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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Trends Parasitol. Author manuscript; available in PMC Jan 1, 2012.
Published in final edited form as:
PMCID: PMC3014499
NIHMSID: NIHMS245181

A simple fibril and lectin model for cyst walls of Entamoeba and perhaps Giardia

Abstract

Cyst walls of Entamoeba and Giardia protect them from environmental insults, stomach acids, and intestinal proteases. Each cyst wall contains a sugar homopolymer: chitin in Entamoeba and a unique N-acetylgalactosamine (GalNAc) homopolymer in Giardia. Entamoeba cyst wall proteins include Jacob lectins (carbohydrate-binding proteins) that cross-link chitin, chitinases that degrade chitin, and Jessie lectins that make walls impermeable. Giardia cyst wall proteins are also lectins that bind fibrils of the GalNAc homopolymer. While many of the details remain to be determined for the Giardia cyst wall, current data suggests a relatively simple fibril and lectin model for the Entamoeba cyst wall.

Why cyst walls are important

Entamoeba histolytica, the cause of amebic dysentery and liver abscess, and Giardia lamblia, a cause of diarrhea, are both spread by the fecal-oral route (Figure 1). Tetranucleate cysts of Entamoeba and Giardia are the diagnostic and infectious stage of each parasite [1,2]. The cyst wall, which is hard and impermeable to small molecules, protects Entamoeba and Giardia from lysis by environmental insults such as osmotic shock in fresh water, stomach acid, and duodenal proteases. Conversely, the cyst wall is quickly broken when parasites excyst and colonize the epithelium of the colon (Entamoeba) and duodenum (Giardia).

Figure 1
The diagnostic and infectious form of Giardia and Entamoeba is the quadranucleate cyst. After ingestion, the cyst wall is broken in the lumen of the duodenum (Giardia) or the colon (Entamoeba), and trophozoites, the dividing and adherent forms, are released. ...

Major issues for each organism are the composition of the cyst wall, how it is constructed, and how it is broken when the parasites excyst. Other important issues that will not be extensively discussed are the stimuli that trigger encystation and excystation, signals that mediate these stimuli, Golgi apparatus function, and the mechanisms whereby encysting protists secrete vast amounts of proteins and carbohydrates onto their surfaces [36]. Recent progress has been made on understanding how the Entamoeba cyst wall becomes impermeable to small molecules and how cyst wall proteins of Giardia bind to fibrils of the GalNAc homopolymer.

The major structural polymer of the Entamoeba cyst wall is chitin

The Entamoeba cyst wall has a single layer and contains a single sugar polymer, chitin, which is composed of β-1,4-linked N-acetylglucosamine (GlcNAc) (See Box 1 for descriptions of relevant glycans in Entamoeba and Giardia) [7]. Indeed Entamoeba is the only protist identified to date that makes chitin. The Entamoeba enzymes that make chitin (chitin synthase), modify chitin (chitin deacetylase), and degrade chitin (chitinases) all share common ancestry with the Saccharomyces enzymes (Figure 2(a)) [811]. The Entamoeba chitin synthase 2 complements a double mutant of Saccharomyces chitin synthases 1 and 3 [8]. The Entamoeba chitin deacetylase, expressed as a recombinant protein in Saccharomyces, deacetylates chitooligosaccharides in vitro [9]. About 25% of the chitin in the Entamoeba cyst wall is deacetylated in vivo, resulting in a positively charged polymer resistant to chitinases that is called chitosan. Entamoeba chitinases expressed as recombinant proteins in bacteria cleave chitooligosaccharides [10,11]. In addition, some Entamoeba chitinases have an N-terminal chitin-binding domain (CBD) that is unique to the parasite.

Glycobiology as it applies to the cyst walls of Entamoeba and Giardia

Glycobiology is the study of (i) carbohydrate polymers (for example, cellulose and chitin that are present in plant and fungal walls, respectively), (ii) sugars that are added to glycoproteins (for example, asparagine-linked glycans (N-glycans) or glycosylphosphatidylinositol (GPI) anchors), (iii) the enzymes that make and degrade the carbohydrate polymers (for example, chitin synthase and chitinase) and add or remove sugars from glycoproteins (for example, glycosyltransferases or glycosylhydrolases), and (iv) the proteins that bind carbohydrates that are called lectins (e.g. the Gal/GalNAc lectin of Entamoeba). Sugar homopolymers are described by the sugars they contain and the linkages between sugars (for example, chitin is composed of β-1,4-linked GlcNAc).

