Identification and features of PTEN genes in E. histolytica

A genome-wide survey of PTEN in the genome of E. histolytica HM-1:IMSS reference strain (AmoebaDB, http://amoebadb.org) by BLASTP analysis using human PTEN (P60484) as a query, revealed that E. histolytica possesses 6 possible PTEN or PTEN-like proteins that contain PTEN phosphatase domain, and show different domain configurations (Fig 1A) [20]. We tentatively designated them firstly in an ascending order of the number of recognizable domains and secondly in a descending order of the overall length (EhPTEN1, EHI_197010; EhPTEN2, EHI_098450; EhPTEN3, EHI_131070; EhPTEN4, EHI_054460; EhPTEN5, EHI_041900; EhPTEN6, EHI_010360). Our previous transcriptome data [24–26] verified that one protein (EhPTEN1, EHI_197010) is highly expressed in the trophozoite stage in both E. histolytica HM-1: IMSS cl6 and G3 strains, while the 5 other PTENs are expressed at relatively low levels (Fig 1B). RNA seq data from our laboratory [27] indicate the abundance (FPKM) of six EhPTEN is as follow: EhPTEN1-6, 173.7, 25.8, 9.7, 6.7, 17.8, and 7.0 in HM-1:IMSS cl-6 transfected with HA control vector; 62.0, 11.6, 5.9, 2.0, 9.6, and 7.6 in G3 strain transfected with psAP2-Gunma mock vector. EhPTEN1 shows 39% mutual identity to human PTEN at the amino acid level (S1 Table). Multiple sequence alignment by ClustalW program (http://clustalw.ddbj.nig.ac.jp) shows that the key catalytic residues in the phosphatase domain (H-C-K/R-A-G-K-G-R) needed for lipid and protein phosphatase activity [4,28] are well conserved in EhPTEN1 (Fig 1C). In addition, the PtdIns(4,5)P₂-binding motif (K/R-x4-K/R-x-K/R-K/R-R, PDM domain), which is predicted to regulate the recruitment of protein to the plasma membrane, located at the amino terminus, is also conserved in EhPTEN1 [6,29]. The cytosolic localization signal (D-G-F-x-L-D-L, CLS), where mutation of phenylalanine was shown to induce nuclear localization [30], as well as threonine and isoleucine responsible for TI loop formation in an extension of the active site pocket are also conserved (Fig 1C) [28]. EhPTEN1 also possesses C2 domain which has an affinity for membrane phospholipids and helps PTEN to be recruited to the cell membrane [28]. The homology of the first 190 amino acids in the amino terminus of PTEN to the actin binding protein tensin may suggest the involvement of PTEN in the regulation of actin dynamics. InterPro domain search annotates the region of a.a. 550–680 of EhPTEN1 as a domain of unknown function (DUF457). The two PEST sequences, rich in proline, glutamic acid, serine, and threonine, at the C terminus of human PTEN, are not conserved in EhPTEN1. Although PEST sequences are known to enhance proteolytic sensitivity, the regulation of EhPTEN1 functions may differ from the human PTEN [31]. EhPTEN1 also lacks PDZ-binding motif (T/S-x-V) located at the C terminus in human PTEN and facilitates the protein-protein interactions [6,32]. The human PTEN was shown to interact with several PDZ domain-containing proteins, such as membrane-associated guanylate kinase inverted (MAGI) and Na⁺/H⁺ exchanger regulatory factor (NHERF) in a PDZ-dependent manner [8,9]. Interaction of MAGI to the PTEN C-terminal PDZ domain facilitates targeting of PTEN to the plasma membrane, thereby enhancing its activity [9]. While NHERF regulates the activation of the PI3K–AKT pathway by binding and recruitment of PTEN to platelet-derived growth factor receptor (PDGFR) [8]. However, the E. histolytica genome apparently does not encode proteins with similarity to NHERF or MAGI, as indicated by BLAST search. Therefore, these PDZ domain binding proteins may be unique to mammals, which represents a major difference between mammalian PTEN and EhPTEN1. The ~400 a.a. carboxyl-terminal extension which is absent in human ortholog and rich in charged amino acids could be involved in regulating its protein-protein interactions.

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Fig 1. Structural features and sequence alignments of PTEN in Entameoba histolytica.

(A) Domain organization of PTEN from human and E. histolytica. PDM [PtdIns(4,5)P₂-binding motif], Ptase (Phosphatase tensin-type domain), C2 (C2 tensin-type domain), DUF547 (Domain of unknown function), PDZ-BM (PDZ-binding motif), PEST (proline, glutamine, serine, threonine sequence), red triangle indicates the nuclear localization sequence. (B) Relative mRNA expression of PTEN homologs in E. histolytica trophozoites HM1: IMSS cl6 and G3 strains. The steady-state mRNA levels of PTEN isotypes in E. histolytica were created using the data from our previous work [24–26] where transcriptome of trophozoites of E. histolytica HM-1:IMSS cl6 reference strain was analyzed using DNA microarrays technology [25]. The levels of steady-state mRNA of PTEN isotypes are shown after normalization against that of RNA polymerase II. (C) Multiple amino acid sequence alignment of human PTEN (P60484), Xenopus laevis (AAD46165.1), Drosophila melanogaster (Q9Y0B6), Dictyostelium discoideum PTEN (Q8T9S), and EhPTEN1 (XP_653141.2) was constructed by using clustalw algorithm (http://clustalw.ddbj.nig.ac.jp). PTEN phosphatase domain and C2 domain are shown with blue and yellow backgrounds, respectively. The green rectangle corresponds to the PtdIns(4,5)P₂-binding motif (PDM domain). Amino acid residues implicated for PtdIns(3,4,5)P₃ catalysis are marked with a red rectangle. Cytosolic localization signal and restudies important for TI loop formation are indicated in black and blue lines, respectively. Note that for human PTEN, only the amino terminal part is shown.

https://doi.org/10.1371/journal.ppat.1010147.g001

Cellular localization and dynamics of EhPTEN1 in the motile E. histolytica trophozoite

