Mechanisms of Giardia lamblia Differentiation into CystsMechanisms of Giardia lamblia...

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS,1092-2172/97/$04.0010

Sept. 1997, p. 294–304 Vol. 61, No. 3

Copyright © 1997, American Society for Microbiology

Mechanisms of Giardia lamblia Differentiation into CystsHUGO D. LUJÁN,1 MICHAEL R. MOWATT,2 AND THEODORE E. NASH2*

Department of Biological Chemistry, School of Medicine, National University of Córdoba, Córdoba, Argentina,1

and Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, Maryland 20892-04252

INTRODUCTION .......................................................................................................................................................294STIMULUS FOR ENCYSTATION AND REGULATION OF ENCYSTATION-SPECIFIC

GENE EXPRESSION.........................................................................................................................................294SYNTHESIS AND INTRACELLULAR TRANSPORT OF CYST WALL COMPONENTS .............................297ASSEMBLY OF THE CYST WALL.........................................................................................................................299CONCLUSION............................................................................................................................................................301REFERENCES ............................................................................................................................................................301

INTRODUCTION

Giardia is a flagellated protozoan that inhabits the uppersmall intestine of its vertebrate host (1, 147) and is the mostcommon cause of defined waterborne diarrhea in the UnitedStates (18, 81). It is also an increasingly significant problem forbackpackers and children in day care centers (38, 148) and isan enteric pathogen transmitted by sexual contact among ho-mosexual men (79). Infection is initiated by ingestion of cystsfollowed by excystation and colonization of the small intestineby the trophozoites, which are responsible for the manifesta-tions of the disease (1, 18, 147). Trophozoites, like most intes-tinal parasitic protozoa (77, 83, 101, 104), undergo fundamen-tal biological changes to survive outside the intestine of theirhost by differentiating into infective cysts. Cyst formation, orencystation, is essential for the transmission of Giardia (18,127, 152) and as such represents a target for blocking thedissemination of the parasite.

Because Giardia belongs to the earliest branch of the eu-karyotic lineage (54, 136), an understanding of its fundamentalcellular mechanisms would shed light on the evolution of thesemechanisms in higher eukaryotes. Therefore, the process ofcyst formation or encystation represents one of the most prim-itive developmental responses of eukaryotes to environmentalsignals.

The Giardia encystation process can be divided into threedistinct parts: (i) the stimulus for encystation and the regula-tion of encystation-specific gene expression, (ii) the synthesisand intracellular transport of cyst wall components, and (iii)the assembly of the extracellular cell wall.

STIMULUS FOR ENCYSTATION AND REGULATION OFENCYSTATION-SPECIFIC GENE EXPRESSION

Since the initial work of Gillin et al. in 1987 (45), whichdemonstrated Giardia encystation in vitro for the first time,several molecular and cellular biological aspects of this processhave been elucidated (32, 42, 44, 87, 90, 93, 97, 98, 105).Nevertheless, the molecular basis for the induction of encys-tation remained undefined for a long time, primarily becauseseveral different conditions were known to trigger trophozoite

differentiation in vitro (45, 66, 124, 131). Several reports sug-gested that bile salts were responsible for Giardia encystation(45, 66, 124, 131); however, other studies showed that encys-tation is possible in the absence of bile (143). Therefore, thenature of the stimulus which induces Giardia trophozoites todifferentiate into cysts remained unclear. For instance, encys-tation may be induced by (i) primary bile salts added to TYI-S-33 medium without bile (pH 7.1) (45); (ii) bile deprivation ina nitrogen atmosphere (143); (iii) bovine bile (5 to 10 mg/ml)(pH 7.0) (124); (iv) glycocholate (12 mM) plus either penta-decanoic acid or myristic acid (0.5 to 1 mM) (pH 7.8) (124); (v)human bile (2.5%, vol/vol), porcine bile (0.5 mg/ml), or bovinebile (5 mg/ml) (pH 7.8) for 70 h (131); (vi) sodium glycocholate(10 mM) plus myristic acid (0.5 mM) and oleic acid (0.1 mM)(pH 7.8) (44); (vii) preencystation in TYI-S-33 without bile for3 days and encystation with porcine bile (0.5%) plus lactic acidhemicalcium salt (5 mM) (pH 7.8) (44); (viii) bovine bile (10mg/ml) (pH 7.8) for 24 h, with return to TYI-S-33 medium for96 h (66); or (ix) trophozoites routinely grown in the absenceof bile and encysted with porcine bile (0.25%) plus lactic acidhemicalcium salt (0.55 mg/ml) (pH 7.8) for 48 h (44). Theefficiency of these media to generate viable cysts in vitro variessignificantly. However, comparison is difficult because differentparameters were used by different investigators to assess suchcapability. For instance, the ability of cysts to resist an osmoticshock, infect susceptible animals, excyst in vitro, and take up orexclude fluorescent dyes was used to determine cyst viability.By using a comparative study with encystation medium con-taining porcine bile (0.25%) plus lactic acid hemicalcium salt(0.55 mg/ml) (pH 7.8), Campbell and Faubert (12) demon-strated that encystation efficiency also differed among fourclones derived from the same Giardia WB isolate. However,they observed no difference in cyst excretion when gerbils wereacutely infected with those clones. Moreover, supporting theimportance of bile on the induction of encystation, Reiner etal. (124) reported that the inability of the WB-derived cloneA6 to encyst in vitro correlates with a deficiency of bile saltuptake by this subline. However, since this clone also encystsnormally during experimental infection in laboratory animals(124), encystation of Giardia in a bile-rich culture medium maynot have general physiological significance.

