Abstract
The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a “wattle and daub” model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).
Highlights
The infectious and diagnostic form of Entamoeba histolytica (Eh), the trophozoites of which cause amebic dysentery and liver abscess, is the cyst, which contains four nuclei surrounded by a chitin-containing wall [1,2,3,4]
In addition to chitin that is partially deacetylated to form chitosan, mass spectroscopy showed the Entamoeba invadens (Ei) cyst wall contains three lectin families, members of which contain one or more Cys-rich chitin-binding domains (CBDs) that are unique to Entamoeba [8,9,10,11]
We hypothesize that Jacob lectins cross-link chitin fibrils, because all of the Jacob lectins have tandemly arranged CBDs, as do peritrophins, which are the major protein in chitin-based walls around the insect blood meal [12]
Summary
The infectious and diagnostic form of Entamoeba histolytica (Eh), the trophozoites of which cause amebic dysentery and liver abscess, is the cyst, which contains four nuclei surrounded by a chitin-containing wall [1,2,3,4]. Each Jessie lectin contains a single N-terminal 8-Cys CBD like that of Ei chitinase, a low complexity spacer, and a unique C-terminal domain of unknown function [10]. Recombinant Jessie lectins self-aggregated and caused transfected bacteria to agglutinate These results suggest a ‘‘wattle and daub’’ model of the Ei cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie lectins). Chitin is made in secretory vesicles prior to its deposition into the cyst wall Chitin and the chitin-binding lectins present in the cyst wall (Jacob, Jessie, and chitinase) were all absent from Ei trophozoites (data not shown). There was little or no Jessie lectin in the Ei cyst wall at 48 hrs
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