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Evidence for a "wattle and daub" model of the cyst wall of entamoeba.

Chatterjee A, Ghosh SK, Jang K, Bullitt E, Moore L, Robbins PW, Samuelson J - PLoS Pathog. (2009)

Bottom Line: 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 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA, USA.

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).

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Chitin-binding is associated with the N-terminal Cys-rich domain of Jessie3.Left panel: Coomassie blue stained SDS-PAGE shows an MBP-fusion-protein containing the CBD of Jessie3, which was purified with an amylose resin. A lower mol wt band is likely MBP alone, as only the MBP-Jessie3 CBD fusion-protein binds to chitin (right lane). Left center panel: A Coomassie-stained MBP-fusion-protein containing the C-terminal unique domain of Jessie3, which was purified with the amylose resin (left lane), fails to bind to chitin (right lane). Right center panel: A Western blot with polyclonal rabbit antibodies to Jessie3 shows that an MBP-fusion-protein containing full-length Jessie3 self-aggregates, so that it is difficult to purify on the amylose resin (left lane). However, the MBP-full-length Jessie3 fusion-protein binds to chitin (right lane). Right panel: Polyclonal rabbit anti-Jessie3 antibodies bind weakly to trophozoites of Ei (left lane) but bind strongly to an ∼60-kDa protein in encysting Ei (the expected size of Jessie3) (right lane). Lower molecular weight bands may reflect a cleavage product between the N-terminal CBD and the C-terminal unique domain, as we have previously shown that Jacob lectins of encysting Ei are often cleaved between CBDs [9]. There was no binding of a control non-immune sera to trophozoite or cyst proteins (not shown).
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ppat-1000498-g005: Chitin-binding is associated with the N-terminal Cys-rich domain of Jessie3.Left panel: Coomassie blue stained SDS-PAGE shows an MBP-fusion-protein containing the CBD of Jessie3, which was purified with an amylose resin. A lower mol wt band is likely MBP alone, as only the MBP-Jessie3 CBD fusion-protein binds to chitin (right lane). Left center panel: A Coomassie-stained MBP-fusion-protein containing the C-terminal unique domain of Jessie3, which was purified with the amylose resin (left lane), fails to bind to chitin (right lane). Right center panel: A Western blot with polyclonal rabbit antibodies to Jessie3 shows that an MBP-fusion-protein containing full-length Jessie3 self-aggregates, so that it is difficult to purify on the amylose resin (left lane). However, the MBP-full-length Jessie3 fusion-protein binds to chitin (right lane). Right panel: Polyclonal rabbit anti-Jessie3 antibodies bind weakly to trophozoites of Ei (left lane) but bind strongly to an ∼60-kDa protein in encysting Ei (the expected size of Jessie3) (right lane). Lower molecular weight bands may reflect a cleavage product between the N-terminal CBD and the C-terminal unique domain, as we have previously shown that Jacob lectins of encysting Ei are often cleaved between CBDs [9]. There was no binding of a control non-immune sera to trophozoite or cyst proteins (not shown).

Mentions: To better understand the domain structure of the Jessie3 lectin, we expressed full-length Eh Jessie3, the putative N-terminal CBD domain, and the unique C-terminal domain as maltose-binding protein (MBP)-fusion proteins in the periplasm of Escherichia coli [26]. The MBP-fusions with the N-terminal CBD or the full-length Jessie3 each bound chitin beads (Fig. 5), while the MBP-fusion with the unique C-terminal domain of Jessie3 did not bind chitin. These results are consistent with our previous demonstration that the N-terminal domain of the Eh Jessie3 lectin, when expressed as an epitope-tagged protein in transfected Eh, is sufficient for chitin-binding [10].


Evidence for a "wattle and daub" model of the cyst wall of entamoeba.

Chatterjee A, Ghosh SK, Jang K, Bullitt E, Moore L, Robbins PW, Samuelson J - PLoS Pathog. (2009)

Chitin-binding is associated with the N-terminal Cys-rich domain of Jessie3.Left panel: Coomassie blue stained SDS-PAGE shows an MBP-fusion-protein containing the CBD of Jessie3, which was purified with an amylose resin. A lower mol wt band is likely MBP alone, as only the MBP-Jessie3 CBD fusion-protein binds to chitin (right lane). Left center panel: A Coomassie-stained MBP-fusion-protein containing the C-terminal unique domain of Jessie3, which was purified with the amylose resin (left lane), fails to bind to chitin (right lane). Right center panel: A Western blot with polyclonal rabbit antibodies to Jessie3 shows that an MBP-fusion-protein containing full-length Jessie3 self-aggregates, so that it is difficult to purify on the amylose resin (left lane). However, the MBP-full-length Jessie3 fusion-protein binds to chitin (right lane). Right panel: Polyclonal rabbit anti-Jessie3 antibodies bind weakly to trophozoites of Ei (left lane) but bind strongly to an ∼60-kDa protein in encysting Ei (the expected size of Jessie3) (right lane). Lower molecular weight bands may reflect a cleavage product between the N-terminal CBD and the C-terminal unique domain, as we have previously shown that Jacob lectins of encysting Ei are often cleaved between CBDs [9]. There was no binding of a control non-immune sera to trophozoite or cyst proteins (not shown).
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Related In: Results  -  Collection

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ppat-1000498-g005: Chitin-binding is associated with the N-terminal Cys-rich domain of Jessie3.Left panel: Coomassie blue stained SDS-PAGE shows an MBP-fusion-protein containing the CBD of Jessie3, which was purified with an amylose resin. A lower mol wt band is likely MBP alone, as only the MBP-Jessie3 CBD fusion-protein binds to chitin (right lane). Left center panel: A Coomassie-stained MBP-fusion-protein containing the C-terminal unique domain of Jessie3, which was purified with the amylose resin (left lane), fails to bind to chitin (right lane). Right center panel: A Western blot with polyclonal rabbit antibodies to Jessie3 shows that an MBP-fusion-protein containing full-length Jessie3 self-aggregates, so that it is difficult to purify on the amylose resin (left lane). However, the MBP-full-length Jessie3 fusion-protein binds to chitin (right lane). Right panel: Polyclonal rabbit anti-Jessie3 antibodies bind weakly to trophozoites of Ei (left lane) but bind strongly to an ∼60-kDa protein in encysting Ei (the expected size of Jessie3) (right lane). Lower molecular weight bands may reflect a cleavage product between the N-terminal CBD and the C-terminal unique domain, as we have previously shown that Jacob lectins of encysting Ei are often cleaved between CBDs [9]. There was no binding of a control non-immune sera to trophozoite or cyst proteins (not shown).
Mentions: To better understand the domain structure of the Jessie3 lectin, we expressed full-length Eh Jessie3, the putative N-terminal CBD domain, and the unique C-terminal domain as maltose-binding protein (MBP)-fusion proteins in the periplasm of Escherichia coli [26]. The MBP-fusions with the N-terminal CBD or the full-length Jessie3 each bound chitin beads (Fig. 5), while the MBP-fusion with the unique C-terminal domain of Jessie3 did not bind chitin. These results are consistent with our previous demonstration that the N-terminal domain of the Eh Jessie3 lectin, when expressed as an epitope-tagged protein in transfected Eh, is sufficient for chitin-binding [10].

Bottom Line: 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 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA, USA.

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).

Show MeSH
Related in: MedlinePlus