Experimental manipulation of ehmp8-1 and ehmp8-2 expression alters the transcript profiles of pathogenic and non-pathogenic amoeba clones

Recently, ehmp8-1 was shown to be expressed in the non-pathogenic clone A1^np at almost the same level as in the pathogenic clone B2^p (reads: 2028 vs 2228; [4]). The expression level of ehmp8-2 in clone A1^np is only about half that of ehmp8-1 (reads: 1098), while ehmp8-2 is almost not expressed at all in clone B2^p (reads: 7.4) [4].

To characterize the metallopeptidases, transfectants of the non-pathogenic clone A1^np were generated in which the expression of ehmp8-1 (A1^npMP8-1^Si), ehmp8-2 (A1^npMP8-2^Si) or both (A1^npMP8-1+2^Si) was silenced. Furthermore, transfectants of the pathogenic clone B2^p were generated in which ehmp8-1 (B2^pMP8-1^Si) was silenced or ehmp8-2 (B2^pMP8-2^OE) was overexpressed. Real-time quantitative PCR (qRT-PCR) confirmed the successful silencing and overexpression of the corresponding genes in the transfectants (Fig 1A and 1B and S1 Prism).

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Fig 1.

mRNA expression profile of ehmp8-1 (A) and ehmp8-2 (B) in A1^np and B2^p silencing and overexpression transfectants by means of RT-qPCR. RNA isolated from trophozoites was transcribed into cDNA and used for qPCR with SYBR Green to determine the relative mRNA concentration of ehmp8-1 (A) and ehmp8-2 (B) of the silencing transfectants A1^npMP8-1^Si, A1^npMP8-2^Si, A1^npMP8-1+2^Si, B^pMP8-1^Si and the overexpression transfectant B2^pMP8-2^OE. Controls: Non-transfected A1^np or B2^p trophozoites. Actin was used as a calibrator and controls were normalized to 1. n = 2–4 (in duplicate). ns: not significant, **p < 0.01, ***p < 0.001 (non-parametric ANOVA test and Mann-Whitney U test).

https://doi.org/10.1371/journal.ppat.1011745.g001

Transcriptome analyses of the transfectants showed that silencing had a significant effect on the expression of other genes (padj < 0.05, fold change > 1.8). Silencing of ehmp8-1 in the non-pathogenic clone A1^np (A1^npMP8-1^Si transfectant) resulted in altered expression of 216 genes (38 upregulated, 178 downregulated), silencing of ehmp8-2 (A1^npMP8-2^Si transfectant) resulted in altered expression of 347 genes (70 upregulated, 277 downregulated), and silencing of both metallopeptidase genes (A1^npMP8-1+2^Si transfectant) resulted in altered expression of 58 genes (12 upregulated, 46 downregulated) (Figs 2, 3A–3C and S1 and S1–S3 Tables). Depending on the transfectant, between 3.8 and 4.7 times more genes were downregulated than upregulated. Gene ontology (GO) analyses revealed that silencing of ehmp8-1 significantly affected five GO-biological processes (GO-BP) terms, including “obsolete electron transport” and “cell redox homeostasis” (p < 0.01) (Fig 4A and S4 Table). Within the GO term “cellular component” (GO-CC), there are only “plasma membrane” and “cell periphery” significantly affected (p < 0.01) (S4 Table). The GO term "molecular function” (GP-MF) contains nine significant terms, mainly involved in oxidoreductase and antioxidant activity (p < 0.01) (S4 Table).

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Fig 2. Venn diagram of differentially expressed genes in metallopeptidase silencing transfectants of non-pathogenic clone A1^np (A1^npMP8-1^Si, A1^npMP8-2^Si, A1^npMP8-1+2^Si).

Padj < 0.05, fold change > 1.8.

https://doi.org/10.1371/journal.ppat.1011745.g002

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Fig 3. Heatmap of significantly differentially expressed genes (> 1.8 fold, padj < 0.05) in the different E. histolytica transfectants silencing or overexpressing ehmp8-1, ehmp8-2 or both compared to the corresponding controls. A maximum of 20 up- or downregulated genes with the highest fold change are shown.

A. A1^np (control) versus A1^npMP8-1^Si, B. A1^np (control) versus A1^npMP8-2^Si, C. A1^np (control) versus A1^npMP8-1+2^Si, D. B2^p (control) versus B2^pMP8-1^Si.

https://doi.org/10.1371/journal.ppat.1011745.g003

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Fig 4.

GO-BP analysis of differential expressed genes in (A) A1^npMP8-1^Si transfectant, (B) A1_npMP8-2^Si transfectant and (C) A1^npMP8-1+2^Si transfectant.

https://doi.org/10.1371/journal.ppat.1011745.g004

Silencing of ehmp8-2 has a significant influence on GO-BP terms “obsolete electron transport”, “cytoskeleton organization” and “organelle organization” (Fig 4B and S5 Table). Within GO-CC, only “chromosome” is significantly affected (p = 0.01), and GO-MF includes seven significant terms e.g., “FMN reductase activity”, “oxidoreductase activity, acting on the CH-NH group of donors” and “actin binding” (p < 0.01) (S5 Table). When both ehmp8-1 and ehmp8-2 are silenced, four GO-BP terms are significantly affected including “adhesion” and “amino acid metabolism” (Fig 4C and S6 Table). Within GO-CC no significantly regulated term was identified. For GO-MF an influence of twelve terms was observed, including”GTP binding” and “metallopeptidase activity” (S6 Table). However, it is important to note that about 47% of the differential expressed genes encode hypothetical proteins (S1–S3 Tables).

Looking at the genes individually, ehhp127 was found to be the second most overexpressed gene in A1^npEhMP8-1^Si transfectants (22-fold change, padj 1,67E-19; Figs 3A and S1 and S1 Table). Furthermore, the expression of genes encoding proteins that protect against oxidative stress, such as peroxiredoxin, NADPH-dependent FMN reductase domain-containing protein, NADPH-dependent FMN reductase domain-containing protein, and iron-containing superoxide dismutase, are significantly upregulated. In addition, genes encoding the peptidases CAAX prenyl protease and EhCP-A7 are significantly upregulated whereas ehcp-a4 is downregulated (Figs 3A and S1 and S1 Table). The 20 most downregulated genes also included four aig genes (between 5.3- and 6.7-fold) (Figs 3A and S1 and Tables 1 and S1).

