Life Science (Tokyo, Japan). Lipofectamine, Plus reagent, and geneticin (G418) were sourced from Invitrogen. PlasMem Bright Red was purchased from Dojindo (Tokyo, Japan). CellTracker Orange was purchased from Thermo Fisher Scientific (Massachusetts, USA). Unless otherwise specified, restriction enzymes and DNA-modifying enzymes were obtained from New England Biolabs (Massachusetts, USA). Luria-Bertani (LB) medium was procured from BD Difco (New Jersey, USA). Other general reagents were purchased from Wako Pure Chemical (Tokyo, Japan), unless noted otherwise.
Phylogenetic analysis, multiple sequence alignment, and 3D protein construction
For phylogenetic analysis, we collected ~706 protein sequences identified by the Foldseek search (https://search.foldseek.com/search) using the CL6EHI_096680 protein sequence (GAT93065.1) as a query, the PDB100 20240101 as the database, and 3Di/AA as the chosen mode [94]. Protein sequence IDs were stated as Protein Data Bank (PDB) ID. After removing duplicated proteins, around 402 proteins remained for further analysis. Sequences were aligned using the MUSCLE algorithm in Molecular Evolutionary Genetics Analysis (MEGA) 12 software. The maximum-likelihood (ML) analysis was implemented in the MEGA 12 with 100 standard bootstrap replications [95]. Other parameters were set as default. Trees were constructed using iTOL ver 7.2 [96]. Meanwhile, for important residue prediction, a small multiple sequence alignment was performed using Clustal Omega [97]. The 3D protein structure was constructed using AlphaFold3 [37] and visualized using open-source PyMOL [98].
Establishment of E. histolytica transformants
The plasmid construction for expressing EhVFT (CL6EHI_096680, NCBI Accession No. GAT93065.1) with an HA tag was tried using five different construct designs based on the HA tag position. However, only three were used for localization and domain importance analysis (see Fig 3a). The coding regions were amplified by PCR from E. histolytica cl6 cDNA using specifically designed primers (see S1 Table). The resulting PCR products and pEhExHA vector plasmids were digested with either XhoI and XmaI (for N-terminal tagging) or BglII (for C-terminal tagging), as appropriate, and then ligated [49], resulting in the constructs of pEhEx- EhVFT-HA-GPI, pEhEx-EhVFT-HA-ΔGPI, pEhEx-HA-EhVFT-ΔSPΔGPI, pEhEx-SP-HA-EhVFT, pEhEx-EhVFT-HA, and pEhExHA mock. The pEhEx-SP-HA-EhVFT and pEhEx-EhVFT-HA constructs were excluded from analysis due to their Endoplasmic Reticulum localization. For antisense small RNA-mediated transcriptional silencing of EhVFT genes in the G3 strain, 416 bp fragments from the N-terminal coding regions were amplified by PCR from cDNA using sense and antisense primers containing StuI and SacI restriction sites. After digestion, the insert was ligated with the psAP2-Gunma vector, resulting in the psAP2-EhVFTgs construct [91,99,25]. pEhExHA vector-based plasmids were introduced into E. histolytica HM-1:IMSS cl6 trophozoites, while those derived from the psAP2-Gunma vector were introduced into the G3 strain via lipofection, following previously described methods [49]. Transformants were started to be exposed with 1 or 2 μg/ml G418 on the next day. Depending on the number of cells remaining and the expression level, the concentration was gradually increased up to 10 μg/ml. To assess gene silencing, reverse transcriptase PCR was conducted to evaluate mRNA levels of the silenced genes. Total RNA was extracted from logarithmic-phase trophozoites using TRIzol reagent (Life Technologies, California, USA). DNase-treated RNA was used for cDNA synthesis with the Superscript III First-Strand Synthesis System (Thermo Fisher Scientific, Massachusetts, USA), using oligo(dT) primers, according to the manufacturer’s instructions. DNA amplification was performed using the ExTaq PCR system and the primer sets listed in the S1 Table.
