# Trafficking and organization of cyst wall components into a robust biphasic structure in Entamoeba

## TL;DR

This study reveals how components of the Entamoeba cyst wall are organized into a two-layered structure during parasite development.

## Contribution

The study identifies the temporal and spatial dynamics of cyst wall component synthesis and localization, revealing a biphasic structure.

## Key findings

- Jacob1/3 lectins are post-translationally modified and localized to the cyst wall before Jessie3a and Chitinase1/4.
- Jessie lectin localization is disrupted by a chitinase inhibitor, indicating a distinct mechanism compared to Jacob lectins.
- Immunoelectron microscopy confirms a biphasic structure of the Entamoeba cyst wall with inner and outer layers.

## Abstract

Entamoeba histolytica, a protozoan parasite, causes amebiasis. Amebiasis is mainly transmitted by oral ingestion of cysts. Cysts are produced in the large intestine of humans from proliferative trophozoites by a cell differentiation process called encystation. The Entamoeba cyst wall consists of chitins and proteins that include chitinase and Jacob and Jessie lectins. During encystation, these components are synthesized and layered around encysting Entamoeba cells. The structures of these components are well studied; however, the detailed timings of their synthesis (the transcription of the encoding genes and the translation of the resulting mRNAs) and of changes in their localization during encystation are poorly understood. Here, we performed quantitative RT-PCR and an approach combining western blotting and immunofluorescence, confocal, and immunoelectron microscopy to analyze Entamoeba invadens cells that were sampled with short-time intervals during encystation. A chitinase inhibitor, D-B-09, which disrupts the compression of chitin fibers was used to analyze component interaction with chitin fibers. All genes encoding cyst wall proteins were stage-specifically transcribed and translated, and post-translationally modified forms of Jacob1/3 were trafficked to the cyst wall before Jessie3a and Chitinase1/4 were simultaneously localized in the cyst wall. The trafficking of Jacob lectins to the cyst wall and their co-localization with chitin fibers in encysting cells were not affected by D-B-09, while the localization of Jessie protein was impaired, indicating that localizations of Jacob and Jessie lectins are spatially different positions via different modes in cyst wall. These results indicate that cyst wall components are functionally linked and that they play different roles during Entamoeba cyst wall formation. Immunoelectron microscopy confirmed the immunofluorescence and confocal microscopy results. Importantly, immunoelectron microscopy also indicated that the Entamoeba cyst wall consists of a biphasic structure of electron-light (inner) and electron-dense (outer) areas.

Amebiasis is caused by Entamoeba histolytica infection and is mainly transmitted by oral ingestion of cysts. The Entamoeba cyst wall consists of chitins and proteins, including chitinase, and Jacob and Jessie lectins. During cyst formation, all these components are synthesized and layered around encysting Entamoeba cells. However, the detail sequence of their synthesis, including gene transcription and mRNA translation, and of their changes in localization during this process remain to be determined. Here, we investigated the temporal and spatial profiles of these events. These analyses revealed that Jacob1 and Jacob3 are post-translationally modified and co-localized with chitin fibers before Jessie3a and Chitinase1/4 localize in cyst wall. Notably, in contrast to Jacob lectins, localization of Jessie lectin in cyst wall was impaired by a chitinase inhibitor that disrupts the compression of chitin fibers. Subcellular localizations of cyst wall components were revealed by immunoelectron microscopy, which provided evidence that the Entamoeba cyst wall consists of a biphasic structure. All components therefore have different roles in Entamoeba cyst wall formation.