To examine the cellular localization of EhPTEN1 in trophozoites, we established a transformant line expressing EhPTEN1 with the GFP-tag at the amino terminus (GFP-EhPTEN1). The expression of GFP-EhPTEN1 or GFP (control) in transformant trophozoites was verified by immunoblot analysis using anti-GFP antibody. A single band corresponding to non-truncated GFP fusion protein with an expected molecular mass of GFP-EhPTEN1 (90 kDa plus 26 kDa for the GFP tag) was observed in the GFP-EhPTEN1-expressing transformant (Fig 2A). Live imaging analysis revealed that GFP-EhPTEN1 was localized throughout the cytosol (Fig 2B and S1 Movie). The line intensity plots across the GFP-EhPTEN1 overexpressing trophozoites further demonstrated the enrichment of GFP-EhPTEN1 along the extended pseudopod of the motile trophozoite (Fig 2B) (Two additional images are also show in S1A and S1B Fig). The normalized average fluorescence intensities at the leading regions of pseudopods were nearly 1.5-fold higher in GFP-EhPTEN1 overexpressing trophozoites compared to GFP-expressing mock transformants (Fig 2C). Furthermore, the intensity line plot of GFP-expressing control strain showed no accumulation of GFP signal on pseudopods (S2 Fig and S2 Movie). These findings confirm the enrichment of GFP-EhPTEN1 in the pseudopod-like protrusive structures. Although the levels of expression of GFP-EhPTEN1 and GFP varied in the respective population, the accumulation of GFP-EhPTEN1 in the pseudopod like structure at the initial stage of trogocytosis and phagocytosis (S4 Movie) was observed even in the cell that expressed GFP-EhPTEN1 at low levels (showing only dim fluorescence). Similarly, immunofluorescence imaging of HA-EhPTEN1 overexpressing trophozoites using anti-HA antibody revealed that HA-EhPTEN1 was localized in the cytoplasm in steady-state, and enriched in pseudopods when the fluorescence signal across the line traversing the cell was measured (S3C Fig). HA-EhPTEN1 localization was also evaluated by measuring the fluorescence signal in the different portion of the cell, confirming that HA-EhPTEN1 is highly concentrated in the pseudopods, compared to other parts of the cell (S3D Fig). The migration (motility) of the GFP-EhPTEN1 overexpressing trophozoites using the montage of time-lapse imaging was 0.54±0.09 μm/sec (mean±S.D.), which was significantly greater than that of control GFP expressing transformants (0.27±0.08 μm/sec) (Fig 2D). We also investigated the effect of repression of EhPTEN1 gene expression and found that EhPTEN1 gene silencing reduced migration (see below).

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Fig 2. Expression and localization of GFP-EhPTEN1 in motile trophozoites.

(A) Immunoblot of GFP-EhPTEN1 and GFP (control) in E. histolytica transformants. Approximately 30 μg of total lysates from mock-transfected control (GFP) and GFP-EhPTEN1-expressing transformant (GFP-EhPTEN1) were subjected to SDS-PAGE and immunoblot analysis using anti-GFP antibody and anti-CS1 antibody. Arrow indicates GFP-EhPTEN1. (B) Live imaging montage showing a time series of motile trophozoites expressing GFP-EhPTEN1. The pseudopodal localization of GFP-EhPTEN1 is indicated by white arrow. The line intensity plot shows GFP-EhPTEN1 intensity in pseudopods vs. cytoplasm with the distance. (Scale bar, 10 μm) (C) Relative fluorescence intensities were quantified in the pseudopod regions of GFP-EhPTEN1 and GFP control expressing trophozoites then normalized to the fluorescence intensities in the total cells. Data points in the graph show the mean and error bars represent standard deviation for 30 cells. Statistical significance was examined with t-test (***P<0.001). (D) Cell motility of GFP and GFP-EhPTEN1 transfected strains. Time-lapse images of the transformant trophozoites were collected every sec for 5 min using CQ1 and 30 cells were selected randomly for analysis by CellPathfinder software. The average of three independent experiments is shown. Statistical significance was examined with Dunnet test (**P< 0.05).

https://doi.org/10.1371/journal.ppat.1010147.g002

Localization of EhPTEN1 during trogocytosis and phagocytosis

The fact that EhPTEN1 was previously identified as a PtdIns(3)P-binding effector and suggested to be involved in the phagosome biogenesis [23], prompted further characterization of the role of EhPTEN1 in host cell internalization. To examine the role of EhPTEN1 in ingestion of mammalian cells, we first examined trogocytosis (i.e., nibbling or chewing of a part of a live cell) of Chinese hamster ovary (CHO) cells by GFP-EhPTEN1 and GFP expressing transformant lines. We co-cultured trophozoites of the two transformant lines with live CHO cells that had been stained with CellTracker Orange. Time-lapse imaging of trogocytosis of CHO cells by the amoebae (Fig 3A and S3 Movie) revealed that GFP-EhPTEN1 was accumulated in the region that covers, but not always in close proximity to, the tunnel-like structure, which is the extended neck (or tube)-like structure connecting the enclosed (or being enclosed) trogosome and the remaining portion of the target cell that is partially ingested (Fig 3A–3D). Upon completion of closure of the trogosome, GFP-EhPTEN1 appeared to be dissociated from the region around the trogosome and the tunnel-like structure (Fig 3A–3E). The quantification of the fluorescence intensity in a cross section of the cell confirmed the dynamism of GFP-EhPETN1 during trogocytosis (Fig 3B–3E). In contrast, at the very early phase of trogocytosis, GFP-EhPTEN1 was not concentrated on the newly formed trogocytic cup.

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Fig 3. Localization of GFP-EhPTEN1 during trogocytosis.

(A) Time series montage showing the localization of GFP-EhPTEN1 during trogocytosis of live CHO cells by amoebic trophozoites. The site of trogocytosis is marked with arrow. (Scale bar, 10 μm). (B) Analysis of GFP-EhPTEN1 intensity along the line drawn at the initial phase of CHO internalization soon after attachment. (C) The plot showing the intensity of GFP-EhPTEN1 along the line drawn reveals its enrichment in the tunnel formed during amoebic trogocytosis. (D) The graph shows the intensity of GFP-EhPTEN1 at the late phase of trogocytosis soon after closure of the trogocytic cup (E) The graph shows the intensity of GFP-EhPTEN1 after the closure of the trogocytic cup.

https://doi.org/10.1371/journal.ppat.1010147.g003

The dynamics of GFP-EhPTEN1 in a course of phagocytosis (i.e., internalization with a single bite, not multiple bites) of dead CHO cells was also examined. The live imaging of GFP-EhPTEN1 expressing trophozoites co-cultured with pre-killed CHO cells (Fig 4 and S4 Movie) showed an enrichment of GFP-EhPTEN1 at the tip of the leading edge of the phagocytic cup during the internalization of dead host cells until phagosome closure (Fig 4A and 4B). Soon after closure of the phagosome, GFP-EhPTEN1 was concentrated on the closing side of the phagosome (Fig 4A–4C), and rapidly disappeared soon after (Fig 4D). The fluorescence intensity line plot of a cross section (as indicated by arrows) of the cell also reinforced the observation (Fig 4A–4D). As control, GFP-expressing mock strain showed no observable concentration of GFP signal in a course of CHO ingestion (S4 Fig and S5 Movie).