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Table 1. Aig1 genes with significantly differential expression in A1^npMP8-1^Si, A1^npMP8-2^Si, A1^npMP8-1+2^Si, B2^pMP8-1^Si, and A1^npHP127^OE transfectants compared to respective controls.

https://doi.org/10.1371/journal.ppat.1011745.t001

The second most upregulated gene in the A1^npEhMP8-2^Si transfectant (EHI_180390; fold change 8.7, padj 4.9E-10) also encodes a protein of the AIG1 family (Figs 3B and S1 and Tables 1 and S2). However, a total of 8 genes encoding AIG1 family proteins are downregulated, four of them in the 20 most downregulated genes (Figs 3B and S1 and Tables 1 and S2). In addition, as in A1^npEhMP8-1^Si transfectants, a number of genes that could encode for antioxidants (NADPH-dependent FMN reductase domain containing protein, iron-sulfur flavoprotein, iron hydrogenase, peroxiredoxin) are upregulated. It is also striking that many genes encoding proteins related to the cytoskeleton and surface proteins are downregulated (surface antigen ariel1, myotubularin, calponin homology domain protein, formin homology 2 family protein, caldesmon, actin-binding protein, Gal/GalNAc lectin 35 kDa subunit, Gal/GalNAc lectin 170 kDa subunit, filopodin, villin, formin homology 2 family protein, villidin, actinin-like protein) (Figs 3B and S1 and S2 Table).

Looking at the A1^npEhMP8-1+2^Si silencing transfectants it was surprising that simultaneous silencing of both metallopeptidase genes had the least effect on the gene expression profile and that few genes were found in the intersection of all three silencing transfectants (Fig 2). However, it was very striking that 20 of the 48 downregulated genes belonged to the aig1 family, twelve of which were among the 20 most highly regulated genes (Figs 3C and S1 and Tables 1 and S3). In addition, the genes encoding the large and small subunits of the Gal/GalNAc lectin and the cysteine peptidase EhCP-B2 were also downregulated (S1 Fig and S3 Table).

Silencing of ehmp8-1 in the pathogenic clone B2^p regulates the expression of only a small number of genes. The expression of a total of 26 genes was significantly affected, of which 9 were upregulated and 17 were downregulated in their expression (Figs 3D and S1 and S7 Table). Again, two of the nine upregulated genes are aig1 genes, while one aig1 gene was downregulated (Tables 1 and S7 and Figs 3D and S1).

Silencing and overexpression of metallopeptidase genes affect cytopathic, cysteine peptidase and hemolytic activities

To obtain information on the effect of silencing and overexpression on the phenotype of E. histolytica, the motility, growth, phagocytosis rate, hemolytic activity, cytopathic activity, and cysteine peptidase activity of the transfectants were analyzed. Although silencing affects the expression of a number of genes, both silencing and overexpression have little effect on trophozoite motility, with significantly increased motility (padj = 0.0347) observed for A1^npEhMP8-2^Si transfectants (Fig 5A–5C and S2 Prism). To determine the growth rate of the different transfectants, the division rate was determined in 24 h periods (Fig 5D–5F and S3 Prism). In contrast to the motility, silencing of ehmp8-1 resulted in significant inhibition of growth in both non-pathogenic A1^np and pathogenic B2^p trophozoites (padj = 0.0008, p < 0.0001) (Fig 5D and 5E and S3 Prism). Similarly, overexpression of ehmp8-2 in B2^p resulted in significantly faster growth (p = 0.0031) (Fig 5F and S3 Prism). Significantly decreased erythrophagocytosis (p = 0.0414) was observed only for B2^pEhMP8-2^OE transfectants (Fig 6A–6C and S4 Prism). Hemolytic activity is significantly reduced in A1^npEhMP8-1^Si (padj = 0.0165) and A1^npEhMP8-1+2 (padj < 0.0001) (Fig 6D and S5 Prism), whereas no effect was observed in B2^pEhMP8-1^Si transfectants (Fig 6E and S5 Prism). However, B2^p trophozoites already have very low hemolytic activity (Fig 6E and S5 Prism). Overexpression of the ehmp8-2 gene in pathogenic B2^p trophozoites led to a significant increase in hemolytic activity (B2^pMP8-2^OE, p = 0.0024) (Fig 6F and S5 Prism).

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Fig 5.

Determination of motility (A-C) and growth (D-F) of silencing transfectants A1^npMP8-1^Si, A1^npMP8-2^Si, A1^npMP8-1+2^Si and B2^pMP8-1^Si and overexpression transfectant B2^pMP8-2^OE. Non-transfected A1^np and B2^p trophozoites were used as controls for silencing transfectants and B2^p trophozoites transfected with the control plasmid pNC were used as control for overexpression transfectants. To determine motility (A-C), the accumulated distance (μm) was measured after 10 min. For each transfectant/control 60 amoebae were analysed. For each experiment, 3 biological replicates were used and the speed of movement was determined for 20 amoebae. To determine growth rate (D-F), 500 trophozoites of each clone were seeded into a 24-well plate, and the cells were counted every 24 h over 72 h. Experiments were performed four times in triplicate. Significance was determined using one-way ANOVA (A, D) and unpaired t test (B/C; E/F) (ns: not significant, *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001).

https://doi.org/10.1371/journal.ppat.1011745.g005

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Fig 6.

Determination of erythrophagocytosis (A-C) and hemolytic activity (D-F) of silencing transfectants A1^npMP8-1^Si, A1^npMP8-2^Si, A1^npMP8-1+2^Si and B2^pMP8-1^Si and overexpression transfectant B2^pMP8-2^OE. Non-transfected A1^np and B2^p trophozoites were used as controls for silencing transfectants and B2^p trophozoites transfected with the control plasmid pNC were used as control for overexpression transfectants. To determine erythrophagocytosis (A-C), trophozoites (2 x 10⁵) and erythrocytes (2 x 10⁸) were incubated for 30 min at 37°C, non-phagocytosed erythrocytes were lysed, then the amoebae were lysed in 1% Triton-X-100 and absorbance was measured at 405 nm. The mean value of the controls was defined as 100% and the measured OD_{405 nm} values of the samples were related to it. At least six biological replicates were performed. For hemolytic activity (D-F), 1.25 x 10⁵ trophozoites were mixed with 2.5 x 10⁸ erythrocytes in 1 ml of PBS and incubated at 37°C for 1 h. After incubation, the cells were sedimented, and the hemoglobin released in the supernatant was measured at 530 nm. Separately incubated erythrocytes and trophozoites were used as negative controls. To determine 100% hemoglobin release, 2.5 x 10⁸ erythrocytes were lysed in 1 ml of water. Experiments were performed at least 3 times in quadruplicate. Significance was determined using one-way ANOVA (A, D) and unpaired t test (B/C; E/F) (ns: not significant, *p < 0.05, **p < 0.01, **** p < 0.0001).