To explain the effects of high concentrations of bile on theinduction of encystation, Gillin et al. (44) proposed that tro-phozoites commonly live attached to the epithelial cells of thegut, underneath a mucus layer, in an environment which ispoor in bile and has a neutral pH. Thus, encystation would be

* Corresponding author. Mailing address: Laboratory of ParasiticDiseases, National Institute of Allergy and Infectious Diseases, Na-tional Institutes of Health, Building 4, Room 126, 9000 Rockville Pike,Bethesda, MD 20892-0425. Phone: (301) 496-6920. Fax: (301) 402-2689. E-mail: [email protected].

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induced when the trophozoites gain access to the slightly al-kaline, bile-rich milieu of the intestinal lumen (44). Unfortu-nately, this hypothesis was based on erroneous assumptions.For instance, no reported data indicate that attached tropho-zoites are covered with a mucus layer which separates theorganism from the bile-rich environment of the intestinal lu-men. In contrast, optical and electron microscopic examina-tions of infected intestines from humans and several animalshave shown that nonencysting trophozoites can be found at-tached to the epithelial cells, swimming free in the lumen, ortrapped and immobilized in strands of mucus (113, 118). More-over, it was suggested that the increased secretion of mucusthat occurs during infection could be an important mechanismof defense against Giardia either to rapidly expel the trappedcells or to prevent the parasites from making direct contactwith the epithelial cells of the gut (113, 118).

To analyze the nature of the stimulus that induces Giardiatrophozoites to differentiate into cysts, we studied the distri-bution of Giardia along the small intestine of chronically in-fected mice with severe combined immunodeficiency (SCIDmice) (10). Our results indicated that the maximum number ofcells localize to the jejunum (86). Few cells were found ineither the duodenum (where the pH is acidic) or the lastportion of the ileum (where most of the nutrients have beenabsorbed). Analysis of the stage of differentiation of the cellsrevealed that nonencysting trophozoites are the most abundantcell type in all the sections of the small intestine that westudied. Trophozoites in the process of encystation were ob-served in the proximal ileum, and cysts were observed in thedistal ileum and in the large intestine, which is in agreementwith previous results obtained with other animal models (12,45, 113). This predilection of the Giardia organisms for thejejunum suggested that trophozoites require a high concentra-tion of nutrients, especially those that the parasite is unable tosynthesize. Giardia, like most eukaryotic cells, requires exoge-nous lipids for proliferation (41, 64, 85, 91). It is well knownthat trophozoites are unable to synthesize cholesterol de novo(64), although the initial steps of the mevalonate pathway arepresent in these organisms. For instance, trophozoites can gen-erate isoprenoid compounds, such as farnesol and geranylge-raniol (89), and ubiquinone (23) from mevalonate, a necessaryprecursor of cholesterol in eukaryotes (47). To multiply andcolonize the midjejunum of mammals, trophozoites might de-pend on preformed biliary lipids present in the upper smallintestine (14). Since the absorption of lipids in humans occursalmost completely in the jejunum (16, 74, 162), we reasonedthat as trophozoites travel down the intestine, they might con-front a lipid-poor environment, which could trigger their dif-ferentiation into cysts, the nonproliferative stage of the para-site.

In a recent report (88), we clearly demonstrated that cystformation is induced in vitro when trophozoites are starved ofcholesterol by growth in a lipoprotein-deficient medium. Fourcloned lines that were derived from two independent Giardiaisolates were tested, and they all formed cysts similarly (includ-ing clone WB/A6, which is unable to encyst in the bile-richculture medium [124]). Furthermore, as would be expectedfrom our model, the addition of either cholesterol, low-densitylipoproteins (LDL), or very low density lipoproteins to thelipoprotein-deficient culture medium inhibited encystation. Incontrast, high-density lipoproteins, phospholipids, bile salts,and fatty acids had little or no effect.

A secondary role for bile salts and alkaline pH in Giardiaencystation was also shown (88). The previously demonstratedeffects of high concentrations of bile salts on encystation werebest explained as a means of preventing the uptake of choles-

terol, not in terms of a direct effect on the trophozoites. Thisconclusion is supported by the following observations: (i) Gi-ardia can grow in the absence of bile salts (19, 69, 103, 160),indicating that these compounds are not essential for the tro-phozoites; (ii) trace amounts of bile salts stimulate the growthof Giardia because they solubilize lipids, increasing their avail-ability to the cells (30, 31, 52, 53, 91); and (iii) paradoxically,high concentrations of bile salts inhibit cholesterol uptake andtherefore favor encystation (88). It is known that primary bilesalts are synthesized from cholesterol in the liver, conjugatedto either glycine or taurine, and stored in the gallbladder (13,56, 84, 134). During digestion, these compounds are releasedinto the proximal duodenum to solubilize lipids and favor theirabsorption. Fat absorption is very efficient and occurs almostcompletely in the jejunum (5, 74). Later, bile salts are reab-sorbed by an active transport system in the distal ileum (149),indicating that Giardia encystation occurs most probably with-out the direct involvement of bile salt molecules.

With regard to the effect of the pH, we demonstrated thatcholesterol starvation induces encystation within a broad rangeof pH (pH 6 to 8), but encystation was optimal at a slightlyalkaline pH (pH 7.6 to 7.8) (88), in agreement with the resultsobtained with the bile-rich encystation medium (45).