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Fig 4. Localization of GFP-EhPTEN1 phagocytosis of pre-killed CHO cells.

(A) Montage of live trophozoite expressing GFP-EhPTEN1 ingesting pre-killed CHO cells by phagocytosis. (Scale bar, 10 μm). (B) Analysis of intensity of GFP-EhPTEN1 across the phagocytic cup along the line drawn. (C) The plot showing the intensity of GFP-EhPTEN1 along the line drawn across the newly formed phagosome. (D) The graph shows the intensity of GFP-EhPTEN1 after phagosome maturation.

https://doi.org/10.1371/journal.ppat.1010147.g004

Effect of overexpression of EhPTEN1 on trogocytosis and phagocytosis

The dynamism of GFP-EhPTEN1, as revealed by live imaging, suggests that EhPTEN1 plays a role in the early to middle stages of trogo- and phagocytosis. We examined the effect of GFP-EhPTEN1 overexpression on the efficiency (i.e., speed and volume of internalization of prey) of trogocytosis and phagocytosis. GFP-EhPTEN1 expressing and GFP-expressing mock transformant strains were incubated with either live or pre-killed CHO cells that had been stained with CellTracker Orange to allow trogocytosis or phagocytosis, respectively. Internalization of CHO cells by the amoebae was measured by CQ1 confocal quantitative image cytometer (Figs 5 and 6). Three parameters were measured and compared between GFP-EhPTEN1 expressing and GFP-expressing mock transformant strains: the number of CHO-containing trogosomes or phagosomes per ameba (Figs 5A and 6A), the volume of all CHO-containing trogosomes or phagosomes per ameba (Figs 5B and 6B), and the percentage of the amebae that ingested CHOs (Figs 5C and 6C). GFP-EhPTEN1 overexpression caused statistically significant reduction in all three parameters in both trogocytosis and phagocytosis.

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Fig 5. Effect of GFP-EhPTEN1 expression on trogocytosis.

(A) Trophozoites of GFP mock transfected and GFP-EhPTEN1 expressing strains were incubated with live CHO cells that have been stained with CellTracker Orange to evaluate trogocytosis. The images were taken on CQ1 as described in Materials and methods and analyzed to calculate the average numbers of CHO cell-containing trogosomes per amoeba. (B) The volume of the ingested CHO cells was calculated using three-dimensionally reconstituted data. (C) The percentage of amoeba trophozoites that ingested live CHO cells. Experiments were conducted three times independently in triplicates and a representative data set is shown. Statistical significance was examined with t-test (*P<0.05, **P<0.01). Error bars indicate standard deviations of two biological replicates.

https://doi.org/10.1371/journal.ppat.1010147.g005

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Fig 6. Effect of GFP-EhPTEN1 expression on phagocytosis.

(A) Trophozoites of GFP mock transfected and GFP-EhPTEN1 expressing strains were incubated with heat killed CHO cells that have been stained with CellTracker Orange to evaluate phagocytosis. The images were taken on CQ1 as described in Materials and methods and analyzed to calculate the average numbers of CHO cell-containing phagosomes per amoeba. (B) The volume of the ingested CHO cells was calculated using three-dimensionally reconstituted data. (C) The percentage of amoeba trophozoites that ingested pre-killed CHO cells. Experiments were conducted three times independently in triplicates and a representative data set is shown. Statistical significance was examined with t-test (*P< 0.05). Error bars indicate standard deviations of two biological replicates.

https://doi.org/10.1371/journal.ppat.1010147.g006

Gene silencing of EhPTEN1 enhances trogocytosis and phagocytosis in E. histolytica

Conversely, we attempted to verify if repression of EhPTEN1 gene expression by antisense small RNA-mediated transcriptional gene silencing [33] causes reverse phenotypes: enhancement of trogocytosis and phagocytosis. Small antisense RNA-based transcriptional gene silencing, used in this study, was robustly usable only in G3, but not in its parental strain HM-1:IMSS [33]. The silencing of the EhPTEN1 gene expression was confirmed by RT-PCR and the level of silencing was estimated to be approximately 77.0±9.2% compared to the mock control (G3 transfected with the empty psAP2-Gunma vector) (Fig 7A and 7B). Non-specific off-target gene silencing of other EhPTEN genes (EhPTEN2-6) was ruled out, except for EhPTEN2, which showed a slight reduction in EhPTN1 gene silenced strain, validating gene-specific silencing (S5 Fig). The RNA pol II transcript level was also unaffected. EhPTEN1 gene silenced and psAP2 mock transformants were cultivated with live or dead CHO cells and images were captured every 10 min for 1 hr. As expected, EhPTEN1 gene silenced strain showed an enhancement of trogocytosis and phagocytosis (Fig 8). All three parameters to evaluate trogocytosis and phagocytosis, as above, i.e., the number of CHO-containing trogosomes or phagosomes per ameba (Fig 8A and 8B), the volume of all CHO-containing trogosomes or phagosomes per ameba (Fig 8C and 8D), and the percentage of the amebae that ingested CHOs (Fig 8E and 8F) was significantly increased in EhPTEN1gene silenced strain compared to the psAP2 mock control stain. For instance, the volume of trogosomes and phagosomes increased by around 1.4-fold for trogocytosis and 2-fold for phagocytosis, respectively, in EhPTEN1 gene silenced strain at later time points of coincubation (at 40–60 mins). Together with the results of EhPTEN1 overexpression, shown above, these data indicate that EhPTEN1 serves as a negative regulator of trogocytosis and phagocytosis.

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Fig 7. Establishment and phenotypes of EhPTEN1 gene silenced strain.