https://doi.org/10.1371/journal.ppat.1011745.g006

Silencing of ehmp8-1 or ehmp8-2 alone in both clones A1^np and B2^p resulted in a significant reduction in cytopathic activity (cell monolayer destruction) between 33%– 53% (A1^npEhMP8-1^Si, padj = 0.0007; A1^npEhMP8-2^Si, padj = 0.0002; B2^pEhMP8-1^Si, p = 0.0014) (Fig 7A and 7B and S6 and S7 Prism). However, silencing of both metallopeptidase genes in A1^np trophozoites had no effect on cytopathic activity (Fig 7A and S6 Prism). Surprisingly, overexpression of ehmp8-2 in B2^p trophozoites (B2^pMP8-2^OE) also led to a significant reduction of the monolayer destruction (p = 0.0014) (Fig 7C and S7 Prism). Furthermore, silencing led to a significant reduction in cysteine peptidase activity in all silencing transfectants examined (A1^npEhMP8-1^Si, padj = 0.01; A1^npEhMP8-2^Si, padj < 0.0001; A1^npEhMP8-1+2^Si, padj < 0.0001; B2^pEhMP8-1^Si, p = 0.0012) (Fig 7D and 7E and S8 Prism). The cysteine peptidase activity in A1^np transfectants decreased from 10.3±1.4 (control) to 7.4±2.7 mU/mg (A1^npEhMP8-1^Si), 4.3±1.9 mU/mg (A1^npEhMP8-2^Si), and 6.2±2.4 mU/mg (A1^npEhMP8-1+2^Si) (Fig 7D and S8 Prism). The cysteine peptidase activity of B2^p trophozoites was 60±15 mU/mg and decreased to 43±11 mU/mg after silencing of ehmp8-1 (Fig 7E and S8 Prism). In contrast, overexpression of ehmp8-2 in B2^p trophozoites resulted in a significant increase in cysteine peptidase activity (B2^pMP8-2^OE, p < 0.0001) (Fig 7F and S8 Prism). Substrate gel electrophoresis confirmed the results of the activity measurement. The overall decrease in activity of the A1^npMP8-1^Si transfectant compared to the control is too small to be detected in a substrate gel. However, for the A1^npMP8-2^Si transfectant, though, there is a clear decrease in the intensity of the EhCP-A1 and EhCPA-7 bands, and for the A1^npMP8-1+2^Si transfectant, all bands show a decrease in intensity. A similar effect was observed for the B2^pMP-1^Si transfectants. In particular, the activity bands of EhCP-A7 show a reduced in intensity (Fig 7G). However, the regulation seems to occur at the translational level, as the expression of ehcp-a1, ehcp-a2, ehcp-a5 and ehcp-a7 was not significantly altered in the different transfectants compared to the controls. The only exception is ehcp-a7, which is significantly upregulated two-fold in the A1^npMP8-1^Si transfectant compared to the control (padj = 0.023) (S1, S2, S3, S7 Tables).

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Fig 7.

Determination of cytopathic activity (A-C) and cysteine peptidase activity (D-G) of silencing transfectants A1^npMP8-1^Si, A1^npMP8-2^Si, A1^npMP8-1+2^Si and B2^pMP8-1^Si and overexpression transfectant B2^pMP8-2^OE. Non-transfected A1^np and B2^p trophozoites were used as controls for silencing transfectants and B2^p trophozoites transfected with the control plasmid pNC were used as control for overexpression transfectants. To determine cytopathic activity (A-C), HepG2 cells (1 x 10⁵) were seeded in 24 well plates, cultured for 48 h and stained with BCECF. Subsequently, 1 x 10⁵ trophozoites were added to the cells in 500 μl DMEM medium and incubated at 37° for 1 h. Afterwards, the cells were lysed, centrifuged and the supernatant was measured at 485 nm absorbance and 535 emission. The negative control was set at 100%. Experiments were performed at least 3 times in triplicate. Cysteine peptidase activity (D-G) was determined using Z-Arg-Arg-pNA as substrate. Experiments were performed at least 6 times in duplicate. Significance was determined using one-way ANOVA (A, D) and unpaired t test (B/C; E/F) (ns: not significant, **p < 0.01, *** p < 0.001, **** p < 0.0001). (G) Determination of cysteine peptidase activity using substrate gel electrophoresis. 4 μg of amoeba extracts from controls (A1^np/B2^p) and transfectants (A1^npMP8-1^Si, A1^npMP8-2^Si, A1^npMP8-1+2^Si, B2^pMP8-1^Si) were separated on SDS-Page co-polymerized with gelatine. To visualize the cysteine peptidase activity, the gels were stained with Coomassie Blue.

https://doi.org/10.1371/journal.ppat.1011745.g007

EhMP8-1 and EhMP8-2 are localized in trophozoite vesicles

To determine the localization of the two metallopeptidases, non-pathogenic A1^np trophozoites were transiently transfected with plasmids allowing translation of the respective protein fused to a c-Myc tag at the C-terminus. Immunofluorescence assays with wild-type B2^p trophozoites used as controls show that the α-myc antibody did not detect any protein in the cytoplasm of saponin-treated cells or on the surface of trophozoites not treated with saponin (S2 Fig).

In the A1^np transfectants A1^npMP8-1^Myc and A1^npMP8-2^Myc, both metallopeptidases were detected in vesicle-like structures within the trophozoites. They were mainly localized in vesicles in the endoplasm of trophozoites (Fig 8A–8D). Neither EhMP8-1 nor EhMP8-2 were detected on the cell surface of trophozoites (S4A–S4C Fig).

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Fig 8. Microscopic analysis of A1^npMP8-1^Myc and A1^npMP8-2^Myc transfectants for localization of the metallopeptidases EhMP8-1 and EhMP8-2.