Gillin et al. (45) also showed that lactic acid added to thebile-rich medium favored encystation and suggested that prod-ucts of the bacterial metabolism in the lower intestine mayhave important effects on the trophozoites. Nevertheless, it isinteresting that the hemicalcium salt of lactic acid was used intheir experiments (45, 124). When the addition of exogenouslactic acid to trophozoites cultured in cholesterol-deficient me-dium was tested, cyst production was slightly improved byaddition of the hemicalcium salt (10% of the control withoutaddition); however, the sodium salt, the lithium salt, and thefree-acid form of lactic acid had no effect on encystation (94).These results indicated that calcium, not lactic acid, promotesencystation. When the hemicalcium salt of lactic acid is addedto the encystation medium, a white precipitate forms, whichsuggests either that the parasite is deprived of some moleculeessential for proliferation or, alternatively, that calcium per seor a calcium complex can have a direct effect on the plasmamembrane of the trophozoites that facilitate encystation.

All these data indicate that cholesterol starvation is neces-sary and sufficient for the stimulation of Giardia encystation invitro and most probably in the intestine of mammalian hostsbut that other factors may also play minor roles in this process.Based on these results, we have constructed the followingmodel for encystation. Trophozoites colonize the midjejunumof mammals (44, 94, 113, 118) and multiply, taking up choles-terol from this environment, which is rich in dietary and biliarycholesterol (5, 14, 162). As the trophozoites travel down theintestine, they encounter an environment poor in cholesterol,which in turn triggers parasite differentiation. This model issupported by studies with different animal models, which dem-onstrated that the process of encystation occurs almost exclu-sively in the lower portions of the ileum and in the largeintestine (44, 118).

We still do not know how cholesterol deprivation inducesthe expression of encystation-specific genes. Although the pre-vious results represented an important step forward in under-standing this process, several questions remain unanswered;these include the questions of how Giardia senses low levels ofcholesterol in the environment and how this sensing mecha-nism correlates with the regulation of gene expression duringencystation.

Two non-mutually exclusive models can be proposed: (i)cholesterol-induced changes in plasma membrane fluidity of

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trophozoites, which trigger a second-messenger pathway, and(ii) cholesterol-mediated regulation of transcription.

The first model theorizes that cholesterol deprivation in-duces changes in the plasma membrane fluidity of trophozo-ites, with subsequent activation of signal transduction pathwaysthat culminate in the expression of encystation-specific genes.The mechanism that is used by both prokaryotic and eukary-otic organisms in responding to environmental stimuli com-monly involves a two-component signal transduction system(17, 57, 141). These systems usually consist of at least twoproteins, a sensor-transmitter protein located in the plasmamembrane and one or more intracellular regulatory proteinsthat transmit the signal to specific genes. The sensor-transmit-ter protein is usually an integral transmembrane protein withcytoplasmic and extracytoplasmic domains (141). Environmen-tal stimuli such as changes in pH, temperature, membrane lipidcomposition and fluidity, redox potential, ligand interactions,and the presence of specific ions can produce conformationalor topological changes in sensor molecules, triggering thetransmission of a particular signal into the cell interior (17, 57).The physical state of the plasma membrane (membrane fluid-ity) is a parameter that can be influenced by the concentrationof cholesterol but also by the pH and calcium (22). Modifica-tions of either the cholesterol content of the membranes or thecholesterol/phospholipid ratio can dramatically influence manymembrane-related activities, such as membrane permeabilityand enzyme, receptor, and sensor functions (15, 22, 25, 29, 60,68, 78, 115, 122, 140). Cholesterol depletion in Giardia culturemedium could effect changes in the fluidity of the trophozoiteplasma membrane (a phenomenon that can be facilitated byalkaline pH and high calcium concentrations), which in turnmay play an important role in the transduction of signals intothe cell nuclei.

Additional support for this model comes from recent workby Ellis et al. (24), who demonstrated differences in the lipidcomposition between encysting and nonencysting trophozoitesand who demonstrated activation during Giardia differentia-tion of an enzyme involved in the regulation of membranefluidity in higher eukaryotes, fatty acid desaturase.

The second hypothesis is based on known data about cho-lesterol regulation of transcription. In mammalian cells, theLDL receptor mediates the uptake of the cholesterol-rich LDLfrom serum (46). Cholesterol uptake from the medium cou-pled with the intracellular production of cholesterol is regu-lated perfectly by feedback repression of the genes that encodethe LDL receptor as well as a series of enzymes that areinvolved in cholesterol synthesis along the mevalonate pathway(47). In these cells, transcription of these genes is repressed inthe presence of cholesterol whereas sterol depletion leads toincreased transcription (47). Giardia, however, is unable tosynthesize cholesterol de novo (64), and several lines of evi-dence suggest that Giardia does not use an LDL receptor totake up cholesterol from its medium (89). The intracellularconcentration of cholesterol in Giardia may be directly depen-dent on the external concentration of this compound (31).Therefore, the high levels of cholesterol available to the par-asite in either the serum, bile-rich culture medium or the uppersmall intestine might repress the transcription of encystation-specific genes. When the environmental cholesterol concentra-tion reaches a critical low level, transcription of the genesneeded for encystation may be activated (88).