(A) Confirmation of gene silencing by RT-PCR analysis of psAP2-Gunma mock transfected and EhPTEN1 gene silenced strain (EhPTEN1gs) strain. Transcripts of EhPTEN1 and RNA polymerase II genes were amplified by RT-PCR from cDNA isolated from the transformants and examined by agarose gel electrophoresis. (B) Relative levels of EhPTEN1 transcripts by qRT-PCR analysis in EhPTEN1gs and psAP2 mock strains. The transcript levels were normalized against RNA polymerase II and are shown in percentage relative to the transcript level in mock control strain. Data shown are the means ± standard deviations of two biological replicates. Statistical comparison is made by t-test (**P<0.01). (C) Growth kinetics of psAP2 mock and EhPTEN1gs transformants s during 96 h incubation in BI-S-33 medium. Data shown are the means ± standard deviations of three biological replicates. Statistical comparison is made by t-test (*P<0.05, **P<0.01, ***P<0.001). (D) Cell motility of psAP2 mock transfected and EhPTEN1 gene silenced strains. The indicated transformant trophozoites were pre-stained with CellTracker green and time-lapse images were collected every sec for 2 min using CQ1 and 30 cells were selected randomly for analysis by CellPathfinder software. The experiments were performed three times independently. The average of three independent experiments is shown. Statistical significance was examined with Dunnet test (**P<0.05).

https://doi.org/10.1371/journal.ppat.1010147.g007

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Fig 8. The effect of gene silencing of EhPTEN1 on trogocytosis and phagocytosis.

(A, C, and E) Trophozoites of psAP2 mock and EhPTEN1gs strains were prestained with CellTracker Blue were incubated with live CHO cells that have been stained with CellTracker Orange to evaluate trogocytosis. The images were taken on CQ1 as described in Materials and methods. (B, D, and F) Trophozoites of psAP2 mock and EhPTEN1gs strains were prestained with CellTracker Blue were incubated with heat killed CHO cells that have been stained with CellTracker Orange to evaluate phagocytosis. The images were taken on CQ1 as described in Materials and methods. (A) The average numbers of CHO cell-containing trogosomes per amoeba. (B) The average numbers of CHO cell-containing phagosomes per amoeba. (C) The volume of the trogosomes were calculated using three-dimensionally reconstituted data. (D) The volume of the phagosomes were calculated using three-dimensionally reconstituted data. (E) The percentage of amoeba trophozoites that ingested live CHO cells. (F) The percentage of amoeba trophozoites that ingested pre-killed CHO cells. Experiments were conducted three times independently in triplicates. Statistical significance was examined with t-test (*P<0.05, **P<0.01). Error bars indicate standard deviations of three biological replicates.

https://doi.org/10.1371/journal.ppat.1010147.g008

EhPTEN1 is a positive regulator for the fluid-phase and receptor-mediated endocytosis in E. histolytica

To investigate the role of EhPTEN1 in pinocytosis of the fluid-phase marker and receptor-mediated endocytosis, we examined the internalization of RITC dextran and transferrin. Pinocytosis was analyzed by measuring, on a fluorometer, the fluorescence intensity of fluid-phase marker, RITC dextran, which was internalized after incubation of amoebic transformants with RITC dextran at 35°C for up to 1 hr. Overexpression of GFP-EhPTEN1 cause approximately 30% increase in pinocytosis in comparison to GFP mock control (53±5.3 or 28±13% at time 30 or 60 min, respectively; p<0.05, Fig 9A). Conversely, EhPTEN1 gene silenced strain showed an approximately 30% decrease in pinocytosis at 30–60 min, as compared to psAP2 mock control cells (30±8.2 or 25±5.1% decrease at time 30 or 60 min, respectively, p<0.05, Fig 9B).

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Fig 9. Effect of EhPTEN1 on pinocytosis.

(A) The effect of GFP-EhPTEN1 expression on pinocytosis. Trophozoites of GFP mock transfected and GFP-EhPTEN1 expressing strains were assayed for RITC dextran uptake in a time-dependent manner. (B) The effect of pinocytosis upon EhPTEN1 silencing in comparison to psAP2 mock control. Trophozoites of mock and EhPTEN1gs strains were incubated in BI-S-33 medium containing RITC dextran and assayed for its uptake for indicated time points. Experiments were conducted three times independently and statistical significance was examined with t-test (*P<0.05). Error bars indicate standard deviations of three biological replicates.

https://doi.org/10.1371/journal.ppat.1010147.g009

We next examined internalization of transferrin conjugated with AlexaFluor 568 by CQ1 image cytometer. Transferrin is presumed to be internalized via receptor-mediated endocytosis. The volume of endosomes that contained transferin-AlexaFluor 568 increased by 30–50% in GFP-EhPTEN1 overexpressing trophozoites compared to GFP mock control at all time points up to 1 hr (p<0.05, Fig 10A). Conversely, transferrin endocytosis decreased by 30–40% in EhPTEN1 gene silenced strain compared to the psAP2 mock strain at 40–60 mins (p<0.05, Fig 10B). These data indicate that EhPTEN1 positively regulates pinocytosis of the fluid-phase maker and receptor-mediated endocytosis in E. histolytica.

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Fig 10. Effect of EhPTEN1 on endocytosis.

(A) The effect of GFP-EhPTEN1 expression on endocytosis. Trophozoites of GFP mock transfected and GFP-EhPTEN1 expressing strains were incubated in BI-S-33 medium containing transferrin and images were taken every 10 min for 1 hr by CQ1 as described in Materials and methods. The volume of endosomes was calculated using three-dimensionally reconstituted data. (B) The effect of EhPTEN1 gene silencing on endocytosis. Images of psAP2 mock and EhPTEN1gs transformant trophozoites that have been co-cultivated with transferrin were taken every 10 min for 1 hr by CQ1 as described in Materials and methods. The volume of endosomes was calculated using three-dimensionally reconstituted data. All experiments were conducted three times independently and statistical significance was examined with t-test (*P<0.05). Error bars indicate standard deviations of three biological replicates.

https://doi.org/10.1371/journal.ppat.1010147.g010

EhPTEN1 is essential for optimum growth and migration of E. histolytica

The biological role of EhPTEN1 in trogo-, phagocytosis, and endocytosis was clearly demonstrated as above. To investigate other physiological roles of EhPTEN1 in E. histolytica, the growth kinetic was monitored in EhPTEN1 gene silenced and control strains. EhPTEN1 gene silencing caused significant growth defect: the population doubling time of EhPTEN1 gene silenced and control strains was 28.1±0.41 and 19.1±0.52 hr, respectively (P<0.05; Fig 7C). We next examined the migration of the trophozoites of EhPTEN1 gene silenced and control strains using time lapse imaging by CQ1. The velocity of motility was >60% reduced in EhPTEN1 gene silenced strain (0.16±0.07 μm /min) compared to the psAP2 mock control (0.44±0.08 μm /min) (Fig 7D).

