The Entamoeba histolytica TBP and TRF1 transcription factors are GAAC-box binding proteins, which display differential gene expression under different stress stimuli and during the interaction with mammalian cells

Abstract

Background: Entamoeba histolytica is the protozoan parasite responsible for human amebiasis. It causes up to 100,000 deaths worldwide each year. This parasite has two closely related basal transcription factors, the TATA-box binding protein (EhTBP) and the TBP-related factor 1 (EhTRF1). TBP binds to the canonical TATTTAAA-box, as well as to different TATA variants. TRF1 also binds to the TATTTAAA-box. However, their binding capacity to diverse core promoter elements, including the GAAC-element, and their role in gene regulation in this parasite remains unknown.

Methods: EMSA experiments were performed to determine the binding capacity of recombinant TBP and TRF1 to TATA variants, GAAC and GAAC-like boxes. For the functional analysis under different stress stimuli (e.g. growth curve, serum depletion, heat-shock, and UV-irradiation) and during the interaction with mammalian cells (erythrocytes, MDCK cell monolayers, and hepatocytes of hamsters), RT-qPCR, and gene knockdown were performed.

Results: Both transcription factors bound to the different TATA variants tested, as well as to the GAAC-boxes, suggesting that they are GAAC-box-binding proteins. The K _D values determined for TBP and TRF1 for the different TATA variants and GAAC-box were in the range of 10^-12 M to 10^-11 M. During the death phase of growth or in serum depletion, Ehtbp mRNA levels significantly increased, whereas the mRNA level of Ehtrf1 did not change under these conditions. Ehtrf1 gene expression was negatively regulated by UV-irradiation and heat-shock stress, with no changes in Ehtbp gene expression. Moreover, Ehtrf1 gene also showed a negative regulation during erythrophagocytosis, liver abscess formation, and a transient expression level increase at the initial phase of MDCK cell destruction. Finally, the Ehtbp gene knockdown displayed a drastic decrease in the efficiency of erythrophagocytosis in G3 trophozoites.

Conclusions: To our knowledge, this study reveals that these basal transcription factors are able to bind multiple core promoter elements. However, their immediate change in gene expression level in response to different stimuli, as well as during the interaction with mammalian cells, and the diminishing of erythrophagocytosis by silencing the Ehtbp gene indicate the different physiological roles of these transcription factors in E. histolytica.

Keywords: Different stress stimuli; Differential gene expression; EhTBP; EhTRF1; Entamoeba histolytica; GAAC-box mutation; Gene knockdown.

Fig. 1

EhTBP and EhTRF1 have DNA-binding activity to different TATA variants in vitro. Recombinant EhTBP and EhTRF1 were expressed in E. coli BL21(DE3) pLysS, purified by IMAC and analyzed by electrophoresis as described in Methods. a SDS-PAGE (12%) gel analysis of extracts from bacteria transformed with pRSET A (Lane 2), pcEhtrf1 (Lanes 3 and 4), pEhtbp (Lanes 6 and 7). Protein expression was induced with 1 mM IPTG for rEhTRF1 and rEhTBP (Lanes 4, and 7, respectively). rEhTRF1 and rEhTBP purified proteins (arrows) are shown in Lanes 5 and 8, respectively. b Western blot of the gel shown in a using anti-His tag monoclonal antibodies. EMSA assay to determine the DNA-binding activity of purified rEhTRF1 and rEhTBP using different probes: (c) TATTTAAA(1); (d) TAgTgAAA(2); (e) TATgTAAA(5); (f) gAgTTAAA(6); (g) TAcTcAAA(7); (h) cAcTcAAA(8). Abbreviations: UC, unspecific competitor; SC, specific competitor

Fig. 2

EhTRF1 and EhTBP are GAAC-box binding proteins (GBPs). a EMSA using the GAAC-box. Formation of rEhTRF1-GAAC and rEhTBP-GAAC complexes was competed by the unlabeled GAAC-box and TATTTAAA-box (Lanes 3 and 4, and 8 and 9, respectively). b EMSA using the GAAC-like box. The rEhTBP or rEhTRF1 with GAAC-like box forming complex (Lanes 2 and 6). Specific competition with 200-fold molar excess of unlabeled probe (Lanes 3 and 7) or unspecific competition with poly(dI-dC) or (dA-dT)_{18 mer} oligonucleotide (Lanes 4 and 8, and Lanes 5 and 9, respectively). c, d Mutated GAAC-box was unable to compete the formation of rEhTRF1-GAAC-box and rEhTBP-GAAC-box complexes (Lane 4). e, f Quantification of the GAAC-box protein complexes as a function of increasing concentrations of rEhTRF1 (e) or rEhTBP (f) by EMSA experiments