Preliminary results from nuclear run-on experiments indi-cated that the expression of encystation-specific genes is reg-ulated primarily at the level of transcription (165). In highereukaryotes, control of gene expression at the level of transcrip-tion is very common. Signals that are generated outside the

cells are transduced to the nucleus through a series of complexinteractions which can either amplify or attenuate gene expres-sion by altering the interaction of transcription factors withtheir cognate targets (151). Although the molecular interac-tions between transcription factors and their cognate geneticsequences are in most cases extremely complex (109, 119, 151),regulation of transcription by cholesterol is relatively simple(46, 47). In mammalian cells, the regulation occurs at the levelof DNA by an element that is 10 bp long, called the sterolregulatory element (SRE) (40, 59). This element is located inthe promoter region of the genes encoding both the LDLreceptor and cholesterol-synthesizing enzymes (7). Recently,two transcription factors which bind to this element mediatingsterol-regulated gene expression were isolated by DNA affinitychromatography of nuclear extracts and named SREBP 1 andSREBP 2 (7, 38, 40, 58, 59, 112, 121, 129, 130, 138, 139, 154,155). These two proteins have been cloned. Their cDNA se-quences showed that they are members of the basic helix-loop-helix-leucine zipper family of transcription factors (40). Bothare synthesized as large (;125-kDa) precursor proteins whichare found as integral membrane proteins of the endoplasmicreticulum (ER). Upon cholesterol starvation, the precursorsare cleaved, releasing an active transcription factor with DNA-binding capability that translocates to the nucleus and activatesgene expression. Active SREBP is degraded rapidly in the nu-cleus in mammalian cells, allowing an instant downregulationof gene expression after a rise in sterol concentration.

Characterization and analysis of two cyst wall protein (CWP1and CWP2) genes, which will be described more fully below,have allowed a more complete understanding of encystation inGiardia. Analysis of the steady-state level of CWP mRNAindicated a coordinate regulation (nearly 140-fold increase)during encystation (Fig. 1A). Interestingly, chromosomal anal-ysis with probes specific for CWP1 and CWP2 demonstratedthat these genes are located in different chromosomes (Fig.1B), indicating that they are not part of the same operon andthat their expression must be regulated at the level of tran-scription or mRNA stability. Moreover, the promoter regions

FIG. 1. CWP1 and CWP2 are encoded by single-copy genes located on G.lamblia chromosomes III and IV, respectively. (A) Southern hybridization of 2mg of G. lamblia genomic DNA digested with BamHI, EcoRI, or SalI with theoligonucleotide probes oMM103[104] for CWP1 and oMM 133[89] for CWP2.Size markers (in kilobase pairs) are shown to the left of the autoradiogram. (B)Southern hybridization of G. lamblia chromosomes separated by orthogonal-field-alternation gel electrophoresis. The locations of the ethidium bromide-stained chromosomes are shown in the right panel, with chromosome designa-tions. Hybridization of the blotted chromosomes with the CWP probes is shownin the left panel. Final posthybridization washes in 0.13 SSC–0.1% SDS wereperformed at 65°C.

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of these two CWP genes share several characteristics which arenot present in any other Giardia gene expressed constitutively(94). For instance, there is a domain of 14 nucleotides locatedin the short 120-nucleotide 59 untranslated region of the CWPgenes (TTCTGGCTAACAGT in CWP1, and TTCTGACAAATAGT in CWP2) that, although different from the SRE of mam-malian genes, might be responsible for responding to the lowlevel of cholesterol in this parasite. Future progress towardunderstanding the molecular basis for the coordinated devel-opmental regulation of these genes will be greatly facilitated bytwo techniques developed recently, nuclear run-on transcrip-tion (106) and DNA transfection (163, 164, 166). Studies inprogress involving these techniques will determine whether acholesterol-regulated process similar to that in mammaliancells occurs during the induction of encystation in Giardia.

SYNTHESIS AND INTRACELLULAR TRANSPORTOF CYST WALL COMPONENTS

Once initiated, encystation proceeds with the synthesis, pro-cessing, and transport of cyst wall constituents and their as-sembly into a protective cyst wall. The mechanisms that controlprotein transport in Giardia are not well understood. In highereukaryotes, for instance, proteins destined for secretion arefolded, assembled, and glycosylated as they are transportedfrom the ER through the Golgi complex to the cell surface (8,128). Some cells are specialized to store secretory materialswithin cytoplasmic organelles called secretory granules (8).These secretory granules release their contents only when cellsare stimulated by an external factor called a secretatogue. Thiskind of secretion is called regulated secretion, and its pathwayis called the regulated pathway (8). The process involved in theformation of the secretory granule in higher eukaryotes can besummarized as three distinct events (2, 102, 150): (i) the se-lective condensation of secretory proteins which aggregate toform a dense core, (ii) the selection of the membranes whichenvelope the aggregate, and (iii) the budding and release of thenascent secretory granule. On the other hand, proteins leavingthe cell by the “constitutive” pathway are not concentrated insecretory granules but are constantly secreted without stimu-lation (8).

Evidence for continuous protein secretion by Giardia is thetransport to the plasma membrane and the release into theculture medium of variant-specific surface proteins (92, 107,108). Regulated protein secretion is exemplified by the forma-tion of the cyst wall during encystation, which is characterizedby the appearance in the trophozoite cytoplasm of those denseencystation-specific vesicles (ESVs) (32). Because most of thecells that specialize in regulated secretion contain secretorygranules during their entire lifetime (2, 8), the biogenesis ofthe secretory granules in higher eukaryotes is difficult to study.The ability of Giardia to regulate the formation of these gran-ules during encystation makes this parasite an excellent modelto study how and where the secretory granules form, howproteins are sorted to and concentrated within these special-ized organelles, and why other proteins are excluded.

