Structural and functional characterization of the divergent Entamoeba Src using Src inhibitor-1

Abstract

Background: The abundant number of kinases that Entamoeba histolytica possesses allows us to assume that the regulation of cellular functions by phosphorylation-dephosphorylation processes is very important. However, the kinases responsible for the phosphorylation in Entamoeba spp. vary in the structure of their domains and, therefore, could be responsible for the unusual biological characteristics of this parasite. In higher eukaryotes, Src kinases share conserved structural domains and are very important in the regulation of the actin cytoskeleton. In both Entamoeba histolytica and Entamoeba invadens, the major Src kinase homologue of higher eukaryotes lacks SH3 and SH2 domains, but does have KELCH domains; the latter are part of actin cross-linking proteins in higher eukaryotic cells.

Methods: The function of the EhSrc protein kinase of Entamoeba spp. was evaluated using Src inhibitor-1, microscopy assays, Src kinase activity and western blot. In addition, to define the potential inhibitory mechanism of Src-inhibitor-1 for the amoebic EhSrc protein kinase, molecular dynamic simulations using NAnoscale Molecular Dynamics (NAMD2) program and docking studies were performed with MOE software.

Results: We demonstrate that Src inhibitor-1 is able to prevent the activity of EhSrc protein kinase, most likely by binding to the catalytic domain, which affects cell morphology via the disruption of actin cytoskeleton remodeling and the formation of phagocytic structures without an effect on cell adhesion. Furthermore, in E. invadens, Src inhibitor-1 inhibited the encystment process by blocking RhoA GTPase activity, a small GTPase protein of Rho family.

Conclusions: Even though the EhSrc molecule of Entamoeba is not a typical Src, because its divergent amino acid sequence, it is a critical factor in the biology of this parasite via the regulation of actin cytoskeleton remodeling via RhoA GTPase activation. Based on this, we conclude that EhSrc could become a target molecule for the future design of drugs that can prevent the transmission of the disease.

Keywords: Actin cytoskeleton; EhSrc; Entamoeba spp.; Src inhibitor-1.

Fig. 1

EhSrc is highly divergent from its closest homologues. a Alignment of the conserved domain among EhSrc (EAL46348.1), EiSrc (XP_004258856.1) and human Src (NP_938033). Identical amino acid residues are in dark gray and similar amino acid residues in light gray. b Phylogenetic tree of Src proteins of E. histolytica, E. invadens, Homo sapiens, Mus musculus and C. elegans. EhSrc protein of E. histolytica shows the highest homology with the Src protein of E. invadens, and these two proteins are poorly related with Src proteins from mammals as well as the nematode C. elegans. c Structural domains of typical Src and EhSrc proteins from E. histolytica and E. invadens are shown. The typical Src is composed of an amino-terminal myristoylation sequence (M), a unique region (U), Src-homology-2 (SH2) and SH3 protein-interaction domains; the Src of Entamoeba lacks these domains with the exception of the kinase domain

Fig. 2

Homology structural modeling of EhSrc. a 3D model of EhSrc. b The structural alignment of EhSrc kinase domain in red and 2SRC kinase domain in cyan. c RMSD of EhSrc at 100 ns of trajectory on MD simulation. d Rg of EhSrc at 100 ns of trajectory on MD simulation. e RMSF of EhSrc at 100 ns of MD simulation

Fig. 3

Src inhibitor-1 inhibits EhSrc protein activation of E. histolytica in silico and in vivo. a Predicted binding site of Src inhibitor-1 in the EhSrc protein of E. histolytica. b Confocal microscopy analysis of trophozoites with or without Src inhibitor-1 (30 μM) treatment. Actin was stained with rhodamine-phalloidin (red) (1/25), the nucleus was identified with DAPI 1/300 (blue) and p-Src (green) was detected with an anti-p-Src antibody (1/50). c Detection by western blot of phosphorylated tyrosine (p-Tyr), actin (α-actin) and EhSrc in EhSrc immunoprecipitated complex from treated and non-treated trophozoites with anti-phospho-tyrosine (1/1000), anti-actin (1/5000), and anti-EhSrc (1/5000) antibodies. d The kinase activity of DMSO- and Src inhibitor-1-treated trophozoites was determined using a Pro-Fluor® Src-Family kinase assays (Promega, Fitchburg, WI, USA) according to manufacturer’s instructions; the kinase activity from DMSO-treated trophozoites was defined as 100%. Values for each group represent mean ± SEM results from three independent experiments

Fig. 4

EhSrc interacts with actin during actin cytoskeleton reorganization. a Confocal microscopy analysis of trophozoites adhered to FN; actin was stained with rhodamine-phalloidin (red) (1/25), and Src (green) was detected with EhSrc antibody (1/250) and FITC-labeled goat anti- rabbit IgG (1/100). The white arrows in the merged image indicate the colocalization sites. At the bottom of panel (b) and to the right of panel (c), histograms of green and red intensities corresponding to selected areas (white boxes in confocal images) are shown. Areas of colocalization are marked with yellow arrows; areas where no colocalization was found are marked with green arrows. In each panel (b and c), the dispersion values indicating the results of the colocalization analyzes​​ are shown. d Predicted binding site of EhSrc and actin of E. histolytica. Image processing was performed with the Zen Blue software (v. 2012); the Pearson correlation coefficient was calculated with the SPSS software (v. 24). Abbreviation: R, Pearson correlation coefficient. Scale-bars: 10 μm

Fig. 5

EhSrc participates in the formation of structures involved in erythrophagocytosis but not in amoebic adhesion. a Upper panel: scanning electron microscopy; lower panel: transmission electron microscopy analyses of trophozoites of E. histolytica with or without Src inhibitor-1 treatment adhered to nylon grids. b Trophozoites of E. histolytica with or without Src inhibitor-1 treatment interacted with human erythrocytes for 5 min (upper panel) or 15 min (lower panel), analyzed by scanning electron microscopy

Fig. 6

Src inhibitor-1 blocks encystment process and GTP-RhoA activity. a Percentage of cyst formation inhibition by Src inhibitor-1 treatment (30 μM) during 96 h. Calcofluor m2r staining of cysts produced in each condition. b E. invadens trophozoites were induced to encyst in the presence of Src inhibitor-1 or vehicle (DMSO); after 12 h of encystment, the level of RhoA-GTP was determined with a G-LISA RhoA activation assay kit (Kit no. BK121, Cytoskeleton, Inc., Denver, CO, USA), according to the manufacturer’s instructions