Fig. 3

Graphical representation of the data obtained in the quantification of DNA-protein complexes shown in Fig. 2 (e and f). a, d Data corresponds to the natural logarithm of the radioactivity present in the DNA-protein complexes (ln S_x) as a function of the protein concentration x. Dots correspond to experimental data. The continuous line is the graph predicted by the second-degree polynomial function. b, e Mathematical relationships shown by the natural logarithm of the fraction (F) of DNA probe in GAAC-box-protein complexes and the rEhTRF1/GAAC-box molar ratios (b) and rEhTBP/GAAC-box molar ratio (e). Dots correspond to experimental data. Continuous line is the graph predicted by the second-degree polynomial function. c, f Mathematical relationships shown by the reciprocal of F (1/F) and the reciprocal of the total active uncomplexed protein P (1/P) for rEhTRF1 and rEhTBP, respectively. Dots correspond to experimental data. The continuous line is the graph predicted by the linear eq. 1/F = K_D (1/P) + 1, where its slope corresponds to the K_D value. The arrow indicates the complex with GAAC-like box

Fig. 4

Ehtbp is upregulated during nutrient deprivation. a Growth curve of trophozoites at 12, 18, 36, 72, 96 and 120 h. b, c Relative gene expression levels of Ehtbp (b) and Ehtrf1 (c) at different times of growth curve. Statistical significance (Student’s t-test) defined as *P < 0.05, **P < 0.001. d, e Images of trophozoites grown in TYI-S-33 medium for 12 h at 37 °C with serum (d) or without serum (e). Photomicrographs were obtained with a magnification of 200×. f RT-qPCR analysis of Ehtbp and Ehtrf1 genes. Statistical significance was determined using the Student’s t-test (****P < 0.0001). Scale-bar: 10 μm

Fig. 5

Immunolocalization of EhTBP during the growth curve and serum depletion. a Cells were grown at the indicated times and immunostained with rabbit polyclonal anti-rEhTBP antibodies (green channel). DNA was stained with DAPI (red channel). b Trophozoites were grown in the presence or absence of serum and processed as in (a). Scale-bar: 10 μm

Fig. 6

Ehtrf1 is downregulated in response to UV irradiation and heat-shock stress. UV-irradiation a-e Relative gene expression of Ehtbp (a), Ehtrf1 (b), Ehrad54 (c), Ehblm (d) and Ehpcna (e) normalized with the endogenous gene control Eh40Ss2. f Gene expression analysis of Ehtbp and Ehtrf1 by RT-qPCR in trophozoites cultured at 37 °C and 42 °C as described. g As a control of heat-shock response, the expression level of Ehhsp70 gene was determined. No change in morphology was observed in both cases. Significant values are marked with asterisks (Student’s t-test, *P < 0.05, **P < 0.001, ***P < 0.0005)

Fig. 7

Gene expression analyses of Ehtrf1 and Ehtbp genes of E. histolytica trophozoites during erythrophagocytosis and MDCK monolayer destruction and amoebic abscess formation. a-d Erythrophagocytosis assay. a Representative photomicrographs showing the accumulation of ingested erythrocytes in trophozoites at different time points. Photomicrographs were taken at 200× magnification. b Graph showing the average number of ingested erythrocytes by trophozoites. c, d Gene expression levels of Ehtbp (c) and Ehtrf1 (d). e-g MDCK cell monolayer destruction by trophozoites. e Images of representative culture plates of stained MDCK cell monolayers destroyed by live trophozoites interacting for 5, 15 and 30 min. An intact MDCK cell monolayer is shown (0 min) for comparison. f, g RT-qPCR analysis of Ehtbp and Ehtrf1 genes, respectively. Statistical analysis with significant difference are marked with asterisks (Student’s t-test, *P < 0.05, **P < 0.001, ***P < 0.0003). Scale-bar: 10 μm

Fig. 8

Gene expression analyses of Ehtrf1 and Ehtbp genes of E. histolytica trophozoites during amoebic abscess formation. a, b Images of normal liver (NL) and with a number of amoebic liver abscesses (ALA), respectively. c Gene expression analysis of Ehtbp and Ehtrf1 genes in trophozoites grown in normal conditions (NT) or isolated from amoebic liver abscesses (ALA). Statistical analysis with significant difference is marked with an asterisk (Student’s t-test, *P < 0.05)

Fig. 9

Ehtbp gene knockdown using G3 trophozoites. a Gene expression analysis of Ehtbp in knocked down (KD) cells grown at 3, 5, 10 μg/ml G418, using transfected cells with empty vector as a control. b Gene expression analysis of Ehtrf1 gene in Ehtbp knockdown cells. c Number of ingested erythrocytes at 2, 5, 15 and 30 min in control G3 cells and 10 μg/ml G418 Ehtbp knockdown cells. d Gene expression analysis of two genes containing the GAAC motif (Ehhgl, Ehrho) in Ehtbp KD cells. Significant difference was measured using Student’s t-test and marked with asterisks (*P < 0.05, **P < 0.002, ***P < 0.0004)