Like other members of the order Diplomonadida, Giardia isdevoid of a stack of flattened cisternae which is the hallmark ofthe Golgi apparatus (114, 128). This is a fundamental eukary-otic organelle essential for protein processing and secretion(49, 51, 117, 120). It has been documented, however, thatnonencysting trophozoites secrete nonglycosylated proteins,such as the variant-specific surface proteins, by mechanismssimilar to that of higher eukaryotes (87). In two elegant ultra-structural studies (97, 98), McCaffery et al. found small trans-port vesicles and a series of smooth tubular elements dispersed

throughout the cytoplasm of both encysting and nonencystingparasites and proposed that these elements were part of theGolgi complex. However, since no Golgi-specific markers wereused in these investigations, it was not possible to differentiatethose structures from the smooth ER or transitional elements.By using N-(e-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)(NBD)-ceramide, a fluorescent lipid analog that stains theGolgi apparatus in other cells, we were able to label the Golgiapparatus only in encysting trophozoites (87). During encysta-tion, moreover, Giardia trophozoites underwent a develop-mental induction of Golgi enzyme activities (87). These obser-vations indicate an intimate relationship among thecomposition, structure, and function of the Golgi apparatus.Since one of the principal functions of the Golgi apparatus isthe biosynthesis of complex carbohydrate portions of lipids andproteins (114, 128), cells lacking such enzymes or expressingthem at very low levels may also lack a characteristic Golgistructure. A more extensive Golgi function may be crucial forthe biogenesis of the Giardia cyst wall during encystation, whenthe carbohydrate-rich extracellular cyst wall must rapidly besynthesized. Nevertheless, brefeldin A, a fungal metabolitethat inhibits protein transport and disassembles the Golgi ap-paratus in mammalian cells (70, 116), inhibited protein secre-tion in both nonencysting and encysting trophozoites (87). Thissuggests that despite the absence of Golgi structure in nonen-cysting trophozoites, the intracellular pathway used by proteinswithin these cells resembles that of higher eukaryotes.

Interestingly, the induction of the Golgi structure and car-bohydrate-processing enzymatic activities (Golgi function) inGiardia also correlates with the appearance of ESVs (32, 87),which transport cyst wall components to the plasma membraneof the encysting cell and release their contents to the cellexterior during cyst wall formation (42, 43).

Although reports have clearly established the appearance ofcyst-specific antigens within ESVs during encystation (11, 22,23, 45, 123, 125, 144, 158), only recently have the structure andbiosynthesis of two components of the ESVs been establishedin our laboratory (90, 105). Our approach toward identifyingand characterizing these molecules involved the production ofmonoclonal antibodies (MAbs) to encystation-specific mole-cules. We developed a panel of monoclonal antibodies to bothcyst walls and ESVs and used them to clone genes upregulatedduring encystation. Two of the MAbs are protein componentsof the cyst wall (CWP1 and CWP2 [see above]) (90, 105), andthe other is BiP/GRP78 (20, 50), a molecular chaperone of theER. In addition to serving as diagnostic reagents, the MAbswere powerful tools for the identification of cyst wall compo-nents and for the study of cyst wall biosynthesis and proteintrafficking during encystation.

The CWP1 and CWP2 genes predict acidic and leucine-richproteins with molecular weights of 26,000 and 39,000, respec-tively, targeted to the secretory pathway by amino-terminalsignal peptides. Interestingly, the two proteins share severalcharacteristics. CWP2 and CWP1 possess 61% identity in theamino acid sequence in the overlapping region (90). Bothcontain a cysteine-rich region and five tandem copies of aleucine-rich repeat, and both colocalize within ESVs of encyst-ing trophozoites and in the cyst wall of mature cysts (Fig. 2).Besides being structurally similar, the proteins are inducedwith identical kinetics during encystation. The steady-state lev-els of CWP1 and CWP2 transcripts increased to a maximum ofnearly 140 times that observed in nonencysting trophozoites(90). In contrast, glutamate dehydrogenase (a housekeepingenzyme [163]) mRNA levels remained constant (90, 105). Theseobservations suggest that the dramatic increase in steady-stateCWP1 and CWP2 mRNA levels observed during encystation is



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probably controlled at the level of transcription and/or mRNAturnover. Clearly, the mechanisms responsible for effecting theincrease in CWP mRNA levels as well as for controlling CWPsynthesis remain to be established (see above). The fact thatcholesterol is now known to play a central role in the activationof these genes opens a new approach to further study thisphenomenon.

The ability of CWP1 and CWP2 to form disulfide bonds andtheir colocalization in the ESVs and the cyst wall prompted usto investigate the possibility that the ;65-kDa species ob-served in immunoblots of nonreduced encysting trophozoiteproteins by using MAbs represented a complex of the two cystwall proteins (90). In contrast to their clearly distinguishablepatterns of reactivity in immunoblots, the anti-CWP1 and anti-CWP2 MAbs exhibited identical immunoprecipitation profiles:;26 and ;39 kDa. When supernatants from immunoprecipi-tations were subsequently precipitated with the opposite MAb,the same two bands were detected but at significantly reducedlevels, indicating that CWP1 and CWP2 formed a stable com-plex with each other within 5 min of their synthesis (90).

The most striking difference between these two CWPs is thepresence of a 121-amino-acid carboxy-terminal extension inCWP2. The alkaline nature of that tail (pI 5 12.23) predicts ahigh net positive charge in this domain at physiological pH,suggesting an electrostatic predilection for an anionic receptormolecule. It is known that many proteins contain clusters ofbasic amino acids that can bind to acidic phospholipids, whichare located preferentially on the cytoplasmic surface of cellularmembranes (55, 135, 168) or acidic proteins (9, 80). Severalextrinsic proteins probably use electrostatic interactions formembrane attachment (9, 55, 80, 168). For CWP2, an anionic“receptor” might be luminally disposed at the membranes of

the ER or at a post-ER compartment, allowing it to be con-centrated at specific regions of these membranes (the secondstep in granule formation as described above).

