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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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Recombinant Jessie 3 lectins self-aggregate.Fluorescence microscopy (A to D) and negative staining (E) of bacteria transformed with MBP-fusion-proteins expressing full-length Jessie3 (green in A) or the unique C-terminal unique domain of Jessie3 (green in B) agglutinate to form large clumps of bacteria. In addition, biofilms containing MBP-full length Jessie3 are formed (green in D and immunostained with gold in E). Antibodies to the unique C-terminal domain of Jessie 3 are conjugated to Alexafluor in (A, B, and D) or are detected with immunogold in (E). Antibodies to the N-terminal CBD of Jessie3 (green in C) show that MBP-fusion-proteins containing this domain do not self-aggregate on a macro-scale and do not agglutinate bacteria. Bar (A to D) is 2 microns. Bar (E) is 500 nm.
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ppat-1000498-g006: Recombinant Jessie 3 lectins self-aggregate.Fluorescence microscopy (A to D) and negative staining (E) of bacteria transformed with MBP-fusion-proteins expressing full-length Jessie3 (green in A) or the unique C-terminal unique domain of Jessie3 (green in B) agglutinate to form large clumps of bacteria. In addition, biofilms containing MBP-full length Jessie3 are formed (green in D and immunostained with gold in E). Antibodies to the unique C-terminal domain of Jessie 3 are conjugated to Alexafluor in (A, B, and D) or are detected with immunogold in (E). Antibodies to the N-terminal CBD of Jessie3 (green in C) show that MBP-fusion-proteins containing this domain do not self-aggregate on a macro-scale and do not agglutinate bacteria. Bar (A to D) is 2 microns. Bar (E) is 500 nm.

Mentions: However, an alternative function for the unique C-terminal domain of Jessie3 was suggested by the finding that transformed bacteria expressing full-length Eh Jessie3 agglutinate in solution. After centrifugation in a microfuge at high speed, these bacteria aggregated into a solid pellet, which did not dissociate with vortexing or pipetting. Transformed bacteria expressing MBP-fusions containing only the unique C-terminal domain of Jessie3 also agglutinated in solution and aggregated into an insoluble pellet after centrifugation. Anti-Jessie3 antibodies and fluorescence microscopy showed agglutinated E. coli secrete large amounts of the MBP-Jessie3 fusion-proteins, which accumulated in sheets or biofilms on the surface of the slide or cover slip (Figs. 6A, 6B, and 6D). Negative staining showed secreted MBP-full-length Jessie3 fusion-proteins also formed large planar aggregates that labeled with gold-conjugated secondary antibodies after primary antibody labeling of the Jessie3 lectin (Fig. 6E). In contrast, bacteria expressing MBP only, MBP fused to the N-terminal CBD of Jessie3, or MBP fused to multiple CBDs of the Jacob lectin did not agglutinate bacteria in solution, did not form a solid pellet after centrifugation, and failed to make biofilms on slide surfaces (Fig. 6C and data not shown). These results strongly suggest that the unique C-terminal domain of Eh Jessie3 is an important contributor to bacterial agglutination in solution and insoluble pellet-formation after centrifugation.


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)

Recombinant Jessie 3 lectins self-aggregate.Fluorescence microscopy (A to D) and negative staining (E) of bacteria transformed with MBP-fusion-proteins expressing full-length Jessie3 (green in A) or the unique C-terminal unique domain of Jessie3 (green in B) agglutinate to form large clumps of bacteria. In addition, biofilms containing MBP-full length Jessie3 are formed (green in D and immunostained with gold in E). Antibodies to the unique C-terminal domain of Jessie 3 are conjugated to Alexafluor in (A, B, and D) or are detected with immunogold in (E). Antibodies to the N-terminal CBD of Jessie3 (green in C) show that MBP-fusion-proteins containing this domain do not self-aggregate on a macro-scale and do not agglutinate bacteria. Bar (A to D) is 2 microns. Bar (E) is 500 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698119&req=5

ppat-1000498-g006: Recombinant Jessie 3 lectins self-aggregate.Fluorescence microscopy (A to D) and negative staining (E) of bacteria transformed with MBP-fusion-proteins expressing full-length Jessie3 (green in A) or the unique C-terminal unique domain of Jessie3 (green in B) agglutinate to form large clumps of bacteria. In addition, biofilms containing MBP-full length Jessie3 are formed (green in D and immunostained with gold in E). Antibodies to the unique C-terminal domain of Jessie 3 are conjugated to Alexafluor in (A, B, and D) or are detected with immunogold in (E). Antibodies to the N-terminal CBD of Jessie3 (green in C) show that MBP-fusion-proteins containing this domain do not self-aggregate on a macro-scale and do not agglutinate bacteria. Bar (A to D) is 2 microns. Bar (E) is 500 nm.
Mentions: However, an alternative function for the unique C-terminal domain of Jessie3 was suggested by the finding that transformed bacteria expressing full-length Eh Jessie3 agglutinate in solution. After centrifugation in a microfuge at high speed, these bacteria aggregated into a solid pellet, which did not dissociate with vortexing or pipetting. Transformed bacteria expressing MBP-fusions containing only the unique C-terminal domain of Jessie3 also agglutinated in solution and aggregated into an insoluble pellet after centrifugation. Anti-Jessie3 antibodies and fluorescence microscopy showed agglutinated E. coli secrete large amounts of the MBP-Jessie3 fusion-proteins, which accumulated in sheets or biofilms on the surface of the slide or cover slip (Figs. 6A, 6B, and 6D). Negative staining showed secreted MBP-full-length Jessie3 fusion-proteins also formed large planar aggregates that labeled with gold-conjugated secondary antibodies after primary antibody labeling of the Jessie3 lectin (Fig. 6E). In contrast, bacteria expressing MBP only, MBP fused to the N-terminal CBD of Jessie3, or MBP fused to multiple CBDs of the Jacob lectin did not agglutinate bacteria in solution, did not form a solid pellet after centrifugation, and failed to make biofilms on slide surfaces (Fig. 6C and data not shown). These results strongly suggest that the unique C-terminal domain of Eh Jessie3 is an important contributor to bacterial agglutination in solution and insoluble pellet-formation after centrifugation.

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