The A1^np trophozoites were transfected with the expression plasmids pNCMP8-1^Myc or with pNCMP8-2^Myc, which allowed the production of a metallopeptidase-myc fusion protein, and the myc-tag could be stained with a specific α-myc antibody. For the immunofluorescent analysis, trophozoites were fixed with PFA and permeabilized with saponin. MP8-1^Myc and MP8-2^Myc fusion proteins were stained with ⍺-c-myc primary antibody (1:100) and ⍺-mouse Alexa Fluor 488 (1:400, green). A, C. Single trophozoite of the A1^npMP8-1^Myc (A) and A1^npMP8-2^Myc transfectant (C). Shown are transmitted light images (DIC), staining of nuclei with Hoechst dye, staining of EhMP8-1 and EhMP8-2 with α-Myc antibody, and an overlay of the images (merge). B, D. Single images of confocal micrographs of a trophozoite of the A1^npMP8-1^Myc (B) and A1^npMP8-2^Myc (D) transfectant. The single images show 0.38 μm thick sections through the trophozoites. A total of 42 images were obtained, and only every second image is shown here.

https://doi.org/10.1371/journal.ppat.1011745.g008

Comparison of EhMP8-1 and EhMP8-2 with leishmanolysins and invadolysins of different organisms

Comparing the amino acid sequences of EhMP8-1 and EhMP8-2 with leishmanolysin-like peptidases from different organisms revealed that the percent identity to those of the liver fluke Clonorchis sinensis, the avian protozoan parasite Histomonas meleagridis, and T. vaginalis ranges from 16% to 20%. The identity to leishmanolysin from L. major is 22% to 23%. The highest identities (24% to 28%) were found with various metazoan invadolysins such as those from the hookworm Ancylostoma caninum, the whipworm Trichuris suis, Drosophila melanogaster, Mus musculus, Macaca thibetana, humans, and the soil-dwelling amoeba Dictyostelium discoideum (S8 Table).

Overexpression, but not silencing, of ehhp127 affects the expression of other genes

It has been shown that ehhp127 is almost exclusively expressed in clone B2^p (reads: B2^p-2314.73 vs A1^np 12.01) [4]. In contrast to the silencing of ehmp8-1 and ehmp8-2 genes, the silencing of ehhp127 in B2^p trophozoites (B2^pHP127^Si) had no effect on the expression of other genes (Figs 9A and S3 and S9 Table). However, this is different when ehhp127 was overexpressed in clone A1^np (Figs 9B and S3 and S9 Table). In A1^npHP127^OE transfectants, ehhp127 was overexpressed approximately 5500-fold. Overall, 17 genes were significantly upregulated more than 3-fold and 40 genes were significantly upregulated more than 2-3-fold (padj < 0.05) (Figs 9B and S3 and S10 Table). However, when the expression profile of the control transfectant A1^nppNC is compared to that of non-transfected A1^np trophozoites, we observed 242- and 7-fold repression of the six most differentially expressed genes, whereas in A1^npHP127^OE transfectants, the expression level returns to that of wild-type A1^np [4]. All six genes encode for hypothetical proteins (S10 Table). The reasons for this effect in mock transfection are unknown.

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Fig 9. Heatmap of genes significantly differentially expressed (> 1.8 fold, padj < 0.05) in the different E. histolytica transfectants silencing or overexpressing ehhp127 compared to the corresponding controls.

A maximum of 20 up- and downregulated genes with the highest fold change are shown. A. B2^p (control) versus B2^pHP127^Si, B. A1^nppNC (control) versus A1^npHP127^OE.

https://doi.org/10.1371/journal.ppat.1011745.g009

Only one of the additional differentially expressed genes (ehi_062680), encoding a hypothetical protein, was also found to be significantly differentially expressed between wild-type A1^np and B2^p trophozoites. This gene was 20-fold more highly expressed in B2^p than in A1^np trophozoites [4]. The increase in expression from A1^nppNC to A1^npHP127^OE was 2.5-fold (S10 Table). However, transfection with the control plasmid pNC also appears to affect expression. The expression of a number of genes was up- or down-regulated compared to wild-type A1^np, but also compared to A1^npEh127^OE transfectants (S10 Table). In total, the expression of 86 genes was significantly downregulated. Of these, 43 genes are downregulated >3-fold and another 43 up to 2-3-fold (S10 Table). Again, an effect of the mock transfection can be seen. The expression of 18 genes that were normally not or only weakly expressed in A1^np wild-type trophozoites occurs in A1^nppNC. However, the expression profile of the A1^npHP127^OE transfectants has returned to the low levels seen in A1^np trophozoites. The difference in expression here was between 4200- and 6.7-fold. Except for one aig1 gene and one gene encoding a putative DNA polymerase, these are all genes encoding hypothetical proteins (Table 1). For all other genes that were significantly downregulated in A1^npHP127^OE transfectants, the expression in A1^nppNC control transfectants correlated with that in A1^np wild-type trophozoites [4]. It is therefore likely that this is a specific effect of ehhp127 overexpression. It is striking that, overall, the expression of 11 aig1 genes was significantly downregulated (Table 1). Furthermore, the expression of the genes encoding cysteine peptidases ehcp-a4 and ehcp-a6, genes encoding heat shock proteins (HSP101, DNAJ family protein, HSP70) and genes encoding antioxidants (peroxiredoxin, iron-sulfur flavoprotein) was downregulated in A1^npHP127^OE transfectants (Figs 9B and S3 and S10 Table).

For nine genes, the expression profile of A1^nppNC transfectants compared to A1^npHP127^OE transfectants was comparable to that of A1^np to B2^p trophozoites. In both A1^npHP127^OE transfectants and B2^p trophozoites, these genes are less expressed compared to A1^nppNC control transfectants and A1^np trophozoites, respectively. These nine genes encode for heat shock proteins (EHI_034710, EHI_022620), AIG family proteins (EHI_126560, EHI_126550), DNA mismatch repair protein Msh2 (EHI_123830), splicing factor 3B subunit 1 (EHI_049170) and three hypothetical proteins (EHI_005657, EHI_075640, EHI_075690) (Figs 9B and S3 and S10 Table).