Secretory granules in higher eukaryotes form in the trans-Golgi network (8, 49, 114, 128); however, it is unclear whetherESVs in Giardia form in the trans-Golgi network or by con-densation within the ER (43, 90). In eukaryotic cells, the ERprovides the requisite environment for the folding and oli-gomerization of secretory proteins with the assistance of resi-dent molecular chaperones such as BiP, calnexin, and proteindisulfide isomerase (20). Two recent reports (93, 137) showedthe first visualization of the ER of this protozoan by immuno-electron and immunofluorescence microscopy with polyclonalantibodies and MAbs, respectively. Moreover, our MAb spe-cific for Giardia BiP showed that BiP expression increasedsimultaneously with the increased expression of CWPs duringencystation (93). These results, considered in conjunction withthe profound changes that occur in the secretory pathway dur-ing Giardia encystation (90, 97), point to an important role forBiP during the differentiation of this primitive eukaryote, mostprobably that of functioning as a molecular chaperone of theCWPs. BiP probably maintains these proteins in a solubleform, inhibiting an early and unproductive polymerization. Weproposed that oligomerization or aggregation of CWP1-CWP2-receptor complexes could result in ESV budding (90).The formation of ESVs in this case could be a direct conse-quence of the synthesis of the CWPs, especially CWP2, andtheir trafficking through the developmentally induced secre-tory pathway of encysting trophozoites. Thus, it is important tounderstand whether the basic tail in CWP2 plays a role in CWPsorting into ESVs and, if so, the detailed molecular mechanismby which it occurs.

FIG. 2. CWP1 and CWP2 colocalize to the ESVs and the cyst wall. Laser-scanning confocal immunofluorescence images of encysting trophozoites (bottom panels)and cysts (top panels) are shown. The cells were labeled simultaneously with fluorescein-conjugated anti-CWP1 MAb 5-3C (left panel) and rhodamine-conjugatedanti-CWP2 MAb 7D2 (right panel). Superimposition of left and right panels is shown in the center panels.



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It will also be very interesting to find how many CWPs arepresent in the cyst wall. Several MAbs and polyclonal antibod-ies against Giardia cysts have been generated previously (11,44, 131, 143, 158). A comprehensive analysis of the literatureshows that all cyst-specific antigens recognized by those anti-bodies may be placed into four different groups: proteins withapparent molecular masses of (i) 25 to 30 kDa (CWP1 andCWP2?), (ii) 36 to 40 kDa (CWP2?), (iii) 50 to 65 kDa(CWP1 1 CWP1 5 52 kDa; CWP1 1 CWP2 5 65 kDa?), and(iv) higher than 70 kDa. CWP2 was probably among the anti-gens detected in previously reported studies. By immunoblot-ting experiments with MAb 8C5, McCaffery et al. (98) showeda pattern of expression in encysting trophozoites similar to thatobtained with our MAb 7D2: an early ;44-kDa predominantband and the subsequent appearance of two other lower-mo-lecular-mass bands after 5 h of encystation. Moreover, theseauthors also noted that the antigens recognized by MAb 8C5seemed to be the same as those identified by MAb CGSA-1(29, 36, 39, and 45 kDa), which was described by Ward et al.(158). Under reducing conditions, MAb 8C5 recognized a sin-gle ;26-kDa band in cyst walls that were obtained by boilingwater-resistant cysts in sodium dodecyl sulfate (SDS). Basedon those results and our own observations, it is possible thatthe Giardia cyst wall contains only two cyst proteins, CWP1and CWP2, and that different degrees of posttranslational pro-cessing (e.g., glycosylation, proteolysis, and cross-linking) ac-count for the variation in relative mobilities of the antigensthat is observed during trophozoite encystation.

In addition, the almost identical morphologies and antige-nicities shared by the cyst walls of several different Giardiastrains isolated from different animals, different Giardia spe-cies (e.g., G. lamblia and G. muris), and even different genera(e.g., Spironucleous sp.) (33, 62) suggest the possibility thatthese proteins are common components of the cyst walls of avariety of intestinal parasitic protozoa. For these reasons, theprobes and reagents developed for Giardia could be useful inunderstanding cyst wall formation in related parasites of med-ical and veterinary importance.

ASSEMBLY OF THE CYST WALL

Several explanations have been proposed to clarify themechanisms involved in vesicle release and cyst wall formation

(28, 42, 43). Reports have indicated that cyst wall componentsare released by exocytosis; however, the way in which therelease occurs is still undefined. The difficulties encountered byus and others in observing vesicle release by electron micros-copy may be due to the rapidity and synchrony of this process.Therefore, what is the stimulus for vesicle release, and how isit sensed by the vesicles?