Demonstration of phosphatase activity and substrate specificity of EhPTEN1

To see if EhPTEN1 possesses lipid phosphatase activity, bacterial recombinant EhPTEN1 with the histidine tag at the amino terminus was produced using the pCold I E. coli expression system. SDS-PAGE analysis followed by Coomassie Brilliant Blue staining showed that the purified recombinant EhPTEN1 was apparently homogenous with the predicted molecular mass of 96 kDa including the histidine tag (S6A Fig). Immunoblot analysis of the purified recombinant protein using His-tag antibody confirmed the absence of truncation (S6B Fig). We first examined the enzymatic activities of recombinant EhPTEN1 using a variety of phosphoinositides (PIs) as substrates. EhPTEN1 revealed reasonable activity in a broad pH range with maximum activity obtained at pH 6.0 when the reaction was performed with 50 μM PtdIns(3,4,5)P₃ at 37°C for 40 min (S6C Fig). We then determined the substrate specificity of EhPTEN1, using a panel of di-C8 PIs. EhPTEN1 showed highest activity with PtdIns(3,4,5)P₃ with the apparent specific activity of 8.18±0.78 nmol/min/mg (Fig 11). EhPTEN1 also catalyzed dephosphorylation of PtdIns(3,4)P₂ and PtdIns(3,5)P₂ with 6- or 3-fold lower specific activities, respectively, compared to that toward PtdIns(3,4,5)P₃. The activities against PI monophosphates and PtdIns(4,5)P₂ were relatively low. All these characteristics are similar to those of human PTEN [34,35]. A comparison of kinetic parameters of EhPTEN1 reveals a higher affinity towards PtdIns(3,4,5)P₃ (Km = 92.5 ± 4.72 μM) as compared to PtdIns(3,4)P₂ (292 ± 18.8 μM) and PtdIns(3,5)P₂ (161 ± 20.1 μM) demonstrating that in vitro is as well the preferred substrate (Table 1 and S8 Fig). Furthermore, we produced a mutant form of EhPTEN1, in which the cysteine residue implicated for catalysis is replaced with serine by site-directed mutagenesis (EhPTEN1_C140S) (S10 Fig). Recombinant EhPTEN1_C140S showed markedly lower phosphatase activity towards PtdIns(3,4,5)P₃ when compared to wild type, with the low specific activity (0.77 ± 0.14 nmol/min/mg). Moreover, expression of the inactive mutant GFP-EhPTEN1_C140S did not cause significant changes in pinocytosis, trogocytosis, and phagocytosis of E. histolytica trophozoites, which indicates that the enzymatic activity of EhPTEN1 is responsible for its phenotypes (S9 Fig).

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Fig 11. Substrate specificity and enzymatic activity of EhPTEN1.

Determination of EhPTEN1 Specific Activity. The specific activity of bacterial recombinant EhPTEN1 fusion protein toward a panel of synthetic di-C8-phosphoinositide substrates was determined using a malachite green-based assay for inorganic phosphate. Reactions were carried out in a volume of 25 μl for 40 min at 37^❍C, then terminated by the addition of 100 μl of malachite green reagent as described in Materials and methods. The absorbance at 630 nm was measured and phosphate released was quantified by comparison to a standard curve of inorganic phosphate. The means ± standard deviations of three independent experiments performed in duplicates are shown.

https://doi.org/10.1371/journal.ppat.1010147.g011

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Table 1. Kinetic parameters of EhPTEN1.

https://doi.org/10.1371/journal.ppat.1010147.t001

Demonstration of phospholipid binding of EhPTEN1

The lipid overlay assay using amebic lysates from GFP-EhPTEN1 expressing and GFP-expressing mock transformants showed that EhPTEN1 preferentially bound to PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, and, to a lesser extent, PtdIns(3,5)P₂, and PtdIns(4,5)P₂ (S7 Fig). Furthermore, recombinant His-tagged EhPTEN1 also revealed a similar binding affinity toward a panel of PIs on the membrane, which is similar to the data given for recombinant human PTEN [36]. The binding of EhPTEN1 with diverse immobilized phospholipids may regulate its localization to different cellular compartment where EhPTEN1 exerts its phosphatase activity as has been shown previously that the binding of PtdIns(3)P with C2 domain of human PTEN target its localization to endosomal membranes [36].

Identification and comparison of PTEN sequences

Amino acid sequences of PTEN from E. histolytica and other organisms were gained from AmoebaDB (http://amoebadb.org/amoeba/) and NCBI (https://www.ncbi.nlm.nih.gov) respectively, and aligned using CLUSTAL W program (http://clustalw.ddbj.nig.ac.jp/) to examine the domain configuration and the key residues for phosphatase activity [71].

Organisms, cultivation, and reagents

Trophozoites of E. histolytica clonal strains HM-1:IMSS cl6 and G3 strain were cultured axenically in 6 ml screw-capped Pyrex glass tubes in Diamond’s BI-S-33 (BIS) medium at 35.5°C as described previously [72–74]. CHO cells were grown at 37°C in F12 medium (Invitrogen-Gibco, New York, U.S.A.) supplemented with 10% fetal bovine serum on a 10-cm-diameter tissue culture dish (IWAKI, Shizuoka, Japan). Escherichia coli BL21 (DE3) strain was purchased from Invitrogen (California, USA). Ni²⁺-NTA His-bind slurry was obtained from Novagen (Darmstadt, Germany). Rhodamine B isothiocyanate-Dextran (RITC-Dextran) and anti-GFP antibody were purchased from Sigma-Aldrich (Missouri, USA). The anti-HA 16B12 monoclonal mouse antibody was purchased from Biolegend (San Diego, USA). Anti-His antibody was purchased from Cell Signaling Technology (Massachusetts, USA). Lipofectamine, PLUS reagent, and geneticin (G418) were purchased from Invitrogen. CellTracker Green, Orange, and Blue were purchased from Thermo Fisher Scientific (Massachusetts, USA). Restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs (Massachustts, USA) unless otherwise mentioned. Luria Bertani (LB) medium was purchased from BD Difco (New Jersey, USA). Other common reagents were from Wako Pure Chemical (Tokyo, Japan), unless otherwise stated.