Overexpression of ehhp127 significantly regulated only one GO-BP term, namely, "obsolete electron transport" (S11 Table). No significantly regulated pathway was identified within GO-CC. This is different for GO-MF, where 20 pathways are affected, including “guanyl ribonucleotide binding”, “lysozyme activity”, and “FMN reductase activity” (S11 Table).

Altered expression of ehhp127 has a major impact on motility and cytopathic activity

As for the EhMP8 transfectants, various phenotypic characteristics were also analyzed for the ehhp127 overexpressing and silencing transfectants (A1^npHP127^OE, B2^pHP127^Si).

Silencing and overexpression of ehhp127 had less overall impact and less significant effects on the E. histolytica phenotype. For the silencing transfectants, this was not surprising since no other genes except ehhp127 itself were affected in its expression. What is surprising, however, is that although the expression profile for a number of genes was regulated during overexpression, this only affected motility (Fig 10A and S9 Prism). This was also highly significantly affected in silencing transfectants (Fig 10B and S9 Prism). While overexpression of ehhp127 in A1^np (A1^npHP127^OE) increases the distance traveled from 261±25 μm to 389±84 μm after 10 min (p < 0.0001) (Fig 10A and S9 Prism), silencing of ehhp127 in B2^p (B2^pHP127^Si) led to a decrease in the distance traveled from 449±141 μm/10 min to 270±38μm/10 min (p < 0.0001) (Fig 10B and S9 Prism). Erythrophagocytosis was not affected in the ehhp127 transfectants A1^npHP127^OE and B2^pHP127^Si (Fig 10C and 10D and S10 Prism). However, as with all metallopeptidase transfectants, we observed that silencing of ehhp127 reduced cytopathic activity. While 74±8.6% of the monolayer was destroyed by the control (B2^p trophozoites), silencing of the ehhp127 gene (B2^pHP128^Si) reduces the destruction to 51±10% (p < 0.0001) (Fig 10F and S11 Prism). As previously described [5], overexpression had no significant effect on cysteine peptidase activity (Fig 10G and S12 Prism), whereas silencing of ehhp127 results in a significant reduction of cysteine peptidase activity (190±49 mU/mg vs 123±23 mU/mg, p = 0.024) (Fig 10H and S12 Prism). With respect to hemolytic activity, only a significant reduction in B2^p (2.6±1.2% vs 1.7±1%, p = 0.015) was observed after ehhp127 silencing (Fig 10J and S13 Prism).

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Fig 10.

Determination of motility (A, B), erythrophagocytosis (C, D), cytopathic activity (E, F), cysteine peptidase activity (G, H), and hemolytic activity (I, J) of EhHP127 transfectants (overexpressing transfectant: A1^npHP127^OE, pNC-transfected A1^np trophozoites were used as control; silencing transfectant: B2^pHP127^Si, non-transfected B2^p trophozoites were used as control). A, B. To determine motility, the accumulated distance (μm) was measured after 10 min. For each transfectant/control 30 amoebae (two biological replicates, 15 amoebae each) were analyzed. Significance was determined using the unpaired t test (****p < 0.0001). C, D. To determine erythrophagocytosis, trophozoites (2 x 10⁵) and erythrocytes (2 x 10⁸) were incubated for 30 min at 37°C, non-phagocytosed erythrocytes were lysed, then the amoebae were lysed in 1% Triton-X-100 and absorbance was measured at 405 nm. The mean value of the controls was defined as 100%, and the measured OD_{405 nm} values of the samples were related to 100%. At least five biological replicates were performed per transfectant/control and significance was determined using the unpaired t test (ns: not significant). E, F. To determine cytopathic activity, HepG2 cells (1 x 10⁵) were seeded in 24 well plates, cultured for 48 h and stained with BCECF. Subsequently, 1x10⁵ trophozoites were added to the cells in 500 μl DMEM medium and incubated for 1 h at 37°C. Afterwards, the cells were lysed, centrifuged and the supernatant was measured at 485 nm absorbance and 535 nm emission. The negative control was set at 100%. Experiments were performed at least 3 times in duplicate. Significance was determined using the unpaired t test (ns: not significant, ****p < 0.0001). G, H. Cysteine peptidase activity was determined using Z-Arg-Arg-pNA as substrate. The experiments were performed three times in duplicate for A1^npHP127^OE (G) and five times in duplicate for B2^pHP127^Si (H). Significance was determined using the unpaired t test (ns: not significant, *p < 0.05). I, J. To determine hemolytic activity, 1.25 x 10⁵ trophozoites were mixed with 2.5 x 10⁸ erythrocytes in 1 ml PBS and incubated at 37°C for 1 h. After incubation, the cells were sedimented and the hemoglobin released in the supernatant was measured at 530 nm. Separately incubated erythrocytes and trophozoites were used as negative controls. To determine 100% hemoglobin release, 2.5 x 10⁸ erythrocytes were lysed in 1 ml of water. The experiment was performed 2 times in duplicate (I) and 7 times in triplicate (J). Significance was determined using the unpaired t test (ns: not significant, *p < 0.05).

https://doi.org/10.1371/journal.ppat.1011745.g010

Localization of EhHP127 in vesicles of trophozoites

Similar to the experiments in which the hypothetical protein EhHP127 was overexpressed, amoebae were transiently transfected with a plasmid allowing translation of EhHP127 fused to a c-Myc tag at the C-terminus. In B2^pEhHP127^Myc transfectants, EhHP127^Myc was also localized to trophozoite vesicles. A uniform distribution within the trophozoites was observed (Fig 11A–11C). In western blot analysis, EhHP127 was detected only in the insoluble pellet fraction and not in the soluble fraction (Fig 11D). However, EhHP127 was not detected on the surface of trophozoites (S4D Fig).

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Fig 11. Localization of the hypothetical protein EhHP127.

