Project summary: This is a competitive renewal of 1R01-AI044070, now entitled "Multiple lectin model of the cyst wall of Entamoeba." Entamoeba histolytica (Eh) is a protozoan parasite that causes amebic dysentery and liver abscess. The infectious and diagnostic form of Eh is a chitin-walled cyst, which is spread by the fecal-oral route. Because Eh does not encyst in axenic culture, we have used Entamoeba invadens (Ei) to study cyst wall formation. Our goal is to make as detailed understanding as possible of Ei cyst formation and then to translate this information to the human pathogen Eh. Progress to date includes the following: The Ei cyst wall contains chitin, which is a homopolymer of beta-l,4-GlcNAc. Entamoeba chitin is made by two chitin synthases, one of which functions in fungal mutants. Chitin in the Ei cyst wall is deacetylated to make chitosan, which has a positive charge. Chitin is degraded by an Entamoeba chitinase, which has a unique N-terminal Cys-rich chitin-bindingdomain. Similar 8-Cys chitin-binding domains are present in a novel family of Entamoeba lectins called Jessie. Chitin fibrils in the Ei cyst wall are cross-linked by Jacob lectins, which have unique 6-Cys chitin- binding domains. Jacob lectins are post-translationally modified by addition of unique O-phosphodiester- linked glycans (O-phospho-glycans) and by cleavage at specific sites by endogenous Cys proteases. A plasma membrane Gal/GalNAc lectin binds galactose on Jacob lectins and so attaches the cyst wall to the surface of amebae. Galactose is also present in unique Asn-linked glycans of Entamoeba. Together these results give a model for Entamoeba cyst wall formation that is more detailed and complete than that for any other protist. The Specific Aims are to use molecular and biochemical methods to: Aim 1: Identify structural proteins and enzymes of Ei cyst walls. Aim 2: Determine the structures of cyst wall N-glycans and O-phospho-glycans. Aim 3. Characterize enzymes involved in chitin synthesis and deacetylation and synthesis of UDP-GlcNAc. Aim 4: Translate findings for Ei cyst walls to those of Eh. Relevance: Entamoeba histolytica (Eh) is an important cause of dysentery and liver abscess in developing countries. We are studying the cyst wall of Eh, because cysts are the infectious and diagnostic form. These studies will lead to a better understanding of how Eh causes disease and perhaps to better diagnostics.