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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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Negative stains of bacteria transformed with MBP constructs targeted to the periplasm suggest the unique C-terminal domain of Jessie3 is an important contributor to self-aggregation.Bacteria (B) expressing MBP alone (low magnification in A and high magnification in F) or an MBP-Jacob2 fusion-protein (jac in low magnification in B) release dense quantities of proteins, which do not self-aggregate. In contrast, bacteria expressing an MBP-Jessie3 CBD fusion-protein (J3 CBD in low magnification in C and high magnification in G) release proteins in conical shaped eruptions from the periplasm, and the MBP-Jessie3 CBD fusion-proteins form short, thin linear arrays. Bacteria expressing MBP-Jessie3 unique C-terminal domain fusion-proteins (J3 sad (self-adhering domain) in low magnification in D and high magnification in H) or MBP-full-length Jessie3 (J3 ful in low magnification in E and high magnification in I) release dense aggregates of proteins, and sheets of these MBP-Jessie3 fusion-proteins contain branched aggregates of multiple-stranded linear forms that are thicker than those formed by Jessie3 CBD. Bar (A to E) is 250 nm. Bar (F to I) is 100 nm. Circles and ovals are added to figures to highlight structures of secreted proteins.
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ppat-1000498-g007: Negative stains of bacteria transformed with MBP constructs targeted to the periplasm suggest the unique C-terminal domain of Jessie3 is an important contributor to self-aggregation.Bacteria (B) expressing MBP alone (low magnification in A and high magnification in F) or an MBP-Jacob2 fusion-protein (jac in low magnification in B) release dense quantities of proteins, which do not self-aggregate. In contrast, bacteria expressing an MBP-Jessie3 CBD fusion-protein (J3 CBD in low magnification in C and high magnification in G) release proteins in conical shaped eruptions from the periplasm, and the MBP-Jessie3 CBD fusion-proteins form short, thin linear arrays. Bacteria expressing MBP-Jessie3 unique C-terminal domain fusion-proteins (J3 sad (self-adhering domain) in low magnification in D and high magnification in H) or MBP-full-length Jessie3 (J3 ful in low magnification in E and high magnification in I) release dense aggregates of proteins, and sheets of these MBP-Jessie3 fusion-proteins contain branched aggregates of multiple-stranded linear forms that are thicker than those formed by Jessie3 CBD. Bar (A to E) is 250 nm. Bar (F to I) is 100 nm. Circles and ovals are added to figures to highlight structures of secreted proteins.

Mentions: We used negative staining to get a better view of the aggregated MBP-Jessie3 fusions. The full-length Jessie3 and the unique C-terminal domain of Jessie3 formed dense aggregates on the surface of transformed E. coli and often disrupted the outer bacterial membrane (Figs. 7D and 7E). While these aggregates did not form a higher order crystal structure, MBP-full-length Jessie3 fusion-proteins made linear structures that aggregated into larger branched structures (Fig. 7I). Similar linear and branched structures were formed by MBP-Jessie3 unique C-terminal domain fusion-proteins, although the branches were not so long (Fig. 7H). In contrast, control MBP alone and MBP-Jacob2 lectin fusion-proteins stayed in solution and did not self-aggregate (Figs. 7A, 7B, and 7F). Interestingly, MBP fusion-proteins containing the N-terminal CBD of Jessie3 often formed cone-shaped aggregates, as they were released from the periplasm of E. coli (Fig. 7C). These MBP-Jessie3 CBD fusion-proteins formed smaller self-aggregates and thinner linear forms than the MBP-full length Jessie3 fusion-proteins (Fig. 7G).


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)

Negative stains of bacteria transformed with MBP constructs targeted to the periplasm suggest the unique C-terminal domain of Jessie3 is an important contributor to self-aggregation.Bacteria (B) expressing MBP alone (low magnification in A and high magnification in F) or an MBP-Jacob2 fusion-protein (jac in low magnification in B) release dense quantities of proteins, which do not self-aggregate. In contrast, bacteria expressing an MBP-Jessie3 CBD fusion-protein (J3 CBD in low magnification in C and high magnification in G) release proteins in conical shaped eruptions from the periplasm, and the MBP-Jessie3 CBD fusion-proteins form short, thin linear arrays. Bacteria expressing MBP-Jessie3 unique C-terminal domain fusion-proteins (J3 sad (self-adhering domain) in low magnification in D and high magnification in H) or MBP-full-length Jessie3 (J3 ful in low magnification in E and high magnification in I) release dense aggregates of proteins, and sheets of these MBP-Jessie3 fusion-proteins contain branched aggregates of multiple-stranded linear forms that are thicker than those formed by Jessie3 CBD. Bar (A to E) is 250 nm. Bar (F to I) is 100 nm. Circles and ovals are added to figures to highlight structures of secreted proteins.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2698119&req=5

ppat-1000498-g007: Negative stains of bacteria transformed with MBP constructs targeted to the periplasm suggest the unique C-terminal domain of Jessie3 is an important contributor to self-aggregation.Bacteria (B) expressing MBP alone (low magnification in A and high magnification in F) or an MBP-Jacob2 fusion-protein (jac in low magnification in B) release dense quantities of proteins, which do not self-aggregate. In contrast, bacteria expressing an MBP-Jessie3 CBD fusion-protein (J3 CBD in low magnification in C and high magnification in G) release proteins in conical shaped eruptions from the periplasm, and the MBP-Jessie3 CBD fusion-proteins form short, thin linear arrays. Bacteria expressing MBP-Jessie3 unique C-terminal domain fusion-proteins (J3 sad (self-adhering domain) in low magnification in D and high magnification in H) or MBP-full-length Jessie3 (J3 ful in low magnification in E and high magnification in I) release dense aggregates of proteins, and sheets of these MBP-Jessie3 fusion-proteins contain branched aggregates of multiple-stranded linear forms that are thicker than those formed by Jessie3 CBD. Bar (A to E) is 250 nm. Bar (F to I) is 100 nm. Circles and ovals are added to figures to highlight structures of secreted proteins.
Mentions: We used negative staining to get a better view of the aggregated MBP-Jessie3 fusions. The full-length Jessie3 and the unique C-terminal domain of Jessie3 formed dense aggregates on the surface of transformed E. coli and often disrupted the outer bacterial membrane (Figs. 7D and 7E). While these aggregates did not form a higher order crystal structure, MBP-full-length Jessie3 fusion-proteins made linear structures that aggregated into larger branched structures (Fig. 7I). Similar linear and branched structures were formed by MBP-Jessie3 unique C-terminal domain fusion-proteins, although the branches were not so long (Fig. 7H). In contrast, control MBP alone and MBP-Jacob2 lectin fusion-proteins stayed in solution and did not self-aggregate (Figs. 7A, 7B, and 7F). Interestingly, MBP fusion-proteins containing the N-terminal CBD of Jessie3 often formed cone-shaped aggregates, as they were released from the periplasm of E. coli (Fig. 7C). These MBP-Jessie3 CBD fusion-proteins formed smaller self-aggregates and thinner linear forms than the MBP-full length Jessie3 fusion-proteins (Fig. 7G).

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