B2^p trophozoites were transfected with the expression plasmid pNCHP127^Myc, which allowed the production of an EhHP127-Myc fusion protein, and the myc-tag was stained with a specific α-myc antibody. A-C. For the immunofluorescent analysis, trophozoites were fixed with PFA and permeabilized with saponin. EhHP127^Myc fusion protein was stained with ⍺-c-myc primary antibody (1:100) and α-mouse Alexa Fluor 488 (1:400, green). For co-localization an antibody against the cytoplasmically localized superoxide dismutase (SOD) and α-rabbit Alexa Fluor 594 (1:400, red) was used. Nuclei were stained with Hoechst dye (blue). A. Single trophozoite from the B2^pHP127^Myc transfectant. B. Confocal images of a trophozoite of the B2^pEh127Myc transfectant. Series of 10 sections with a distance of 1.9 μm from top to bottom, covering a major part of the cell. C. Side view of the whole cell with the three modes green, red and merge, for the detection of EHI_127670 and SOD both single and merged. For the side view, a 3D-model was constructed out of 55 single slices using Imaris. D. Western blot of a 13% Tricine-SDS-PAGE using an ⍺-c-myc primary antibody (1:1000) and ⍺-mouse-HRP secondary antibody (1:2000). For the Western blot, B2^p trophozoites were transfected twice independently ([1], [2]) and the pellet fraction (P) and the soluble fraction (S) were prepared from each of the two transfectants.

https://doi.org/10.1371/journal.ppat.1011745.g011

E. histolytica cell culture and generation of transfectants

Cultivation of E. histolytica trophozoites was performed under microaerophilic and axenic conditions at 37°C in plastic culture flasks (Corning, Kaiserslautern, Germany) using TYI-S-33 medium [48]. Non-pathogenic E. histolytica clone A1^np and pathogenic E. histolytica clone B2^p were derived from the cell lines HM-1:IMSS-A and HM-1:IMSS-B [4].

Overexpressing and silencing transfectants were generated as described [4, 5]. In summary, for overexpression of ehmp8-2 (ehi_042870) in clone B2^p and ehhp127 (ehi_127670) in clone A1^np, trophozoites were transfected with the expression plasmid pNC containing the gene of interest under control of the E. histolytica lectin promoter (B2^pMP8-2^OE, A1^npHP127^OE). As a control B2^p or A1^np trophozoites were transfected with the plasmid pNC [4]. Overexpressing transfectants were cultured in TY-I-S-33 medium containing 20 μg/ml G-418 (Gibco, Thermo Fisher Scientific, Schwerte, Germany).

For silencing of ehmp8-1 (ehi_200230) or ehmp8-2 in clone A1^np, trophozoites were transfected with the silencing plasmid pSiA containing ehmp8-1 (267 bp) or ehmp8-2 (1989 bp), respectively, in frame with the trigger region of ehi_169670 (A1^npMP8-1^Si, A1^npMP8-2^Si). For silencing of ehmp8-1 or ehhp127 in clone B2^p, trophozoites were transfected with the silencing plasmid pSiB containing ehmp8-1 (268 bp) or ehhp127 (917 bp) in frame with the trigger region of ehi_074080 (B2^pMP8-1^Si) [5]. The silencing transfectants were grown in TY-I-S-33 medium containing 20 μg/ml G-418 for 3 weeks. After cloning of the transfectants by limited dilution, the cells were cultured without selection for at least 4 months until complete loss of the plasmid. Plasmid loss was proven by culturing the amoebae in the presence of 20 μg/ml G418 [5]. To produce the A1^npEhMP8-1+2^Si transfectant, after successful silencing of ehmp8-1, the A1^npEhMP8-1^Si transfectant was transfected with the silencing plasmid pSiA containing ehmp8-2. Subsequently, selection and cultivation were performed as described above. A1^np and B2^p trophozoites were used as controls. Overexpression and silencing were confirmed by specific qRT-PCR (S12 Table).

For localization studies, ehhp127 (ehi_127670) was expressed under its own promoter using a myc-tag containing expression vector (pNC^Myc) in clone B2^p. The pNC^Myc plasmid is based on the pNC expression plasmid. A myc-tag was integrated into the BamHI restriction site via BamHI/BglII. To generate the pNC^Myc vector, two complementary oligonucleotides encoding the c-Myc-tag (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu) and the restriction sites KpnI, NheI, BamHI, XhoI, and BglII at the 5’-end were hybridized (S12 Table). The resulting DNA fragment was then cloned into the pNC vector via the KpnI restriction site at the 5’ end and the BglII restriction site at the 3’ end.

To generate the plasmid pNC_HP127^Myc, a 500 bp long sequence upstream of the start ATG, presumably containing the promoter region, and the entire open reading frame of ehi_127670 was amplified by PCR using a forward primer containing a KpnI restriction site and the reverse primer containing an BamHI restriction site (S12 Table). DNA from the clone B2^p was used as a template. The amplified insert was cloned into the pNC^Myc vector with KpnI and BamHI restriction sites.

Expression of ehmp8-1 and ehmp8-2 was also performed under their own promoter (pNCMP8-1^Myc, pNCMP8-2^Myc). For this purpose, a 500 bp long sequence upstream of start ATG, which presumably contains the promoter region, and the entire open reading frame of ehmp8-1 (ehi_200230) and (ehmp8-2) ehi_042870 was amplified by PCR using the forward primer containing a KpnI restriction site and the reverse primer containing a BamHI restriction site (S12 Table).

RNA extraction and quantitative real-time PCR (qRT-PCR)

For RNA isolation, trophozoites were harvested, washed twice with sodium phosphate-buffered saline (NaPBS; 4°C; 6.7 mM NaHPO₄, 3.3 mM NaH₂PO₄, 140 mM NaCl, pH 7.2) and lysed with Trizol reagent (QIAzol Lysis reagent, QIAgen, Hilden, Germany). Total RNA isolation and DNA digestion were performed using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA, USA). All steps were performed according to the manufacturer’s instructions. RNA concentration and purity were determined using the NanoDrop 2000 (Thermo Fisher Scientific, Schwerte, Germany). The cDNA required for qRT-PCR was synthesized from the isolated RNA using the SuperScriptIII First-Strand Synthesis System Kit (Invitrogen, Thermo Fisher Scientific, Dreieich, Germany), according to the manufacturer’s instructions.

Sense and antisense primers were designed for qRT-PCR experiments to amplify 80–120 bp fragments of the genes of interest (S12 Table). The Luna Universal qPCR Master Mix kit (New England Biolabs, Frankfurt, Germany) was used to perform qRT-PCR. 2–4 biological replicates were analyzed in duplicate each time. Relative concentrations in gene expression were calculated using the 2^-ΔΔCT method and Rotor Gene software (Rotor Gene 6, Corbett Research). A1^np, B2^p, and B2^ppNC were used as calibrators (control), and were set to 1. Actin was used as a housekeeping gene for normalization.