Establishment of E. histolytica transformants

To construct a plasmid to express EhPTEN1 fused with HA or GFP tag fused at the amino terminus, a DNA fragment corresponding to cDNA encoding EhPTEN1 was amplified by polymerase chain reaction (PCR) from E. histolytica cDNA using a pair of primers listed in S2 Table. The PCR-amplified fragments were digested with XmaI and XhoI and cloned into pEhEx-HA and pEhEx-GFP vectors [21,22] that had been predigested with XmaI and XhoI, to produce pEhExHA-EhPTEN1 and pEhExGFP-EhPTEN1. For antisense small RNA-mediated transcriptional silencing of EhPTEN1 gene, a 420 bp fragment of the protein coding region of EhPTEN1 gene, corresponding to the amino terminus of the protein, was amplified by PCR from cDNA with sense and antisense oligonucleotides containing StuI and SacI restriction sites (S2 Table). The amplified product was digested with StuI and SacI and ligated into the compatible sites of the double digested psAP2-Gunma plasmid [75] to synthesize a gene silencing plasmid designated as psAP2-EhPTEN1. Two plasmids, pEhExHA-EhPTEN1 and pEhExGFP-EhPTEN1, were introduced into the trophozoites of E. histolytica HM-1:IMSS cl6 strain, whereas psAP2-EhPTEN1 was introduced into G3 strain by lipofection as described previously [76]. Transformants were initially selected in the presence of 1 μg/ml G418 until the drug concentration was gradually increased to 10 μg/ml for the EhPTEN1 gene silenced stain and 20 μg/ml for the GFP- and HA-EhPTEN1 overexpressing stains. Finally, all transformants were maintained at 10 or 20 μg/ml G418 in BIS medium.

Reverse transcriptase PCR

Reverse transcriptase PCR was performed to check mRNA levels of EhPTEN1 in EhPTEN1 gene silenced and control strains. Total RNA was extracted from trophozoites of EhPTEN1 gene silenced and control strains that were cultivated in the logarithmic phase using TRIZOL reagent (Life Technologies, California, USA). Approximately one μg of DNase treated total RNA was used for cDNA synthesis using Superscript III First -Strand Synthesis System (Thermo Fisher Scientific, Massachusetts, USA) with reverse transcriptase and oligo (dT) primer according to the manufacture’s protocol. Ex Taq PCR system was used to amplify DNA from the cDNA template using the primer pairs listed in S2 Table. The PCR conditions were as follow: initial denaturation at 98°C for 10 sec; then 25 cycles at 98°C for 10 sec, 55°C for 30 sec, and 72°C for 20 sec; and a final extension at 72°C for 7 min. The PCR products obtained were resolved by agarose gel electrophoresis.

Immunoblot analysis

Trophozoites of amoeba transformants expressing HA-EhPTEN1 or GFP-EhPTEN1 grown in the exponential growth phase were harvested and washed three times with phosphate buffer saline (PBS). After resuspension in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton-X 100, 0.5 mg/ml E-64, and protease inhibitor), the trophozoites were kept on ice for 30 min, followed by centrifugation at 500 × g for 5 min. Approximately 20 μg of the total cell lysates were separated on 10% SDS-PAGE and subsequently electrotransferred onto nitrocellulose membranes. The membranes were incubated in 5% non-fat dried milk in Tris-Buffered Saline and Tween-20 (TBST; 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween-20) for 1 hr at room temperature to block non-specific protein. The blots were reacted with one of the following primary antibodies diluted as indicated: anti-HA 16B12 monoclonal mouse antibody at a dilution of 1:1,000, anti-GFP mouse monoclonal antibody (1:100), and anti-CS1 rabbit polyclonal antisera [77] (1:1,000) at 4°C overnight. The membranes were washed with TBST and further reacted with horseradish peroxidase-conjugated (HRP) anti-mouse or anti-rabbit IgG antisera (1:10,000) at room temperature for 1 hr. After washings with TBST, the specific proteins were visualized with a chemiluminescence HRP Substrate system (Millipore, Massachusetts, USA) using LAS 4000 (Fujifilm Life Science, Cambridge, USA) according to the manufacture’s protocol.

Live cell imaging

Approximately 5×10⁵ trophozoites of the transformant strain expressing GFP-EhPTEN1 were cultured on a 35 mm (in diameter) collagen-coated glass-bottom dish (MatTek Corporation, Massachusetts, USA) in 3 ml of BIS medium under anaerobic conditions. CHO cells were stained with BIS medium containing 10 μM CellTracker Orange for 40 min followed by washing three times with PBS. Approximately 2×10⁴ prestained CHO cells 200 μl BIS were gently overlayed to trophozoites grown on the glass-bottom dish as prepared above. The central part of the dish was then carefully covered with a 1 cm square coverslip and the edge of the coverslip on the slide glass was sealed with nail polish. Live images were captured on Zeiss LSM780 confocal microscope (Carl-Zeiss, Oberkochen, Germany) using a 63× oil immersion objective with default settings on the time series mode and analyzed by ZEN software (Carl-Zeiss). For live imaging of trogocytosis and phagocytosis, approximately twenty cells were analyzed per each cell line in an experiment of three independent experiments. GFP-EhPTEN1 recruitment was estimated by quantitating the average fluorescent intensity per pixel across the line drawn over the cell or that of 11–13 regions of interest on the pseudopods and other parts of the cell.

