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. 2021 Oct 16:2021:5746629.
doi: 10.1155/2021/5746629. eCollection 2021.

Protein Phosphatase PP2C Identification in Entamoeba spp

Affiliations

Protein Phosphatase PP2C Identification in Entamoeba spp

Abril Navarrete-Mena et al. Biomed Res Int. .

Abstract

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Phosphatase activity in Entamoeba spp. (a) Enzymatic activity of the total extract (TE, black bar), membrane fraction (MF, dark grey bar), and cytosolic fraction (CF, light grey bar) of E. histolytica, E. invadens, and E. dispar. (b) Relative value (considering E. histolytica as 100% percent) of phosphatase activity in the MF of E. invadens (dark grey bar) and E. dispar (white bar). (c) Phosphatase activity of the MF of trophozoites from E. histolytica (black bar), E. invadens (dark grey bar), and E. dispar (white bar) was evaluated with tungstate (T), molybdate (M), and orthovanadate (O), specific PTP inhibitors. All experiments were run with p-NPP as the substrate. Differences between the phosphate activity of the distinct extracts were considered significant at p < 0.0001. The data is representative of six independent experiments.
Figure 2
Figure 2
Detection of PP2C in Entamoeba spp. (a) Immunodetection of PP2C using the polyclonal antibody against LmxPP2C in the total extract (TE) (lane 1), membrane fraction (MF) (lane 2), and cytosolic fraction (FC) (lane 3). Recombinant protein LmxPP2C was used as a positive control and anti-α-tubulin as the loading control. The signal was analyzed on the Odyssey Infrared Imaging System. (b) Immunofluorescence assay using polyclonal antibody against LmxPP2C. Scale bars = 20 μm. (c) Phosphatase activity on the MF of E. histolytica (black bar), E. invadens (grey bar), and E. dispar found by using sanguinarine, a specific inhibitor for PP2C. p < 0.05.
Figure 3
Figure 3
Detection of PP2C in the encysting process of E. invadens. (a) Differential Interference Contrast (DIC) column shows the purity of the cysts under permeabilized (P) and nonpermeabilized (NP) conditions at 12 and 24 h after encystment. DAPI was used to show the nucleus; presence of PP2C is observed in red using the anti-LmxPP2C antibody evidenced by secondary antibody coupled to TRITC. (b) Western blot analysis using anti-LmxPP2C antibody on total cyst extracts solubilized with triton. Recombinant protein LmxPP2C was used as a positive control. Scale bars = 10 and 2 μm. The proteins were analyzed on the Odyssey Infrared Imaging System.
Figure 4
Figure 4
PP2C protein phosphatase expression model in Entamoeba spp. (a) Localization of protein phosphatase PP2C in trophozoites. (b) Detection of PP2C in E. invadens trophozoites and cysts by Western blot assays. (c) Immunofluorescence of PP2C in E. invadens encystation. A specific differential staining was used for cysts, calcofluor (blue), propidium iodide (purple) for the cyst nuclei, and FITC (fluorescein isothiocyanate, green) fluorophore.
Figure 5
Figure 5
In silico analysis of the PP2C phosphatase protein in E. invadens (EinPP2C). (a) Table of properties and characteristics of the EinPP2C sequence (EIN_095130). (b) Graphical summary of the alignment of the sequences of the PP2C proteins of E. invadens (EinPP2C, EIN_095130), E. histolytica (EhiPP2C, EHI_197120), E. dispar (EdiPP2C, EDI_122040), and L. mexicana (LmxPP2C, Lmx_25.0750), examined on COBALT. The PP2C of E. invadens is found at positions D458, D479, D648, and D687 (red arrows). The most conserved region of the PP2C domain is depicted in purple. (c) A cladogram is displayed of the sequences of the proteins in the OG5_150267 orthologues group and the sequence of LmxPP2C (OG5_150267). (d) Analysis of the degree of evolutionary conservation was carried out, comparing the hypothetical 3D structure of EinPP2C with the orthologue sequences of the two other species of Entamoeba ssp. and with L. mexicana LmxPP2C, scrutinized on the ConSurf server and visualized on the UCSF Chimera program. The β-sandwich domain (in purple) is the most conserved region of the sequences.

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