Search

Movement and phagocytosis are clue events in colonisation and invasion of tissues by Entamoeba histolytica, the protozoan causative of human amoebiasis. During phagocytosis, EhRab proteins interact with other functional molecules, conducting them to the precise cellular site. The gene encoding EhrabB is located in the complementary chain of the DNA fragment containing Ehcp112 and Ehadh genes, which encode for the proteins of the EhCPADH complex, involved in phagocytosis. This particular genetic organisation suggests that the three corresponding proteins may be functionally related. Here, we studied the relationship of EhRabB with EhCPADH and actin during phagocytosis. First, we obtained the EhRabB 3D structure to carry out docking analysis to predict the interaction sites involved in the EhRabB protein and the EhCPADH complex contact. By confocal microscopy, transmission electron microscopy, and immunoprecipitation assays, we revealed the interaction among these proteins when they move through different vesicles formed during phagocytosis. The role of the actin cytoskeleton in this event was also confirmed using Latrunculin A to interfere with actin polymerisation. This affected the movement of EhRabB and EhCPADH, as well as the rate of phagocytosis. Mutant trophozoites, silenced in EhrabB gene, evidenced the interaction of this molecule with EhCPADH and strengthened the role of actin during erythrophagocytosis., (© 2019 John Wiley & Sons Ltd.)

Entamoeba histolytica trophozoites adhere to epithelium at the cell-cell contact and perturb tight junctions disturbing the transepithelial electrical resistance. Behind tight junctions are the adherens junctions (AJs) that reinforce them and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex, and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded β-cat, E-cad, Dsp l/ll, and Dsg-2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they colocalised with β-cat, E-cad, Dsp l/ll, and Dsg-2. Moreover, our results suggested that rEhCP112 could be internalised by caveolae and clathrin-coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with β-cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E-cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence. [ABSTRACT FROM AUTHOR]