Indirect immunofluorescence assay (IFA)

Approximately 5×10³ trophozoites in 50 μl BIS were transferred to an 8 mm round well on a slide glass (Matsunami Glass Ind, Osaka, Japan). After 30 min incubation in an anaerobic chamber at 35.5°C, 5×10⁴ CHO cells that had been pre-stained with 10 μM CellTracker Blue in 50 μl BIS were added to the well and the mixture was incubated for 15 min. After removing the medium, cells were fixed with PBS containing 3.7% paraformaldehyde at room temperature for 10 min, and subsequently permeabilized with PBS containing 0.2% Triton 100-X and 1% bovine serum albumin (BSA) for 10 min each at room temperature. The cells were then reacted with anti-HA mouse monoclonal antibody (1:1000) for 1 hr at room temperature. Then the sample was reacted with Alexa Flouor-488 conjugated anti-mouse IgG (1:1000) antibody (Thermo Fisher, Massachusetts, USA). The images were then captured using LSM 780 confocal microscope and analyzed by ZEN software (Carl-Zeiss, Oberkochen, Germany).

Trogocytosis and phagocytosis assay using CQ1

We quantified trogocytosis and phagocytosis efficiency using an image cytometer as described previously [27]. Briefly, trophozoites of E. histolytica were incubated in BIS containing 10 μM CellTracker Blue (Thermo Fisher) at 35.5°C for 1 hr. After staining, ameba trophozoites were washed 3 times with PBS and resuspended in OPTI-MEM medium (Thermo Fisher, Massachusetts, USA) containing 15% adult bovine serum (Sigma Aldrich). Approximately 2×10⁴ ameba trophozoites were seeded into a well on a 96-well glass bottom plate (IWAKI, Shizuoka, Japan) and incubated in anaerobic chamber for 40 min. After incubation, about 1×10⁵ live or heat killed CHO cells that have been stained with 10 μM CellTracker Orange were added to the well containing amebae. The images of trogocytosis and phagocytosis were captured on a Confocal Quantitative image cytometer CQ1 (Yokogawa Electric Corporation, Tokyo, Japan) using a 20× objective every 10 min for 1 hr. Five fields each per well for three wells were chosen for image analysis. Images on five planes with 2 μm intervals in Z axis were obtained to calculate the volume of the ingested CHO cells using three-dimensional reconstructed images. For analysis of motility, images of approximately 400 cells were captured from each well and analyzed using CellPathfinder software (Yokogawa Electric Corporation, Tokyo, Japan) according to the manufacture’s protocol. The protocol for quantitative analysis of the images was optimized based on the cell morphology and fluorescence intensity to identify E. histolytica trophozoites and CHO cells in each field. The multiple parameters were measured to evaluate the efficiency of trogocytosis and phagocytosis: the average number of internalized CHO cells per amoeba, the combined volume of internalized CHO cells per amoeba, and the percentage of amebic trophozoites that ingested the target cells in the whole population.

Measurement of fluid-phase and receptor-mediated endocytosis

Approximately 2.5×10⁵ amebic transformants were incubated in BIS medium containing 2 mg/ml fluorescent fluid-phase marker RITC dextran at 35°C for indicated time points. The cells were collected, washed three times with PBS, and resuspended in 250 μl of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Triton-X 100). Fluorescence intensity was measured using a plate reader (SpectraMax Paradigm Multi-Mode, Molecular Devices, California, USA) at an excitation wavelength of 570 nm and an emission wavelength of 610 nm.

Approximately 2×10⁴ amebic transformants were incubated in BIS containing 20 μM CellTracker Blue (Thermo Fisher) at 35.5°C for 1 hr. After staining, approximately 10⁴ amebic transformants resuspended in 100 μl of BIS medium were transferred to a well on a 96-well glass-bottom plate. After incubation at 35.5°C in an anaerobic chamber for 40 min, 0.5 mg/ml of transferrin conjugate with Alexa Fluor 568 was added to the well and images were acquired by CQ1 and analyzed as above.

Migration (motility) assay

Amoebic trophozoites grown in the logarithmic growth phase were harvested and labelled with 20 μM CellTracker Green for 1 hr at 35.5°C. After washing 3 times with PBS, cells were transferred to a well on a 96-well glass-bottom plate and time lapse images were captured on CQ1. The motility of the cells was measured using CellPathfinder software.

Quantitative real-time (qRT) PCR

The relative levels of mRNA of EhPTEN1 gene and RNA polymerase II gene, as an internal standard, were measured by qRT-PCR. PCR reaction was prepared using Fast SYBR Master Mix (Applied Biosystems, California, USA) with 100 ng cDNA and a primer set shown in S2 Table. PCR was conducted using the StepOne Plus Real-Time PCR system (Applied Biosystems, California, USA) with the following cycling conditions: an initial step of denaturation at 95°C for 20 sec, followed by 40 cycles of denaturation at 95°C for 3 sec, annealing and extension at 60°C for 30 sec. The mRNA expression level of EhPTEN1 gene in the transformants was expressed as relative to that in the control transfected with psAP2.

Growth assay of E. histolytica trophozoites

Approximately 10⁴ trophozoites of E. histolytica G3 strain transformed with psAP2-EhPTEN1 and psAP2 (control), grown in the logarithmic phase, were inoculated into 6 ml of fresh BI-S-33 medium containing 10 μg/mL G418, and the parasites were counted every 24 hr on a hemocytometer.