Immunoblot analysis
To check the expression level of the HA-tagged EhVFT, trophozoites of E. histolytica transformants were harvested after ~24 hours of cultivation and were washed with phosphate-buffered saline (PBS). The cells were then lysed by resuspending them in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.2% Triton X-100, 0.5 mg/ml E-64, and 1x concentration of the cOmplete Mini EDTA-free Protease Inhibitor (Roche, Mannheim, Germany). The suspension was sonicated on ice, followed by centrifugation at 500 × g for 5 minutes to remove the unbroken cells. Approximately 10–20 μg of the resulting whole-cell lysate was resolved by electrophoresis on a 5–12% SDS-PAGE gel, and the proteins were subsequently transferred onto PVDF membranes. To prevent nonspecific binding, the membranes were blocked for 30 minutes at room temperature with 5% skim milk in Tris-buffered saline and Tween-20 (TBST; 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween-20). Membranes were then incubated overnight at 4°C with one of the following primary antibodies, diluted in TBST: anti-HA mouse monoclonal antibody (clone 16B12) at a dilution of 1:1,000 or anti-CS1 rabbit polyclonal antibody at a dilution of 1:1,000. After washing three times with TBST, the membranes were incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies—either anti-mouse or anti-rabbit IgG—at a 1:10,000 dilution. Three times final washes with TBST were performed to eliminate excess secondary antibody. Protein bands were then visualized using a chemiluminescent HRP substrate system (Millipore, Massachusetts, USA) and detected using the ChemiDoc Imaging System (Bio-Rad, USA), following the respective manufacturer’s instructions.
Indirect immunofluorescence assay (IFA)
Trophozoites of Entamoeba histolytica in logarithmic growth phase (~24 hours) were suspended in 100 μl of BIS medium and placed into 8 mm round wells on glass slides (Matsunami Glass Ind., Osaka, Japan). The slides were incubated for 15 minutes at 35.5°C within an anaerobic chamber to facilitate cell adherence. Post-incubation, the medium was gently aspirated, and the cells were fixed with 3.7% paraformaldehyde in PBS for 10 minutes at room temperature. Fixed cells were washed three times with PBS and permeabilized using a buffer containing 0.2% saponin and 1% BSA in PBS for 10 minutes at room temperature, unless otherwise indicated. For immunofluorescence, samples were incubated with one of several primary antibodies for 1 hour at room temperature: mouse monoclonal anti-HA (1:500), rabbit monoclonal anti-HA (1:200), mouse monoclonal anti-Igl (EH3015; 1:300) [100], rabbit polyclonal antibodies against EhVFT (1:300), APSK (1:300), CS1 (1:500), Sec13 (1:300), or BiP (1:300). After primary staining, cells were washed three times in 0.1% BSA in PBS and incubated with appropriate secondary antibodies: Alexa Fluor 488-conjugated anti-mouse or anti-rabbit IgG (1:1,000), Alexa Fluor 568-conjugated anti-rabbit IgG (1:1,000), or Hoechst 33342 nuclear dye (1 μg/ml), all from Thermo Fisher Scientific (Massachusetts, USA). Confocal images were captured using an LSM 780 microscope (Carl Zeiss, Oberkochen, Germany) and processed with ZEN software and ImageJ software. Colocalization analysis was performed by setting fluorescence thresholds for green and red channels based on single-stain controls and applying these settings uniformly across all samples. The colocalization coefficient was determined using ZEN’s built-in analysis tools. Data was analyzed using GraphPad Prism ver 10.4.1.
Reverse transcriptase PCR
RNA of E. histolytica was extracted from ~1x106 cells using Trizol reagent (Ambion; Life Technologies, Grand Islands, NY, USA) based on the manufacturer’s protocol. Genomic DNA was removed after treatment with DNase I (Invitrogen). cDNA was synthesized using the SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s protocol. cDNA was then amplified using Ex-Taq polymerase (Takara). Primers are listed in the S1 Table. EhRNA polymerase II gene (EHI_056690) was used as a normalizing control [101–103].
qRT-PCR
To measure the EhVFT’s mRNA level, qRT-PCR was performed using primer sets as listed in the S1 Table. The reactions were assembled in a final volume of 20 μL, consisting of 10 μL of 2 × Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), 0.7 μL of each primer at a concentration of 10 μM, 5 μL of 10x diluted cDNA, and nuclease-free water to adjust the volume. Amplification was conducted using the StepOne Plus Real-Time PCR System (Applied Biosystems) under the following thermal cycling protocol: initial denaturation at 95°C for 20 seconds, followed by 40 cycles of denaturation at 95°C for 3 seconds and annealing and extension at 60°C for 30 seconds. Each reaction was performed in triplicate, including no-template controls to ensure specificity and accuracy. Relative expression levels of the EhVFT gene were quantified using the 2^–ΔΔCt method, with EhRNA polymerase II serving as the internal normalization control [101–103].
