RESUMO
By their active movement and voraux phagocytosis, the trophozoites of Entamoeba histolytica constitute an excellent system to investigate the dynamics of the Endosomal Sorting Complex Required for Transport (ESCRT) protein interactions through phagocytosis. Here, we studied the proteins forming the E. histolytica ESCRT-II complex and their relationship with other phagocytosis-involved molecules. Bioinformatics analysis predicted that EhVps22, EhVps25, and EhVps36 are E. histolytica bona fide orthologues of the ESCRT-II protein families. Recombinant proteins and specific antibodies revealed that ESCRT-II proteins interact with each other, with other ESCRT proteins, and phagocytosis-involved molecules, such as the adhesin (EhADH). Laser confocal microscopy, pull-down assays, and mass spectrometry analysis disclosed that during phagocytosis, ESCRT-II accompanies the red blood cells (RBCs) from their attachment to the trophozoites until their arrival to multivesicular bodies (MVBs), changing their interactive patterns according to the time and place of the process. Knocked-down trophozoites in the Ehvps25 gene presented a 50% lower rate of phagocytosis than the controls and lower efficiency to adhere RBCs. In conclusion, ESCRT-II interacts with other molecules during prey contact and conduction throughout the phagocytic channel and trophozoites membranous system. ESCRT-II proteins are members of the protein chain during vesicle trafficking and are fundamental for the continuity and efficiency of phagocytosis.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Entamoeba histolytica , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Entamoeba histolytica/genética , Proteínas de Protozoários/metabolismo , Fagocitose , Proteínas Recombinantes/metabolismoRESUMO
The Naegleria are ubiquitous free-living amoebae and are characterized by the presence of three phases in their biological cycle: trophozoite, cyst and flagellate. Of this genus, only Naegleria fowleri has been reported as pathogenic to humans. The proteasome is a multi-catalytic complex and is considered to be the most important structure responsible for the degradation of intracellular proteins. This structure is related to the maintenance of cellular homeostasis and, in pathogenic microorganisms, to the modulation of their virulence. Until now, the proteasome and its function have not been described for the Naegleria genus. In the current study, using bioinformatic analysis, protein sequences homologous to those reported for the subunits of the 20S proteasome in other organisms were found, and virtual modelling was used to determine their three-dimensional structure. The presence of structural and catalytic subunits of the 20S proteasome was detected by Western and dot blot assays. Its localization was observed by immunofluorescence microscopy to be mainly in the cytoplasm, and a leading role of the chymotrypsin-like catalytic activity was determined using fluorogenic peptidase assays and specific proteasome inhibitors. Finally, the role of the 20S proteasome in the proliferation and differentiation of Naegleria genus trophozoites was demonstrated.