Because the cyst walls of Entamoeba and Giardia are rich in carbohydrates, one needs to understand all aspects of their glycobiology. Entamoeba is remarkable for its ability to synthesize O-phosphodiester-linked glycans, in which sugars are added to Ser and Thr residues of glycoproteins via a phosphate bond rather than directly to Ser and Thr. Giardia is remarkable for its very short N-glycan that is composed of GlcNAc2 rather than the 14 sugars (Glc3Man9GlcNAc2) present in host N-glycans. In addition, the β-1,3-linked GalNAc homopolymer in the Giardia cyst wall has not been described in any other organism.

Figure 2
A wattle and daub model of the Entamoeba cyst wall. (a) In the wattle stage that comes first, encystation-specific Jacob lectins (olive) are bound by plasma membrane Gal/GalNAc lectin (yellow) that is expressed by both trophozoites and cysts. Some Jacob ...

All of the Entamoeba cyst wall proteins are chitin-binding lectins

Because E. histolytica does not readily encyst in axenic culture, Entamoeba invadens, a pathogen of reptiles, has been used as a model to study cyst wall formation [3]. Mass spectrometry of purified E. invadens cyst walls revealed just three proteins: chitinase and two lectins, Jacob and Jessie, that are unique to Entamoeba and are only expressed during encystation (Figures 2 and and3)3) [12,13].

Figure 3
All of the Entamoeba cyst wall proteins are encystation-specific chitin-binding lectins that have an N-terminal signal peptide (tan) and lack a C-terminal transmembrane helix or GPI anchor. (a) The Entamoeba cyst wall chitinase has a conserved C-half ...

The chitin-binding properties of the Jacob lectin, Jessie lectins, and chitinase were determined by expressing each as an epitope-tagged protein in transformed E. histolytica trophozoites that do not make these cyst-specific wall proteins [14,15]. These proteins were also expressed as maltose-binding protein (MBP) fusion-proteins in the periplasm of E. coli (where disulfide bonds are formed) [16]. In both cases, the recombinant Jacob, Jessie, and chitinases bind to particulate chitin, while MBP alone does not bind chitin. Further, there are no trophozoite proteins that bind to chitin, and two E. invadens chitinases that lack the N-terminal CBD are absent from the cyst wall [11,13].

Cyst wall chitinase and Jessie lectins have a single, unique N-terminal CBD

The single E. histolytica chitinase, as well as the chitinase identified in the cyst wall of E. invadens by mass spectrometry, has a unique N-terminal CBD that is ~60-amino acids long (Figure 3(a)). This N-terminal CBD, which is also present in the Jessie lectin (Figure 3(b)), contains 8 conserved cysteine (Cys) residues that form four disulfide bonds, as well as numerous conserved aromatic amino acids that likely bind the sugar rings of chitin [10,14,16]. Yeast and human chitinases have C-terminal CBDs that do not share common ancestry with that of Entamoeba [17,18].

E. histolytica and E. dispar chitinases have low complexity spacers between the N-terminal CBD and the catalytic domain (Figure 3(a)) [10,14]. The chitinase spacers contain variable numbers of heptapeptide repeats that may be used to distinguish clinical isolates of each Entamoeba species [19,20].

In addition to the N-terminal CBD, Jessie lectins have a unique C-half domain that is self-aggregating, the so-called daub domain [14,16]. The daub function of Jessie lectins was discovered when E. coli expressing either the entire Jessie lectin or just the daub domain as MBP-fusions self-aggregated and formed large biofilms [16]. By negative staining the daub domain of the Jessie lectin makes linear structures that self-aggregate into larger branched structures.