Production of EhPTEN1 recombinant protein

To construct the plasmid for the production of recombinant EhPTEN1 containing a histidine-tag at the amino terminus, the full-length protein coding sequence of EhPTEN1 gene was amplified by PCR using oligonucleotide primers listed in S2 Table. PCR was performed with PrimeSTAR Max DNA polymerase (Takara Bio Inc, Shiga, Japan) with the following parameters: initial incubation at 95°C for 1 min; followed by 30 cycles of denaturation at 98°C for 10 sec; annealing at 55°C for 5 sec; and elongation at 72°C for 15 sec; and a final extension at 72°C for 30 sec. The PCR fragment was digested with BamHI and SalI and ligated into BamHI and SalI double digested pCold-1 vector (Takara Bio Inc, Shiga, Japan) to produce pCold1-EhPTEN1 plasmid. The pCold-1-EhPTEN1 was introduced into E. coli BL21 (DE3) cells by heat shock at 42°C for 45 sec. E. coli BL21 (DE3) strain harboring pCold-1-EhPTEN1 was grown at 37°C in 50 ml of LB medium (BD Difco, New Jersey, USA) in the presence of 100 μg/ml ampicillin. The overnight culture was used to inoculate 500 ml of fresh medium, and the culture was further continued at 37°C with shaking at 220 rpm for approximately 2 hr. When A₆₀₀ absorbance reached 0.6, then 1mM of isopropyl β-D-thio galactopyranoside (IPTG) was added, and cultivation was continued for another 24 hr at 15°C. The E. coli cells from the induced culture were harvested by centrifugation at 75,000 rpm for 20 min at 4°C. The cell pellet was washed three times with PBS, re-suspended in 30 ml of the lysis buffer (50 mM Tris–HCl, pH 8.0, 300 mM NaCl, and 0.1% Triton X-100) containing 100 μg/ml lysozyme, and 1 mM phenylmethyl sulfonyl fluoride (PMSF), and incubated at room temperature for 30 min. After incubation, the mixture was sonicated on ice and centrifuged at 13,000 rpm for 20 min at 4°C. The supernatant was mixed with 1 ml of 50% Ni²⁺-NTA His-bind resin (Qiagen, Hilden, Germany), incubated for 1 hr at 4°C with mild rotatory shaking. The resin that recombinant His-EhPTEN1 bound was washed in a disposal column three times with 5 ml of lysis buffer containing 10–30 mM of imidazole. Bound proteins were eluted with 3 ml each of lysis buffer containing 100–300 mM imidazole to obtain recombinant EhPTEN1. The integrity and the purity of the recombinant protein were confirmed with 10% SDS-PAGE analysis, followed by Coomassie Brilliant Blue staining. Then the protein was concentrated, and the buffer was replaced with 50 mM Tris-HCl, 150 mM NaCl, pH 8.0 using Amicon Ultra 50K centrifugal device (Millipore, Massachusetts, USA). The protein was stored at -30°C with 50% glycerol in small aliquots until further use.

Lipid phosphatase assay

EhPTEN1 enzymatic activity was determined by the method previously described [78]. Di-C8 phosphatidylinositol phosphate(s) (PIPs) (Echelon Bioscience, Salt Laken City, USA) were dissolved in 100 mM MOPS, pH 6.0, solution, flash frozen in liquid nitrogen, and stored at -20°C between uses. For determination of pH optimum, the following buffers were used, 100 mM acetate buffer (pH 4.0, pH 4.5, pH 5.0, pH 5,5), 100 mM MOPS buffer (pH 6.0, pH 6.5, pH 7.0), and 100 mM Tris-HCl (pH 7.5, pH 8.0, pH 8.5, pH 9.0). For determination of substrate specificity, a reaction mixture was composed of 25 μl of 100 mM MOPS pH 6.0 containing 5 μg of recombinant EhPTEN1 and 100 μM PIPs. The reaction was carried out at 37°C for 40 min and the produced phosphate was measured using Malachite Green Reagent (Cell Signaling technology, Massachusetts, USA). After incubation for 15 min at room temperature, the absorbance was measured at a wavelength of 630 nm. The Lineweaver Burk Plot was used to calculate the kinetic parameters of EhPTEN1.

Lipid membrane overlay assay

Approximately 6×10⁶ trophozoites of GFP-EhPTEN1 expressing strain were harvested, washed with PBS, and concentrated by centrifugation. Approximately one hundred μl of lysis buffer (50 mM Tris-HCl, pH 7.5 150 mM NaCl, and 0.1% Triton X-100, 1× complete mini, and 0.5 mg/ml E64) was added to the cell pellet. The mixture was incubated on ice for 30 min and centrifuged at 13000 rpm for 5 min. The supernatant was collected and used as the total lysate. GFP-EhPTEN1 was immunoprecipitated using GFP-Trap Agarose Kit (ChromoTek, Planegg, Germany) according to the manufacturer’s instruction, eluted, and confirmed by immunoblot. Lipid membranes on which a panel of phospholipids were immobilized (PIP strips: P-6001, Echelon Bioscience, Salt Laken City, USA) were blocked with 1% fat free BSA in PBS-T (PBS containing 0.05% Tween 20) for 1 hr at room temperature. The membranes were then incubated with 2 ml of lipid binding solution (1% fat free BSA in PBS-T, 1x complete mini, 0.05 mg/ml E64, 20 μl of eluted lysate) for 3 hr at 4C. After the membrane were washed twice with PBS-T at 4°C, they were reacted with anti-GFP at 1:100 dilution with PBS-T containing 1% fat free BSA (PBS-TB) in for 3 hr at 4C. After incubation with the first antibody, the membranes were further reacted with HRP conjugated anti-mouse IgG rabbit antiserum at 1:10,000 dilution with PBS-TB in at 4°C for 1 hr. The membranes were washed three times with PBS-T at 4°C and the specific proteins were visualized with a chemiluminescence HRP substrate system (Millipore, Massachusetts, USA) using LAS 4000 (Fujifilm Life Science, Cambridge, USA) according to the manufactures’ protocol. Recombinant EhPTEN1 protein was also used except that 1 μg/ml of recombinant protein were incubated on the membrane over night at 4°C and anti-His antibody was used as the first antibody with a dilution of 1:1,000.

Site directed mutagenesis of EhPTEN1

Overlapping primers were designed to replace cysteine at amino acid 140 of the pEhExGFP-EhPTEN1 and pCold1-EhPTEN1 plasmids with serine. The primer sets used are listed in S2 Table. PCR reaction was carried out using 0.2 μM/μl each of the primers, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio Inc, Shiga, Japan) and pEhExGFP-EhPTEN1 or pCold1-EhPTEN1as a template. The PCR parameters were: incubation at 95°C for 1 min; followed by 30 cycles of denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, and elongation at 72°C for 50 sec; and a final extension at 72°C for 30 sec. The PCR fragment was evaluated by agarose gel electrophoresis and introduced into DH5α competent E. coli cells by heat shock at 42°C for 45 sec. Bacterial colonies containing the plasmid that encodes mutated EhPTEN1 (pEhExGFP-EhPTEN1_C140S and pCold-1-EhPTEN1_C140S) were selected from LB plate containing 100 μg/mL ampicillin by DNA sequencing of the plasmids. The pEhExGFP-EhPTEN1_C140S plasmid was introduced into the trophozoites of E. histolytica HM-1:IMSS cl6 strain as described previously [76]. Transformants were initially selected in the presence of 1 μg/ml G418 until the drug concentration was gradually increased to 20 μg/ml. The pCold-1-EhPTEN1_C140S was introduced into E. coli BL21 (DE3) cells to produce the inactive mutant recombinant protein as described previously.