Near the dorsal surface of Giardia, there are a series of“lysosome-like” structures (34, 39, 67) called peripheral vacu-oles (PVs), which in cross section range in shape from circularto oval but when viewed transversely appear as a complexsystem of interconnected tubules or canals. They tend to alignin a single row, separating the granular cytoplasm from theplasma membrane. With the exception of the ventral disk,similar vacuoles have been seen near the dorsal surface (39).The fact that these vacuoles contain acid phosphatase (35) andcan take up ferritin (4) suggested that they could be involved infood degradation. Several authors have suggested that thesevesicles might be involved in the encystation process because oftheir similarity to secretory organelles that are found in othercyst-forming protozoa (34, 39, 67). An important observationthat can help in the interpretation of the function of PVsduring encystation is the fact that, in purified ESVs, CWP2 wasfound predominantly as a ;39-kDa protein (26 kDa from theCWP1-like region plus ;13 kDa from the basic tail). In puri-fied cyst walls, however, only a 26-kDa fragment could befound (90, 98), indicating that proteolytic processing of CWP2occurred before its incorporation into the cyst wall. Since PVsare known to contain proteolytic enzymes (82, 161), they mayparticipate in CWP2 cleavage after interaction between thetwo organelles. Electron microphotographs showed an inti-mate contact between ESVs and PVs (Fig. 3A), which maysupport this model. During cyst wall maturation, cyst wall an-tigens can also be seen inside the PVs (Fig. 3B); this could bedue either to the fusion of ESVs with PVs immediately beforethe release of vesicle content or to endocytosis of cyst wallmolecules during membrane recycling after release (61). Usinga MAb generated against Giardia cysts by Campbell and Faub-ert (MAb 8C5) (11), McCaffery et al. (97, 98) also showed thepresence of cyst wall antigens within PVs of encysting tropho-zoites. These authors suggested that PVs might be involved incyst wall formation and that their function could be related tothe storage or processing of cyst wall materials. Interestingly,

FIG. 3. CWP2 is concentrated in the ESVs before being incorporated into the cyst wall. Immunoelectron microscopic detection of CWP2 in encysting trophozoites(A) and a maturing cyst (B) with MAb 7D2 are shown. (A) An area of an encysting trophozoite revealing CWP2 localization in large electron-dense ESVs in closecontact with the PV. (B) An area of a maturing cyst showing the presence of CWP2 within the PVs. A portion of a 24-h in vitro-derived cyst shows a gold labelthroughout the cyst wall (CW) that surrounds the trophozoite. Lysosome-like PVs are also observed. Electron-dense ESVs containing CWP2 form from a cleft (arrow).Glycogen, which is abundant in encysting trophozoites and cysts, was extracted by the immunostaining procedure. Magnification, 330,600.



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MAb 8C5 binds recombinant CWP2 in Western blots (94). Analternative explanation for the processing of CWP2 is the pos-sibility that, like specific secretory granules of hormone-pro-ducing cells (3), the ESV contains a processing enzyme whichgenerates the final processed product before release.

The biosynthesis and assembly of eukaryotic extracellularsuperstructures such as the plant and fungal cell walls (48, 71,126, 145, 146) and the cyst or spore walls of medically impor-tant intestinal pathogens (101) are not completely understood.How Giardia generates a highly ordered supramolecular cystwall is a fascinating problem that can be taken as a model forthe study of cell wall morphogenesis in general. Synthesis ofthe cyst wall poses interesting topological problems becauseprecursors must be synthesized intracellularly but depositionof the components and all macromolecular organization mustoccur outside the permeability barrier of the cell. Therefore,there must be special mechanisms to convey the precursors andthe machinery for cyst wall assembly to the cell surface whileprohibiting their polymerization within the cell. Several fea-tures of cyst wall assembly are of interest also because thesynthesis, posttranslational modifications, cross-linking, glyco-sylation, and fibrillar deposition of cyst wall proteins might bethe target of chemotherapeutic attack to prevent the transmis-sion of the disease.

Initially, answers to the following questions may help tounderstand cyst wall morphogenesis. Which molecules arepresent in the cyst wall? How do these molecules interact togive rise to the final cyst wall architecture? What is the functionof the individual components in the cyst wall?

Morphologically, the Giardia cyst wall is composed of anouter filamentous portion and an inner membranous portion(27, 28, 132, 133). The inner cyst wall consists of two mem-branes which border the peritrophic space. The outer cyst wallis 0.3 to 0.5 mm thick and is composed of filaments that rangebetween 7 and 20 nm in diameter (26, 27). Erlandsen et al.(28), using a combination of immunogold labeling and high-resolution field emission scanning electron microscopy, haveperformed a chronological study of the cyst wall formation inGiardia. This analysis showed that the process begins with theappearance of cyst wall antigens on small protrusions of thetrophozoite surface, which grow up to form caplike structuresapproximately 100 nm in diameter. Interestingly, these struc-tures were observed covering the entire surface of the tropho-zoite, including the adhesive disk and flagella, and were in-volved in filament formation. Unfortunately, this sophisticatedstudy could not resolve how the cyst wall filaments grow toform the rigid cyst wall. The filamentous nature of the cyst walland its constant thickness (0.25 to 0.30 mm) suggest that itforms from successive layers of cyst wall materials (26, 28).How is this new material added to the wall? In other words,does the cell wall grow by addition of newly secreted materialson top, at the internal surface of the wall, or without organi-zation? Is it possible that successive layers arise from succes-sive waves of vesicles that fuse upon contact with the plasmamembrane? Are all the vesicles released at the same time? Ifthe last is true, why does the trophozoite not increase its sizeupon the addition of the membrane of the vesicles to theplasma membrane of the cell? Does a rapid mechanism ofmembrane recycling take place during or after exocytosis ofthe ESVs (97, 98), or will the membranes of the ESVs nowform the peritrophic space that surrounds the trophozoite inthe mature cysts?

Biochemical analyses suggest that the cyst wall has bothcarbohydrate and protein components (63, 65, 95, 96, 111, 156,157). An interesting feature of CWPs is the presence ofleucine-rich repeats (LRRs). LRRs are found in a functionally

diverse group of proteins related by their ability to participatein protein-protein interactions (72, 73, 153). LRRs are be-lieved to confer conformational flexibility upon the proteins inwhich they reside, thereby promoting protein-protein interac-tions (6, 21, 72, 73, 110, 142, 159).