RNA-seq analysis
Total RNA of psAP2-EhVFTgs and psAP2-mock control trophozoites was subjected to directional library preparation and sequenced on an Illumina NovaSeq 6000 platform (Veritas Genetics, China; Fujifilm, Japan). For sequence read analysis, fastp (version 0.23.2) was used to assess the quality of all reads and remove those of low quality. Each read obtained after filtering was mapped to the reference genome AmoebaDB-EhistolyticaHM1IMSS-Genome using STAR (version 2.7.10a). Mapping reads was done using the default parameter values. Additionally, featureCount (version 2.0.1) was used to calculate the number of counts for each gene, as defined by its gene ID. In this case, counting reads was performed for the gene ID defined by AmoebaDB‒EhistolyticaHM1IMSS release‒54 (https://amoebadb.org/amoeba/app/downloads). Genes which have no count in all samples were removed. After filtering, the total number of reads was normalized to 1 million per sample (CPM, count per million). Normalized read counts were analyzed using edgeR (version 3.40.2) to identify Differentially Expressed Genes (DEGs) between the control and silencing groups. The up-regulated genes are those whose Fold Change (FC) > 2 and q.value < 0.05. Similarly, genes whose Fold Change < 0.5 and q.value < 0.05 were defined as down-regulated genes. Principal component analysis (PCA) and volcano plots were created to visualize the results. PCA plots were used to display sample variation, while volcano plots highlighted differentially expressed genes. Both types of visualizations were generated using the ggplot2 R package (v3.5.1) and GraphPad Prism ver 10.4.1. Gene Ontology (GO) analysis was performed using shinyGO ver 0.77 with STRING.5759.Entamoeba database [104].
Production of rabbit polyclonal antibody against EhVFT
To generate rabbit polyclonal antibodies, recombinant EhVFT (rec-EhVFT) was produced in an Escherichia coli expression system. A truncated version of the CL6EHI_096680 gene, corresponding to nucleotides 58–1128 (encoding amino acids 20–376), was cloned into the pCOLD1 vector and transformed into E. coli BL21 (DE3) cells. The culture was grown in LB medium containing 100 μg/mL ampicillin at 37°C with shaking until the optical density at 600 nm (A600) reached approximately 0.6. Protein expression was induced by adding 0.05 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), followed by incubation at 15°C for 24 hours at 180 rpm. Cells were harvested and resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0; 300 mM NaCl; 0.1% Triton X-100; 100 μg/mL lysozyme; 1 mM PMSF; and 10 mM imidazole), and incubated at room temperature for 30 minutes. Cell lysis was performed using a French Press, and the lysate was centrifuged at 16,000 × g for 40 minutes at 4°C to obtain the soluble protein fraction. For purification, clarified lysates were incubated with a 50% slurry of Ni² ⁺ -NTA resin (Novagen, Darmstadt, Germany) for 1 hour at 4°C with gentle rotation. After extensive washing, bound proteins were eluted using an elution buffer containing 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 0.01% Triton X-100, and 300 mM imidazole. The purity of the eluted rec-EhVFT protein was assessed by SDS-PAGE followed by Coomassie Brilliant Blue staining. Imidazole was removed from the eluate through repeated filtration using Amicon Ultra 10K centrifugal filters. The final purified protein was stored in 20% glycerol at −80°C until further use.
Polyclonal anti-EhVFT (CL6EHI_096680) antibodies were commercially produced by Eurofins Genomics Co., Ltd. (Tokyo, Japan). For immunization, 0.15 mg of recombinant EhVFT was mixed with Freund’s Complete Adjuvant (FCA) and injected subcutaneously into rabbits. This was followed by five booster injections of 0.3 mg each, administered at two-week intervals. The production of specific antibodies was evaluated by comparing sera collected after the final immunization with pre-immune sera, using both ELISA and western blot analyses.
