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

Additional file 1: Figure S1. of Structural and functional characterization of the divergent Entamoeba Src using Src inhibitor-1

Docking analysis of EhSrc protein and actin. (TIFF 538 kb

Additional file 2: Figure S2. of Structural and functional characterization of the divergent Entamoeba Src using Src inhibitor-1

Src inhibitor-1 did not affect adhesion of E. histolytica trophozoites. Trophozoites with or without Src inhibitor-1 treatment adhered to plastic during 15 min. The non-adhered trophozoites were eliminated, and the adhered trophozoites were stained with Sytox green and quantified by fluorescence. (TIFF 584 kb

Additional file 3: Figure S3. of Structural and functional characterization of the divergent Entamoeba Src using Src inhibitor-1

Src inhibitor-1 inhibits phagocytic cup formation during erythrophagocytosis. E. histolytica trophozoites with or without Src inhibitor-1 treatment and incubated with human erythrocytes for 5 min were fixed with paraformaldehyde and stained with rhodamine-phalloidin (red) (1/25). A phagocytic invagination is clearly seen in the non-treated trophozoite (left panel) in comparison with the treated one where such structures cannot be seen (right panel). (TIFF 2135 kb