The LRR consensus sequences of the Giardia CWPs mostclosely resemble those found in the extracellular domain ofplant transmembrane and extracellular matrix proteins (75, 76,110). These LRRs are characterized by absolutely conservedglycine and proline residues, a feature that distinguishes these24-residue LRRs from other 24-residue LRRs, including smallproteoglycans of the mammalian extracellular matrix (76).LRR regions may serve as flexible domains that allow or fa-cilitate the dimerization of the CWPs (90). The presence ofLRRs in G. lamblia cyst walls also suggests a possible role ofthese domains in conferring flexibility and, simultaneously, sta-bility to this superstructure. Since the earlier work of Filice(37), several reports have indicated the flexible nature of thecyst wall (16, 133). The filamentous structure of this wall pos-sesses sufficient flexibility, since trophozoites can move withinthe cyst and deform the cyst wall and also can pass throughindividual pores with diameters smaller than that of the cystitself (132). Additionally, the cyst wall is rigid enough to keepits shape after the excysted trophozoite(s) has escaped (36),when the cyst is immersed in distilled water (1, 2), or duringcyst wall purification (90). Although the involvement of LRRsin cyst wall fibrillar assembly has not been directly demon-strated, several lines of evidence point in that direction. Forinstance, protein-protein interaction is directly attributable tothe LRR region in the Saccharomyces cerevisiae CCR4 protein,a glucose-regulated transcription factor (21). It was shown thatthe LRR region was both necessary and sufficient for interac-tion of CCR4 with at least two other cellular proteins. More-over, a point mutation or a deletion within LRRs in the a-subunit of human platelet glycoprotein Ib resulted in aninability of this protein to bind von Willebrand factor (159).Further characterization of the function of the LRR and struc-tural analysis of CWPs in Giardia will provide new informationnot only about the assembly of the cyst wall but also about theimportance and the evolution of LRRs in nature.

After SDS treatment, a fraction of the cyst wall remainsinsoluble; however, purified cyst walls can be dissolved com-pletely by the addition of disulfide reducing agents (94), indi-cating the presence of disulfide cross-linking of the wall con-stituents. Many components of the mammalian extracellularmatrix (e.g., fibronectin and collagen type VI) exhibit similarcharacteristics (99). Fibronectin dimers are formed rapidly af-ter synthesis by oxidation of cysteine residues, and our datasuggest that this also occurs in CWPs (90).

Manning et al. (96) identified galactosamine as the predom-inant sugar associated with the filamentous component of theGiardia cyst wall and provided compelling data that refuted thepresence of chitin as a major structural component (156). Theabundance of GalNAc, together with the insolubility of the cystwall, suggested the presence of N-acetylgalactosamine in apolymerized form in this structure. CWP1 and CWP2 eachcontain a single N-glycosylation site (90, 105). No publishedevidence supports the presence of N glycosylation in Giardia.Additionally, carbohydrate analysis of a purified variant-spe-cific surface protein of Giardia that contains two potentialN-glycosylation sites showed that it is not glycosylated (92).The profusion of Gal and GalNAc in the cyst wall, the abun-dance of potential sites of O glycosylation in the CWPs (CWP1and CWP2 are rich in serine and threonine; together, these twoamino acids comprise 14% of the residues in each protein),their altered mobility in SDS-polyacrylamide gel electrophore-



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sis late in encystation (90, 105), and the induction of galac-tosamine and GalNAc transferase activities in encysting cellssuggest that the CWPs may be glycosylated. Therefore, futurecarbohydrate studies on purified proteins will generate impor-tant information about the process of glycosylation in Giardiaand its place in the evolution of eukaryotic organisms.

In addition to the glycosylation sites present in CWP1 andCWP2, another interesting feature of both proteins is the pres-ence of several sites for phosphorylation and myristolation anda cysteine-rich carboxy-terminal domain with homology to thecysteine-rich, zinc-binding VSPs of Giardia (90, 105). Whetherlipids and/or phosphate groups are attached to these proteinsis a matter for speculation. However, if myristate is found to beattached to CWPs, it will be an interesting mechanism forprotein sorting during ESV formation or for CWP attachmentto the surface of the cell after their release to the cell exterior(100). In addition, the cysteine-rich domain may share thecoordination of metal ions with the VSP present on the cellsurface of the trophozoite (92, 107, 108), allowing CWPs toremain attached to the surface of the cell after vesicle release.On the other hand, since it was suggested that acid phospha-tase activity is involved in cyst wall disassembly during excys-tation (36), the presence of phosphate moieties in the cyst wallmight be related to the conferring of stability to this extracel-lular superstructure.

CONCLUSION

Giardia belongs to the earliest identified lineage among eu-karyotes; therefore, an understanding of its cellular, biochem-ical, and molecular processes offers a unique insight into theprogression from primitive to more complex eukaryotic cells.Giardia encystation appears to be the most primitive adaptiveresponse developed by eukaryotes to survive under adverseenvironmental conditions. Many questions about this phenom-enon have been answered, but several more must be addressedin the future. It is clear at this point that further elucidation ofthe biological mechanisms used by Giardia will allow us tounderstand more clearly the evolution of fundamental cellularprocesses of higher eukaryotic cells, such as signal transduc-tion, control of transcription and translation, vesicular trans-port, and extracellular matrix formation. Comprehension ofthese mechanisms will also provide important information fordeveloping chemotherapy targeting this parasite-specific met-abolic pathway.

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