Growth kinetic assay of silencing strains
The proliferation of EhVFTgs strains, along with the control strain harboring the mock psAP2-Gunma vector, was assessed over four days. Each culture was initiated by seeding 30,000 trophozoites into 6 mL of fresh BI-S-33 medium containing 10 µg/mL G418 antibiotic. Trophozoite counts were performed every 24 hours using a hemocytometer to monitor growth kinetics. Observations were conducted in triplicate using independently cultured samples. Statistical comparisons were analyzed using a 2-way ANOVA followed by Šídák’s multiple comparisons test for growth kinetics and unpaired t-test for doubling time on GraphPad Prism ver 10.4.1.
Cell migration assay
Trophozoites from the EhVFTgs strain and the psAP2-Gunma mock control were collected and washed with phosphate-buffered saline (PBS). To induce serum starvation, the cells were resuspended in serum-free BI medium and incubated for 3 hours under standard anaerobic conditions. Following starvation, approximately 1.5 × 10⁵ cells were seeded into the upper chamber of an 8-µm pore ThinCerts cell culture insert (Greiner Bio-one, Tokyo, Japan), which was placed in a 12-well plate with a plastic bottom. Complete BIS medium was added to the lower chamber to serve as a chemoattractant. The transwell setup was then incubated at 35.5°C in an anaerobic chamber for 3 hours. After incubation, cells that had migrated through the membrane into the lower chamber were collected and counted using a hemocytometer. The percentage of migrated cells was calculated to assess the migratory capacity of each strain. Statistical comparisons between groups from two independent experiments were analyzed using a 2-way ANOVA followed by Šídák’s multiple comparisons test on GraphPad Prism ver 10.4.1.
Cell motility assay
Approximately 2,500 cells from the EhVFTgs strain and the psAP2-Gunma mock control were seeded into a 12-well glass-bottom plate (IWAKI, Shizuoka, Japan) and incubated overnight at 35.5°C in an anaerobic chamber. The following day, the spent BIS medium was replaced with fresh BIS medium pre-warmed to 37°C and supplemented with PlasMem Bright Red dye at a 1:1000 dilution. The plate was then sealed with adhesive film to maintain the anaerobic conditions. To monitor cell motility, time-lapse confocal imaging was performed using an LSM 780 microscope (Carl Zeiss, Oberkochen, Germany), capturing approximately 150 sequential images per cell over approximately 3 minutes. Two independent experiments were conducted, with each imaging session limited to a maximum of 3 hours to preserve cellular viability and minimize physiological stress. The resulting image sequences were analyzed using ICY version 3 software, utilizing the Motion Profiler and Track Processor plugins to quantify and visualize cell movement dynamics [105]. Statistical comparisons between groups from two independent experiments were analyzed using an unpaired t-test on GraphPad Prism ver 10.4.1.
Phagocytosis and trogocytosis assay
Trophozoites from the EhVFTgs strain, along with psAP2-Gunma mock control, and the EhVFT-HA-GPI ectopic expression strain, along with HA-mock control, were co-cultured with Jurkat cells to assess phagocytosis and trogocytosis. Phagocytosis assays involved heat-killed Jurkat cells (treated at 55°C), whereas trogocytosis was evaluated using live Jurkat cells, both applied at a 1:5 amoeba-to-Jurkat ratio. Prior to co-incubation, Jurkat cells were labeled with 10 nM CellTracker Orange (CTO). The incubation times were limited to 60 minutes for phagocytosis and 120 minutes for trogocytosis.
