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).