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Updated: Jun 17, 2025
Investigating the Phagocytosis of Leishmania using Confocal Microscopy
Published on: July 29, 2021
The Entamoeba histolytica Vps26 (EhVps26) retromeric protein is involved in phagocytosis: Bioinformatic and
Diana Martínez-Valencia1, Cecilia Bañuelos2, Guillermina García-Rivera1
1Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav), Ciudad de México, México.
Abstract:
The retromer is a cellular structure that recruits and recycles proteins inside the cell. In mammalian and yeast, the retromer components have been widely studied, but very little in parasites. In yeast, it is formed by a SNX-BAR membrane remodeling heterodimer and the cargo selecting complex (CSC), composed by three proteins. One of them, the Vps26 protein, possesses a flexible and intrinsically disordered region (IDR), that facilitates interactions with other proteins and contributes to the retromer binding to the endosomal membrane. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, the retromer actively participates during the high mobility and phagocytosis of trophozoites, but the molecular details in these events, are almost unknown. Here, we studied the EhVps26 role in phagocytosis. Bioinformatic analyses of EhVps26 revealed a typical arrestin folding structure of the protein, and a long and charged IDR, as described in other systems. EhVps26 molecular dynamics simulations (MDS) allowed us to predict binding pockets for EhVps35, EhSNX3, and a PX domain-containing protein; these pockets were disorganized in a EhVps26 truncated version lacking the IDR. The AlphaFold2 software predicted the interaction of EhVps26 with EhVps35, EhVps29 and EhSNX3, in a model similar to the reported mammalian crystals. By confocal and transmission electron microscopy, EhVps26 was found in the trophozoites plasma membrane, cytosol, endosomes, and Golgi-like apparatus. During phagocytosis, it followed the erythrocytes pathway, probably participating in cargoes selection and recycling. Ehvps26 gene knocking down evidenced that the EhVps26 protein is necessary for efficient phagocytosis.
Insights
The retromer complex protein EhVps26 is essential for phagocytosis in Entamoeba histolytica, a parasite causing amoebiasis. Its intrinsically disordered region is crucial for protein interactions and cellular functions.
Area of Science:
- Cell Biology
- Parasitology
- Molecular Biology
Background:
- The retromer complex is vital for protein trafficking in eukaryotic cells, mediating cargo recognition and membrane remodeling.
- While retromer components are well-characterized in yeast and mammals, their roles in parasitic protozoa like Entamoeba histolytica remain largely unexplored.
- Entamoeba histolytica, the causative agent of amoebiasis, utilizes retromer-dependent processes during motility and phagocytosis.
Purpose of the Study:
- To investigate the molecular function of the retromer component EhVps26 in the phagocytosis process of Entamoeba histolytica.
- To elucidate the structural and interactional properties of EhVps26, particularly the role of its intrinsically disordered region (IDR).
Main Methods:
- Bioinformatic analyses and molecular dynamics simulations (MDS) were employed to predict EhVps26 structure and binding interactions.
- AlphaFold2 was used to model EhVps26 interactions with other retromer components.
- Confocal and transmission electron microscopy visualized EhVps26 localization within trophozoites.
- Gene knockdown of Ehvps26 was performed to assess its necessity for phagocytosis.
Main Results:
- Bioinformatic analysis revealed EhVps26 possesses a typical arrestin fold and a long, charged IDR.
- MDS predicted binding pockets for EhVps35, EhSNX3, and a PX domain protein, which were disrupted in an IDR-lacking mutant.
- AlphaFold2 modeling predicted interactions of EhVps26 with EhVps35, EhVps29, and EhSNX3, consistent with mammalian retromer structures.
- EhVps26 localized to the plasma membrane, cytosol, endosomes, and Golgi-like structures, and tracked erythrocytes during phagocytosis.
- Knockdown of Ehvps26 significantly impaired efficient phagocytosis in Entamoeba histolytica.
Conclusions:
- The intrinsically disordered region of EhVps26 is critical for its interactions with other retromer components and proper protein function.
- EhVps26 plays an indispensable role in the phagocytic pathway of Entamoeba histolytica, likely involved in cargo selection and membrane recycling.
- These findings provide novel insights into the molecular mechanisms of retromer function in parasitic protozoa and their implications in host-pathogen interactions.