Next generation sequencing

The quality of purified RNA was assessed using the Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, United States) and the Agilent RNA 6000 Pico Reagents Kit (Agilent Technologies). Ribosomal RNA was removed using the QIAseq FastSelect RNA Removal Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA from each sample was prepared for sequencing using the QIAseq Stranded mRNA Library Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Normalized libraries were pooled and sequenced using a NextSeq 500/550 Mid OutputKit v2.5 (Illumina, San Diego, CA, USA) with 150 cycles (2 × 75 bp paired-end) on a NextSeq 550 platform, generating a depth of 5–16 million paired-end reads for each sample. Reads were trimmed and filtered using Trimmomatic [49], and reads were aligned to the E. histolytica transcriptome (AmoebaDB, Release 61, 15 Dec 2022) using RSEM [50] and Bowtie2 [51] software. Differential expression was tested using DEseq2 to normalize the raw data [52].

Determination of motility

24 h before the experiment, 2.5x10⁵ trophozoites were seeded into a T25 culture flask (Sarstedt, Nümbrecht, Germany). For the metallopeptidase transfectants three biological replicates were used and the speed of movement was determined for 20 amoebae each using an Evos FL Auto microscope (Thermo Fisher Scientific). For the Eh127 transfectants two biological replicates were used and the movement was determined for 15 amoebae each. A video was recorded for 10 min (one frame every 5 sec). Manual tracking of the amoebae was performed using ImageJ version 2.0.0-rc-43/1.51d with plugins for manual tracking and chemotaxis.

Erythrophagocytosis assay

Human erythrocytes (blood group 0+ donated from the blood bank of the University Medical Center Hamburg-Eppendorf) and trophozoites to be examined were washed twice with incomplete TY-I-S-33 medium (200 x g, 10 min, 4°C). Subsequently, 2 x 10⁸ erythrocytes and 2 x 10⁵ trophozoites (ratio 1000:1) were added to a 5 ml tube with a total volume of 400 μl incomplete TY-I-S-33 medium. After incubation at 37°C for 30 min, 1 ml each of ddH₂O was added twice and incubated for 1 min to lyse the non-phagocytized erythrocytes. The cells were then centrifuged at 400 x g for 4 min in a 15 ml tube and washed twice with NaPBS. The amoebae were then lysed to release the hemoglobin of the phagocytosed erythrocytes. For this purpose, 1 ml of 1% Triton-X 100 was added to the trophozoites. For photometric measurement, 200 μl of the lysed trophozoites were pipetted into a 96-well plate and measured at 405 nm. The mean value of each control was defined as 100% and the measured OD values were referenced to this value. For the metallopeptidase transfectants at least six biological replicates were performed. For the Eh127 transfectants at least five biological replicates were performed.

Determination of cytopathic activity (monolayer destruction)

To determine the cytopathic activity of E. histolytica, 1 x 10⁵ cells of the hepatocyte cell line HepG2 (Merck, Darmstadt, Germany) were seeded in the wells of a 24-well plate 48 h before the experiment and incubated at 37°C in 2 ml HepG2 cell culture medium (Advanced DMEM; Gibco, Thermo Fisher Scientific, Schwerte, Germany) supplemented with 10% fetal calf serum (FCS; Capricorn, Hamburg, Germany) and 1x penicillin/streptomycin (Capricorn), with the medium changed once after 24 h. After 48 h, a confluent cell monolayer was formed. Trophozoites were seeded into a T12.5 culture flask 24 h prior to the experiment so that a confluent monolayer was formed the next day. At the beginning of the experiment, HepG2 cell culture medium was removed from the HepG2 cells, which were then washed with NaPBS. To stain the HepG2 cells, 200 μL NaPBS + 10 μM of the fluorescent dye BCECF, AM (2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester) was added. The cells were incubated at 37°C for 30 min. Afterwards, BCECF/NaPBS was removed, and the cells were washed twice with NaPBS (37°C). Then, 1 × 10⁵ trophozoites in 500 μl were added to the stained HepG2 cells. As a negative control, the stained HepG2 cells were incubated with 500 μl Advanced DMEM medium, while as a positive control, the stained HepG2 cells were lysed with 500 μl DMEM+1% Triton X-100. The 24-well plate was then incubated at 37°C for 1 h. Subsequently, the plate was then placed on ice for 20 min to dissolve the trophozoites. After washing twice with NaPBS (4°C), 1 ml of 1% Triton X-100 PBS was pipetted into each well to lyse the HepG2 cells, and the plate was incubated at 37°C for 30 min. 150 μl of the supernatant were pipetted into a black 96-well plate (Greiner, Frickenhausen, Germany), which was centrifuged at 1400 x g for 30 sec and measured with a fluorescence plate reader (GENios, TECAN, Fornax Technologies GmbH, Uetikon am See, Switzerland) at an absorbance of 485 nm and an emission of 535 nm. The negative control values were set to 100%. The values of the samples were then related to the negative control. Finally, the result was subtracted from 100% to determine the percentage of cells detached from the cell layer by the trophozoites. Experiments were performed at least 3 times in triplicate.

Determination of cysteine peptidase activity and substrate gel electrophoresis

Amoebae were seeded in T25 cell culture flasks 24 h prior to the start of the experiment to form a monolayer the next day. To prepare the amoeba extracts used for the determination of cysteine peptidase activity and substrate gel electrophoresis, the amoebae were lysed in liquid nitrogen over 3 freeze-thaw cycles and sedimented at 12,000 x g for 15 min at 4°C. The supernatant was used in the corresponding experiments. Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Cysteine peptidase activity was measured in a 96-well plate in a final volume of 200 μl (148 μl CP assay buffer (0.1 M KH₂PO₄, 2 mM EDTA, 1 mM DTT), 2 μl amoeba extract and 50 μl of the synthetic peptide Z-Arg-Arg-pNA (400 μM concentration; Bachem, Bubendorf, Switzerland) as substrate. One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 mmol p-nitroaniline in 1 min. For the metallopeptidase transfectants the experiments were performed at least six times in duplicate and the significance was determined using the unpaired t test. For the overexpressing transfectant A1^npHP127^OE the experiments were performed four times in duplicate and for the silencing transfectant B2^pHP127^Si the experiments were performed five times in duplicate.

