Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis

Abstract

Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMKs). The expression patterns of the E. histolytica TMKs in individual trophozoites and the roles of the TMKs for sensing and responding to extracellular cues were incompletely characterised. Here we provide evidence that single cells express multiple TMKs and that TMK39 and TMK54 likely serve non-redundant cellular functions. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Anti-peptide antibodies were raised against unique regions in the extracellular domains of TMK39, TMK54 and PaTMK, and TMK expression was analysed at the protein level. Flow cytometric assays revealed that populations of trophozoites homogeneously expressed TMK39, TMK54 and PaTMK, while confocal microscopy identified different patterns of cell surface expression for TMK39 and TMK54. The functions of TMK39 and TMK54 were probed by the inducible expression of dominant-negative mutants. While TMK39 co-localised with ingested beads and expression of truncated TMK39 interfered with trophozoite phagocytosis of apoptotic lymphocytes, expression of a truncated TMK54 inhibited growth of amoebae and altered the surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilised for non-redundant functions by the parasite.

Fig. 1

Analysis of transmembrane kinase (TMK) protein expression in Entamoeba histolytica trophozoites.(A) Western blot with anti-peptide antibodies specific for TMK39 and TMK54. Lysate from 5.0 × 10⁶ trophozoites was resolved in a large-well SDS-PAGE gel, transferred to polyvinylidene fluoride (PVDF) membrane and cut into strips. Each strip was incubated with 5 µg/mL of anti-TMK39 (a) or anti-TMK54 (b) that had been pre-incubated with the indicated amount of unconjugated peptide for 1 h at room temperature (RT). Anti-rabbit:AP (Sigma) was used for detection as recommended by the manufacturer. Antibodies recognized single bands of the expected size (127 kDa TMK39 and 100 kDa TMK54). (B) Populations of amebae expressed more than one TMK. Permeabilized trophozoites were stained with antibodies against TMK39, TMK54, TMK96 (PATMK), the Gal/GalNAc lectin or an irrelevant antibody (Negative Control) followed by an anti-rabbit:Cy3 conjugate. Flow cytometry was used to assess staining. Forward scatter (FSC) and side scatter (SSC) were used to gate on intact cells prior to data collection and 10,000 gated events were collected for each sample. The experiment was carried out three times and a representative histogram is shown.

Fig. 2

Transmembrane kinases, TMK39 and TMK54 have discrete distributions at the cell surface. (A) Entamoeba histolytica trophozoites were lysed and fractionated. Trophozoite cytoplasmic (C), inner membrane (IM) and plasma membrane (PM) fractions were prepared for Western blot analysis. The panels were probed with anti-TMK39 (a) or anti-TMK54 (b), and in both instances, anti-Hgl and anti-actin antibodies were used as controls. Both TMK39 and TMK54 were expressed in membrane fractions. (B) Confocal images of non-permeabilized trophozoites are shown after staining with anti-peptide antibodies against TMK39 (a–b) and TMK54 (e–f). Permeabilized cells stained with anti-TMK39 (c) and anti-TMK54 (g) are also shown. As a control, antibodies were preincubated with the corresponding unconjugaetd peptides to competitively inhibit staining and staining with both anti-TMK39 (d) and anti-TMK54 (h) antibodies was abolished.

Fig. 3

Analysis of transmembrane kinase (TMK) function using a dominant-negative approach. (A) Depiction of the kinase domain (KD) deletions of TMK39 and TMK54 in t-39 and t-54, respectively. (B–C) Immunoprecipitation of t-39 and t-54. Amebae were transfected with inducible expression vectors encoding t-39, t-54 or an empty vector control (C). Anti-V5 agarose was used to immunoprecipitate the truncated proteins from whole cell lysate. Western blots of immunoprecipitations are shown, probed with antibodies against TMK39 (B) or TMK54 (C). Heavy chain from the precipitating antibody is visible in every lane. As visualized, full-length wild type proteins co-immunoprecipitated with the truncated proteins in both instances. Separation between panels in B and C indicates different exposure times of blot to film.

Fig. 4

Induction of t-39 and t-54 expression in Entamoeba histolytica trophozoites resulted in different phenotypes with respect to growth. Trophozoites were harvested during log-phase growth and 10,000 cells were seeded into media containing 10 µg/mL tetracycline. Cell numbers were assessed every 24 h for 4 days. For each construct at least two independently transfected clones were analyzed. Each sample was assayed in duplicate and the graph represents the mean of three independent experiments +/− S.D. For t-54 cells P < 0.05 at every time point after 0 h compared with any of the other cell types.

Fig. 5

Expression analysis of the heavy subunit of the Gal/GalNAc lectin (Hgl) in mutant Entamoeba histolytica trophozoites. t-39, t-54 and empty vector transfected cells were induced with 10 µg/mL tetracycline for 24 h, stained with anti-Hgl antibodies and assessed by flow cytometry. As a control, cells were stained with secondary antibody only (No Primary). Prior to data collection forward scatter and side scatter were used to gate for intact cells and 10,000 gated events were collected for each sample. The experiment was repeated more than three times and representative histograms are shown for non-permeabilized (A) and permeabilized (B) cells. P < 0.05 for the mean fluorescence intensity (MFI) of non-permeabilized t-54 cells compared with the MFI of either Control or t-39 cells.

Fig. 6

Co-localization of transmembrane kinase 39 (TMK39) with negatively charged beads during phagocytosis. Multispectral imaging flow cytometry was used to assess co-localization at the population level. Entamoeba histolytica trophozoites were allowed to ingest carboxylate-modified beads, fixed and stained with anti-peptide antibodies. Beads were stained alongside bead-containing cells. Samples were imaged using the Amnis Imagestream imaging cytometer. (A) Representative images are shown for beads stained (as a control) with anti-TMK39 (a), permeabilized, bead-positive cells stained with secondary antibody alone (b), anti-TMK54 (c) or anti-TMK39 (d). (B) ImageStream Data Exploration and Analysis Software (IDEAS) was used to calculate the Bright Detail Similarity score (a measure of co-localization between fluorescence in different channels with higher numbers indicating more co-localization and a score ≥ 3 indicating co-localization) for permeabilized cells stained with anti-TMK39 and anti-TMK54, and results were plotted as mean Bright Detail Similarity (BDS) +/− S.D. for three independent experiments (P-value = 0.039).

Fig. 7

Phagocytosis and pinocytosis in Entamoeba histolytica trophozoites induced to express t-39. (A) t-39 and empty vector transfected control cells were induced for 24 h with 10 µg/mL tetracycline and allowed to ingest carboxylate-modified beads, carboxyfluorescein succinimidyl ester (CFSE) labeled apoptotic Jurkat (aJ) cells, CFSE labeled Ca²⁺ treated erythrocytes or FITC-labeled Dextran. For each vector, at least two independently transfected clones were used in each experiment and the experiments were carried out more than three times. Samples were analyzed by flow cytometry and the graph represents the mean fluorescent intensity (MFI) of t-39 cells plotted as a percentage of control cells. Error bars represent S.D. and P < 0.005 for beads and apoptotic Jurkats compared with either Ca²⁼ treated erythrocytes or dextran. (B) Representative histograms are shown.