Data acquisition was performed using a BD Accuri C6 Plus Flow Cytometer. For flow cytometry analysis, gating strategies were applied to differentiate amoebic cells from Jurkat cells and debris based on forward scatter (FSC) and side scatter (SSC) parameters. A total of 5,000 amoeba events were gated per sample. FlowJo software was used for downstream data analysis. For the phagocytosis assay, the proportion of amoeba cells that had internalized heat-killed Jurkat cells was estimated by detecting an increase in fluorescence intensity in the PE-A channel, characterized by a distinct dual-peak pattern. In contrast, for trogocytosis with live Jurkat cells, the geometric mean fluorescence intensity of the PE-A channel was measured, indicating a fluorescence shift due to partial membrane transfer. Statistical comparisons between experimental and control groups were conducted using a 2-way ANOVA followed by Šídák’s multiple comparisons test on GraphPad Prism ver 10.4.1, based on three independent biological replicates per condition and three trials.
Co-immunoprecipitation (co-IP)
To investigate potential interacting partners of EhVFT (CL6EHI_096680), co-immunoprecipitation (co-IP) assays were performed using trophozoites expressing EhVFT-HA-GPI or EhVFT-HA-ΔGPI. HA mock strain was used as a control. After 24 hours of cultivation, five semi-confluent 10 cm culture plates (IWAKI, Shizuoka, Japan) were harvested and washed three times with PBS. To stabilize protein-protein interactions, the crosslinker dithiobis[succinimidylpropionate] (DSP, Thermo Scientific) was added according to the manufacturer’s instructions. Cells were then lysed in 1 mL of ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5 mg/mL E64) for 10 minutes on ice, followed by centrifugation at 16,100 × g for 5 minutes at 4°C to obtain total lysates. The supernatants were first incubated with 50 µL of Protein G Sepharose 4 Fast Flow (Cytiva, Uppsala, Sweden) beads at 4°C for 1 hour with gentle rotation, followed by centrifugation at 800 × g for 3 minutes at 4°C. The resulting supernatants were then incubated with 50 µL of monoclonal anti-HA agarose antibody produced in mouse, clone HA-7, purified immunoglobulin conjugated to agarose beads (Sigma-Aldrich) at 4°C for 3 hours under rotation. Bead pellets were collected by centrifugation (800 × g, 3 minutes, 4°C), and flow-through fractions were discarded. The beads were washed three times with wash buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100) at 9,300 × g for 1 minute per wash. To elute the bound proteins, beads were incubated overnight at 4°C with Influenza Hemagglutinin (HA) -peptide (Sigma-Aldrich) at a final concentration of 0.2 mg/mL under rotation, followed by centrifugation at 800 × g for 3 minutes at 4°C to collect the eluates. The remaining beads were washed an additional three times, and both eluates and final HA-bead fractions were collected for silver stain and western blot analysis.
Mass spectrometry-based protein sequencing
Protein identification via mass spectrometry of eluted co-IP samples was carried out at the Mass Spectrometry and Proteomics Core Facility, Johns Hopkins University, Maryland, USA. As mentioned previously, in brief, lyophilized protein samples were reconstituted in 40 μL of 20 mM triethylammonium bicarbonate (TEAB; pH 8.0) and subsequently reduced by the addition of 5 μL of 50 mM dithiothreitol (DTT), followed by incubation at 60°C for one hour. After cooling to room temperature, proteins were alkylated by treating them with five μL of 100 mM chloroacetamide for 15 minutes in the dark to prevent disulfide bond reformation. The samples were then diluted with 400 μL of 9 M urea and transferred to 30 kDa molecular weight cut-off (MWCO) spin filters (pre-washed with distilled water) for buffer exchange and concentration. Following sequential washes with urea and TEAB to remove contaminants, each sample was incubated overnight at 37°C with 400 ng of proteolytic enzyme in 300 μL of TEAB for protein digestion. The next day, peptides were collected by centrifugation; the spin filters were additionally rinsed to ensure maximal recovery, and all filtrates were combined. The peptide mixtures were acidified, desalted using Oasis HLB microelution cartridges, vacuum-dried using a SpeedVac, and resuspended in 2% acetonitrile with 0.1% formic acid. The processed peptides were then analyzed on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer for protein sequencing [104].