Unique properties of multivalent Jacob lectins

Jacob lectins, which are present in seven copies in E. invadens and two copies in E. histolytica, contain tandem repeats of a unique ~50-amino acid-long CBD (Figures 3(c) and (d)) [1215]. Each Jacob CBD has six conserved Cys residues that form three disulfide bonds. The relatively short 6-Cys and 8-Cys CBDs of the cyst wall lectins are in contrast to the large Cys-rich domain in the Entamoeba Gal/GalNAc lectin that is an important virulence factor and vaccine candidate [4,21].

Two dimensional protein gels showed E. invadens Jacob lectins are post-translationally modified in two ways that were not expected [13]. First, Jacob lectins are extensively modified in serine- (Ser) and threonine- (Thr) rich spacer regions between CBDs by O-phosphodiester-(O-P) linked glycans attached to these amino acids. These O-P-glycans are distinct from those described on the proteophosphoglycans (PPGs) of trophozoites of E. histolytica, because the cyst sugars also contain rhamnose, while PPGs also contain galactose (Gal) [13,22].

Second, there are numerous cleavages in the spacer regions between CBDs of E. invadens Jacobs resulting in single, pairs, triplets, and quadruplet CBDs rather than the five CBDs in intact Jacobs [13]. Conserved sequences in the spacer domain of Jacob lectins are cleaved by Cys proteinases present in extracts of encysting E. invadens, as well as by human and plant Cys proteinases [4,13].

E. histolytica Jacob1 has two CBDs surrounding a short spacer, while the E. histolytica Jacob2 lectin has an unusually large spacer domain between the second and third CBD [14,15]. This large spacer domain is filled with numerous short repeats that are polymorphic and so may be used to distinguish clinical or field isolates of E. histolytica (the pathogen) and E. dispar (the non-pathogen) [15].

A wattle and daub model of the Entamoeba cyst wall

In numerous ways the cyst wall of Entamoeba resembles walls of simple huts that are made of wattle (sticks and twine) and daub (clay or mud) [13]. The foundation for the Entamoeba cyst wall are plasma membrane Gal/GalNAc lectins that are best known as mediators of adherence to glycans on host epithelial cells (Figure 2(a)) [1,21]. The role here of the Gal/GalNAc lectins is to bind cyst wall glycoproteins that contain Gal but lack their own transmembrane helices or glycosylphosphatidylinositol (GPI) anchors [10,1215]. Indeed in the presence of excess Gal, encysting E. invadens makes chitinase and Jacob lectins, becomes multinucleate, but does not form a wall [12].

The wattle of the Entamoeba cyst wall is made of chitin fibrils that are formed in small vesicles that move to many locations simultaneously on the plasma membrane (Figure 2(a)) [16]. Chitin fibrils are cross-linked by multivalent Jacob lectins that are made early during encystation but in separate vesicles than chitin [12,13,16].

The daub of the Entamoeba cyst wall are Jessie lectins that are made late during encystation and are composed of an N-terminal CBD and a C-terminal self-aggregating or daub domain (Figure 2(b)) [13,14,16]. After the appearance of Jessie lectins in the cyst wall, the wall no longer permeable to small molecules (DAPI and phalloidin) or large molecules (lectins or antibodies) [16]. Even though the Entamoeba cyst wall is made in two stages, it appears as a single layer by transmission microscopy [12,16].

Attempts to humanize the E. invadens cyst wall model

The E. invadens cyst model has been translated to E. histolytica, the human pathogen, in numerous ways. First, genomes of E. histolytica and E. dispar, the human non-pathogen, each predict the full set of cyst wall lectins and the enzymes to make and modify chitin [4,23,24]. Second, recombinant chitin synthase, chitin deacetylase, chitinases, Jacob lectins, and Jessie lectins, all of which have the expected activities, came from E. histolytica [810,1416]. Third, antibodies to recombinant Jacob1 and Jacob2 lectins bind to Entamoeba cysts isolated from human feces [15]. Fourth and conversely, sera from infected persons that recognize the Gal/GalNAc lectin also bind to recombinant Jacob1, Jessie, and chitinase [15]. Major unresolved issues in the cyst wall of Entamoeba are listed in Box 2.