Substrate gel electrophoresis was performed as previously described [4, 53]. Briefly, 4 μg of amoeba extract was separated on a 12% SDS-polyacrylamide gel co-polymerized with 0.1% gelatin. This was followed by incubation with 2.5% Triton X-100 for 1 h, followed by incubation with 100 mM sodium acetate (pH 4.5), 1% Triton X-100, and 20 mM DTT for 3 h at 37°C. Gels were stained with Coomassie to visualize cysteine peptidase activity.

Determination of hemolytic activity

The hemolytic activity assay was performed as described by Biller et al. [3]. For the assay, trophozoites and erythrocytes were mixed at a ratio of 1:2000 (1.25 x 10⁵ amoebae with 2.5 x 10⁸ erythrocytes per ml NaPBS) and then incubated at 37°C for 1 h. After incubation, the cells were sedimented, and the hemoglobin released in the supernatant was measured at 530 nm in a spectrophotometer. Separately incubated erythrocytes and trophozoites were used as negative controls. To determine 100% hemoglobin release, 2.5x10⁸ erythrocytes were lysed in 1 ml of water. The experiments were performed at least three times in quadruplicate for the metallopeptidase transfectants, two times in duplicate for the overexpressing transfectant A1^npHP127^OE and seven times in triplicate for the silencing transfectant B2^pHP127^Si.

Determination of growth rate

To determine growth rate, 500 trophozoites of each amoeba cell line were seeded onto a 24-well plate, and the cells were counted every 24 h over 72 h. Experiments were performed four times in triplicate.

Statistics

The data are presented as box plots showing the minimum, maximum, median, and all individual measurement points. The data were first checked for normal distribution and homogeneity of variance. In the case of normal distribution, the unpaired t-test was used to compare two groups, and the ordinary one-way ANOVA was used to compare three or more groups. For the ANOVA test, the significance threshold after Bonferroni correction was adjusted to the control level for each comparison. If there was no normal distribution, the Mann-Whitney test was used to compare two groups, and the non-parametric ANOVA test was used for three or more groups. Multiple comparison test was performed according to Benjamini and Hochberg. All statistical analyses were performed using Prism 9, Version 9.3.1 (350), December 7, 2021.

Western blot

To prepare amoeba extracts for Western blot, trophozoites were washed twice with NaPBS and sedimented by centrifugation at 400 x g for 2 min at 4°C. To minimize proteolysis, 20 μM trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butan (E64, Sigma-Aldrich, Merck, Taufkirchen, Germany) was added. For lysis the trophozoites were alternately flash frozen in liquid nitrogen for 4 times. The lysates were centrifuged at 40,000 x g for 1 h at 4°C. The supernatants contained PBS-soluble proteins. Pellets were washed twice in ice-cold NaPBS and solubilized in NaPBS supplemented with 1% Triton X-100. Extracts (50 μg/lane) were separated on 13% SDS-PAGE gels under reducing conditions. Proteins were transferred to nitrocellulose membranes by the wet blotting technique, with 25 mM Tris-HCl, 192 mM glycine, 1.3 mM SDS, pH 8.3, and 20% methanol as the transfer buffer. For Western blot analysis, a primary anti-c-myc primary antibody (Sigma-Aldrich) at 1:1000 dilution and a secondary anti-mouse horseradish peroxidase (HRP) antibody (Sigma-Aldrich) at 1:5000 dilution was used. Blots were developed using GE Healthcare Amersham ECL Prime Western Blotting Detection Reagent (fisher scientific, Thermo Fisher Scientific, Schwerte, Germany).

Immunofluorescence analyses

To localize the proteins to be analyzed, transfectants expressing the corresponding c-myc fusion proteins were harvested, washed 1 x with NaPBS, and resuspended in 1 ml NaPBS. After centrifugation at 400 x g at 4°C for 5 min, the supernatant was discarded, the cell sediment was resuspended in 1 ml of 4% paraformaldehyde (PFA; Hatfield, PA, USA) in NaPBS, and the trophozoites were incubated for 30 min at RT on a rolling incubator in the NaPBS/4% PFA solution. This and all subsequent incubation steps were performed on a rolling incubator. After centrifugation at 400 x g for 3 min, the supernatant was discarded, and the trophozoites were resuspended in 500 μl NaPBS/0.05% saponin (for permeabilization of cell membranes (Sigma-Aldrich, Merck, Taufkirchen, Germany) and incubated for 5 minutes. After centrifugation at 400 x g for 3 minutes, the supernatant was discarded and the trophozoites were resuspended in 500 μl 50 μM ammonium chloride solution to block free aldehyde groups and incubated for an additional 15 min at RT. This was followed by two washing steps with 500 μl NaPBS/0.05% saponin or 500 μl NaPBS (400 x g for 3 min) and an incubation for 10 min with 500 μl NaPBS/2% FCS resuspended. An additional washing step was followed by a 1 h incubation at RT with the primary mouse anti-c-myc antibody (500 μl, 1:100, Sigma Aldrich). Further, three washing steps followed as described above. The cell sediments were then incubated in 500 μl NaPBS containing the secondary fluorescently labeled antibody (1:400, anti-mouse alexa fluor 488, Thermo Fisher Scientific, Bremen, Germany) for another 1 h rolling at RT.

For co-localization, an antibody targeting the cytoplasmic iron-containing superoxide dismutase (SOD; dilution 1:200; [54]) and anti-rabbit Alexa Fluor 594 antibody (dilution 1:400; Thermo Fisher Scientific, Bremen, Germany) and an antibody targeting the α-Gal/GalNAc lectin (dilution 1:200; [55]) and anti-rabbit Alexa Fluor 594 antibody (dilution 1:400) were used. After washing the trophozoites again three times, the nuclei were stained by incubation with Hoechst-33342 (1:400; Invitrogen, Thermo Fisher Scientific, Bremen, Germany) diluted in 500 μl NaPBS for 10 min at RT. After a final washing step the trophozoites were resuspended in 50 μl NaPBS and could be stored in the dark at 4°C until analysis. As a control for each experiment, non-transfected A1^np or B2^p trophozoites were incubated with the anti-Myc antibody and the secondary fluorescent anti-mouse Alexa Fluor 488-labelled antibody under the same conditions as described for the transfectants. Microscopy was performed using a Zeiss Axio Imager M2 microscope and an Olympus FluoView1000 confocal microscope.