Isothermal Titration Calorimetry (ITC)
Recombinant EhVFT was prepared following a previously described protocol used for generating anti-EhVFT antibodies, with minor modifications. Cell lysis was performed using a buffer containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 0.5% Triton X-100, 100 μg/mL lysozyme, 1 mM PMSF, and 10 mM imidazole. Bound proteins were eluted using a buffer composed of 50 mM HEPES (pH 7.5), 300 mM NaCl, and 300 mM imidazole. The eluted protein was subjected to sequential extensive dialysis at 4°C to remove imidazole and chelating agents. First, the sample was dialyzed overnight against 3 L of EDTA-containing buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 10 mM EDTA), followed by three consecutive dialysis steps (2 L each, 3 hours per step) against EDTA-free buffer (50 mM HEPES pH 7.5, 300 mM NaCl). A final overnight dialysis was performed against ITC buffer (50 mM HEPES pH 7.5, 300 mM NaCl). Protein purity was assessed by SDS–PAGE followed by Coomassie Brilliant Blue staining.
The purified recombinant EhVFT was used directly for isothermal titration calorimetry (ITC) experiments. ITC measurements were performed using CaCl₂, sodium L-glutamate, and γ-aminobutyric acid (GABA) as candidate ligands. Recombinant EhVFT was loaded into the sample cell, and ligands prepared in the same ITC buffer were loaded into the injection syringe. Experiments were conducted using a standard 13-injection protocol. The instrument was thoroughly washed before, between, and after measurements. MQ–MQ titrations were performed to confirm proper washing, and EDTA–CaCl₂ titrations were used as a positive control to validate instrument performance.
Supporting information
S1 Fig. Bioinformatic analysis of EhVFT (CL6EHI_096680) and its homolog in E. invadens (EIN_328390).
(A) Multiple sequence alignment (MSA) by Clustal Omega and displayed by SnapGene viewer of EHI_096660 genomic sequence (AmoebaDB, 1328 bp, EHI_096680_Gene), EHI_096680 mRNA sequence (AmoebaDB, 1281 bp, EHI_096680_mRNA), and EHI_096680 mRNA sequencing results or CL6EHI_096680 mRNA (1209 bp, CL6EHI_096680_mRNA_Seq) highlights different length of predicted and actual intron. Highlighted in red box is the actual splice donor dinucleotides (GT) and highlighted in green box is the actual splice acceptor dinucleotides (GA). (B) Multiple sequence alignment (MSA) of amino acid sequences among Entamoeba by Clustal Omega. Notably, EHI_096660 (Uniprot ID: C4LWI0, 426 aa) and CL6EHI_096680 (Uniprot ID: A0A175JHL1, 402 aa) show different length of translated amino acid sequences (red box). They have 24 aa differences in the existing databases. Based on the sequencing results, CL6EHI_096680 was the mRNA used in this study. (C) Percent identity matrix of ligand-binding proteins (LBPs) or VFT domains comparing CL6EHI_096680 with representative mammalian and microbial class C GPCR-related proteins. The highest homology was 21.10% by HsCaSR that is consistent with the phylogenetic analysis. HsGABAbR_LBP_1, Homo sapiens GABA type b receptor subunit 1; HsGABAbR_LBP_2, subunit 2; RnMGLUR1, Rattus novergicus metabotropic glutamate receptor 1; HsCaSR, Homo sapiens calcium sensing receptor; DdGRLE, Dictryostelium discoideum GrlE (GABA and glutamate receptor); Atu2422, Agrobacterium tumefaciens GABA binding protein. (D) Percent identity matrix of CL6EHI_096680 homolog proteins among Entamoeba sp. The score ranged around 60 – 80% identical. (E) Multiple sequence alignment of the C-terminal regions of CL6EHI_096680, EIN_328390, and EhIgI2 (EHI_183000) showing hydrophobic amino acid stretch at the C-terminal (green box). (F) Summary of glycosylphosphatidylinositol (GPI) anchor modification site (omega site) prediction using apparent free energy of membrane insertion (ΔGapp, kcal/mol) and online tools NetGPI-1.1. ΔGapp scores were mainly between 0 – 1 which means they might have GPI signal peptides. In addition by NetGPI-1.1, The omega sites were shown in the yellow boxes (E). (G) Signal peptide (SP) sequence prediction of CL6EHI_096680 using SignalP 6.0. It was highly probable that sequence 1 – 19 was standard secretory signal peptides transported by Sec translocon, the cleavage position was between amino acid 19 and 20, and by Signal Peptidase I (Sec/SPI). N, N-terminal domain of SP; H, hydrophobic domain; C, C-terminal domain; and O, other domains.
https://doi.org/10.1371/journal.ppat.1014019.s001
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S2 Fig. Immunofluoresence study of EhVFT-HA-GPI ectopic expression strain stained by rabbit anti-EhVFT (CL6EHI_096680) antibody.