Outstanding questions

Major unresolved questions concerning the Entamoeba cyst walls

  1. What are the three-dimensional structures of the 6-Cys CBDs present in the Entamoeba Jacob lectins and the 8-Cys CBDs at the N-termini of the Jessie lectins and chitinases? Are either similar to the structure of WGA that has been solved [39]?
  2. Can monoclonal antibodies be made to cyst wall proteins that might be used to identify Entamoeba cysts and distinguish cysts of the human pathogen (E. histolytica) from those of the non-pathogen (E. dispar) [24]?
  3. Are chitin fibrils in Entamoeba cyst walls curled and so able to maintain their general shape in the absence of protein (as for fibrils of the GalNAc homopolymer in Giardia cyst wall) [30]?
  4. Does the two-step model for excystation of Giardia, in which host and parasite proteases degrade cyst wall proteins and expose fibrils of the GalNAc homopolymer that are subsequently degraded by parasite glycohydrolases [30], also apply to excystation of Entamoeba?

Unresolved questions concerning the Giardia cyst wall

  1. Is there a plasma membrane lectin that binds to Giardia cyst wall proteins in the same way that the Gal/GalNAc lectin binds to Entamoeba cyst wall glycoproteins [12]? Alternatively, do curled fibrils of the GalNAc homopolymer form their own structure on the surface of encysting Giardia prior to addition of cyst wall proteins [30]?
  2. Do Giardia cyst wall proteins with epidermal growth factor-like repeats [37] bind to and cross-link fibrils of the GalNAc homopolymer in the same way that Jacob lectins with tandemly arrayed CBDs bind to and cross-link chitin fibrils in the Entamoeba cyst wall?
  3. What causes Giardia CWPs to self-aggregate and form the hard, brittle and impermeable cyst wall? Are the Leu-rich repeats or the conserved Cys-rich region of the CWPs involved in daub formation in the Giardia wall? How does formation of disulfide bonds and isopeptide bonds in CWPs contribute to cyst wall structure [43,44]? What are the distinct roles of CWP1 versus CWP2 versus CWP3?
  4. Can monoclonal antibodies be made to cyst wall proteins that might be used to distinguish cysts of Assemblage A and Assemblage B, the two Giardia species that infect humans [4547]?
  5. What are the enzymes that synthesize fibrils of the GalNAc homopolymer and degrade fibrils of the GalNAc homopolymer?

The Giardia cyst wall contains a unique β-1,3-linked GalNAc homopolymer

Because the GlcNAc-binding lectin wheat germ agglutinin (WGA) binds to the cyst wall of Giardia, it was thought for a long time that the protist cyst wall contains chitin [25]. Instead Giardia has a severely truncated Asn-linked glycan that is composed of two GlcNAc that binds WGA [26,27]. The Giardia cyst wall sugar polymer is composed of β-1,3-linked GalNAc, which has never been identified in any other organism [28]. This GalNAc homopolymer is made by an encystation-specific synthase that has been partially purified but has not yet been molecularly identified [29].

The GalNAc homopolymer forms ~5 nm thick fibrils that are coated with oval-shaped protein aggregates in intact cyst walls, which are thin, brittle to sonication, and impermeable to small molecules (Figure 4(a)) [30]. Using a method (strong base) adapted from studies of yeast walls, all of the cyst wall proteins (CWPs) were removed from fibrils of the GalNAc homopolymer (Figure 4(b)) [30,31]. Remarkably, deproteinated fibrils of the GalNAc homopolymer are curled and form a loose lattice that preserves the hollow spherical shape of the cyst wall.

Figure 4
Demonstration of the lectin properties of the Giardia Leu-rich repeat of CWP1 (CWP1LRR) and of an encystation-specific hydrolase of the GalNAc homopolymer. (a) The intact cyst wall of Giardia is composed of fibrils of the GalNAc homopolymer (bright green) ...

Giardia CWPs are lectins that bind fibrils of the GalNAc homopolymer

Three abundant cyst wall proteins (CWP1, CWP2, and CWP3), all of which are encystation-specific, have a series of leucine- (Leu) rich repeats and a C-terminal Cys-rich domain [32,33]. CWP1 and CWP2 are very similar to each other, although CWP2 also contains a C-terminal basic domain that is also rich in Ser and Thr and is cleaved by a specific Cys-protease [34]. CWPs are the target for diagnostic monoclonal antibodies to Giardia in clinical specimens [35], and vaccines against CWPs decrease the release of cysts from mouse models [36]. Additional Giardia cyst wall proteins (EGFCPs) have a tandemly arrayed set of Cys-rich, epidermal growth factor-like repeats [37,38].

Using MBP-fusion proteins similar to those used to demonstrate the chitin-binding activities of Entamoeba Jacob and Jessie lectins [16], intact Giardia CWP1, as well as the Leu-rich repeats of CWP1 (CWP1LRR), were shown to bind to deproteinated fibrils of the GalNAc homopolymer (Figure 4(c) [30]. In contrast, the Cys-rich region of CWP1 (CWP1CRR), failed to bind to the GalNAc homopolymer. These results were unexpected because the CBDs of Entamoeba cyst wall lectins and of WGA are all Cys-rich [1216,39]. Using mass spectrometry, the deproteinated GalNAc polymer was shown to bind native CWP1 and CWP2 present in extracts of encysting Giardia, but does not bind any trophozoite proteins [30].

The Giardia cyst wall is made in two stages

Epitope-tagged CWPs were used in transformed Giardia to show that the C-terminus of CWP2 and the entire CWP3 aggregate and form dense cores within encystation-specific secretory vesicles (ESVs) [40]. As a result, CWP1 and most of CWP2 are sorted into ESVs distinct from those containing the C-terminus of CWP2 and the entire CWP3. CWP1 and most of CWP2 are added early to the surface of encysting Giardia, while the C-terminus of CWP2 and CWP3 are added late to encysting parasites. Inhibition of the Cys-protease that removes the basic tail from CWP2 was also shown to result in cyst walls that are no longer water resistant [40].

With these transformants and with recombinant CWP1 as a probe for the GalNAc homopolymer, it was deduced that CWP2 and CWP3 are made in ESVs that are distinct from vesicles containing the GalNAc homopolymer [30]. Further, the GalNAc homopolymer was found to substantially cover the surface of encysting Giardia, while CWP2 and CWP3 are still present in ESVs.

A two-step (protease and glycohydrolase) model for excystation of Giardia

Excystation of Giardia in vitro is stimulated by conditions that mirror passage through the stomach and duodenum: treatment with acid, with chymotrypsin or trypsin, and then incubation in bicarbonate and bile [41]. An endogenous Cys-protease is also important for Giardia excystation [42]. With heat-killed Giardia cysts, exogenous chymotrypsin was shown to degrade CWPs and makes the cyst wall softer and more permeable to exogenous probes [30]. At the same time, fibrils of the GalNAc homopolymer, which are covered by CWPs in intact cysts, become exposed and are degraded by the encystation-specific glycohydrolase [30]. This model suggests that Giardia takes advantage of intestinal proteases to facilitate escape of trophozoites from the cyst wall. Major unresolved issues in the cyst wall of Giardia are listed in Box 2.

Conclusions

The Entamoeba cyst wall is composed of chitin and encystation-specific chitin-binding lectins that cross-link chitin, degrade chitin, or self-aggregate to form the daub that makes cyst walls impenetrable to small molecules. While Entamoeba chitin synthases, chitin deacetylases, and chitinases each resemble their counterparts in Saccharomyces, none of the enzymes involved in synthesis of the unique GalNAc homopolymer in the Giardia cyst wall have been identified. Although Leu-rich repeats of Giardia CWPs are lectin domains that bind fibrils of the GalNAc homopolymer, most of the details of the assembly and structure of the Giardia cyst wall remain to be determined. In summary, there is substantial experimental data supporting a simple fibril and lectin model for the Entamoeba cyst wall. How well the fibril and lectin model describes the cyst wall of Giardia remains to be determined.

Acknowledgments

This work was supported in part by National Institutes of Health grants AI44070 and AI048082 (to J.S.) and GM31318 (to P.W.R.). Thanks to past and present members of the Samuelson and Robbins labs for their ideas and experiments. Thanks to the anonymous reviewers for their insightful comments.

Role of the funding sources

The funding sources supported some of the experiments upon which this review is based. In contrast, the funding sources had no specific role in writing of the report or in the decision to submit the paper for publication.

Footnotes

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