(A) Immunofluorescence images showing expression of HA-tagged EhVFT and HA mock in trophozoites. Cells were stained with mouse anti-HA (green) and rabbit anti-EhVFT (red) antibodies. Merged images show areas of co-localization (yellow). White arrows indicate representative regions used for fluorescence intensity profiling. DIC and merged channels are provided for structural reference. Signals were compared with and without 0.2% saponin as permeabilizing agent. Observation was done for around 20 images from two independent experiments. Scale bars: 5 µm. (B) Line-scan fluorescence intensity profiles from merged images of saponin positive and (C) negative samples, plotting HA (green) and GPCR (red) signals across the selected regions (white arrows). Peaks indicate localization at the plasma membrane (PM) and vesicular membranes (VM). HA, hemagglutinin; Igl, intermediate subunit of galactose (Gal)- and N-acetyl-D-galactosamine (GalNAc)-inhibitable lectin; and DIC, Differential Interference Contrast.
https://doi.org/10.1371/journal.ppat.1014019.s002
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S3 Fig. General cellular fitness and normal phenotypic evaluation of EhVFTgs trophozoites.
(A) Growth curve of psAP mock control strain (blue) and EhVFTgs strain (red) over a 96-hours period. X-axis showed time-periods (hours, observations were done in every 24 hours) and y-axis showed cell number/ml (log). Experiment was done in three biological replicates. (B) Bar graph showing doubling time of (A) between psAP mock and EhVFTgs strains over 96 hours. Bars represent the mean of doubling time ± SD from three biological replicates. (C) RNAseq results showing count per million (cpm) value of EhVFTgs (CL6EHI_096680) and EhRNA polymerase II (EHI_056690) from three biological replicates. (D) Flow cytometry-based trogocytosis assay of EhVFTgs trophozoites (red) in comparison to control (blue). Data represent mean of trogocytosis percentage ± SD from three technical replicates and three independent experiments. Data was analyzed using 2-way ANOVA. P value: < 0.1234 (ns, not significant); < 0.0332 (*); < 0.0021 (**); < 0.0002 (***); and 0.0001 (****).
https://doi.org/10.1371/journal.ppat.1014019.s003
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S4 Fig. Isothermal Titration Calorimetry (ITC) of rec-EhVFT against various ligand candidates.
(A) SDS-PAGE confirmation for recombinant EhVFT production. The thickest band with size ~43.2 kDa was the rec-EhVFT. MW: molecular weight; M: marker; TL: total lysate; P: pellet; S: supernatant; FT: flowthrough; WB30: washing buffer 30 mM imidazole; WB50: washing buffer 50 mM imidazole; EB300: elution buffer 300 mM imidazole; and EB300 dialysis: after dialysis. (B) Titration of 1 mM Na-L-Glutamate to 10 μM rec-EhVFT showed no binding. (C) Titration of 1 mM CaCl2–10 μM rec-EhVFT showed no binding. (D) Titration of 1 mM GABA to 10 μM rec-EhVFT showed no binding.
https://doi.org/10.1371/journal.ppat.1014019.s004
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S1 Table. Primers used during the experiment.
https://doi.org/10.1371/journal.ppat.1014019.s005
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S2 Table. Complete mass-spectrometry data of EhVFT-HA-GPI co-immunoprecipitation.
https://doi.org/10.1371/journal.ppat.1014019.s006
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S3 Table. Complete mass-spectrometry data of EhVFT-HA-ΔGPI co-immunoprecipitation.
https://doi.org/10.1371/journal.ppat.1014019.s007
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S4 Table. Complete RNA-seq data of EhVFT silencing strains.
https://doi.org/10.1371/journal.ppat.1014019.s008
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S1 Data. Raw data from all independent trials.
https://doi.org/10.1371/journal.ppat.1014019.s009
(XLSX)
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