Identification of Chitin as a Structural Component of Giardia CystsHONORINE D. WARD,' JOSEPH ALROY,2 BOAZ I. LEV,' GERALD T. KEUSCH,' AND
MIERCIO E. A. PEREIRA'*Division of Geographic Medicine, Departments of Medicine' and Pathology,2 Tufts University School of Medicine,
Boston, Massachusetts 02111
Received 8 April 1985/Accepted 3 June 1985
The intestinal parasite Giardia lamblia is a significant cause of diarrheal disease, which is perpetuated by theinfective cyst form of the parasite. Although a rational approach to the control of giardiasis would be to inhibitcyst formation, nothing is known of the chemical composition of the cyst wall or of its biosynthesis. In thesestudies, we have shown that chitin is a major structural component of G. lamblia and G. muris cyst walls. Thisconclusion is based on the finding that chitinase specifically destroys the cyst wall, as revealed by electronmicroscopy. The presence of chitin was also shown directly by lectin binding studies. Of 12 lectins with diversecarbohydrate recognition specificity, only the N-acetylglucosamine-specific lectins wheat germ agglutinin,succinylated wheat germ agglutinin, and tomato lectin bound to cyst walls, as shown by fluorescencemicroscopy and cytochemistry. Wheat germ agglutinin binding was completely abolished by treatment of thecysts with purified chitinase. This effect was specific since it could be prevented by incubating the enzyme withchitin before treatment of the cysts. Treatment of cysts with N-acetyl-,-glucosaminidase partially inhibitedwheat germ agglutinin binding, whereas other glycosidases and proteases had no effect. These findings indicatethat chitin is a major structural component of Giardia cyst walls and raise the possibility that inhibitors of chitinsynthesis may be of use in preventing encystation and thus controlling spread of the disease.
The protozoan parasite Giardia lamblia is a significantglobal cause of endemic and epidemic diarrhea. Transmis-sion of the disease occurs by ingestion of the infective cyst,either in contaminated food or water or by direct person-to-person contact via the orofecal route (10, 15). Excystationtakes place in the stomach or proximal duodenum, releasingthe motile trophozoite, which colonizes the proximal smallintestine and causes the disease. Encystation occurs pre-sumably in the distal small intestine by an unknown mech-anism. Cysts are excreted in the feces into the externalenvironment. Unlike trophozoites, which cannot surviveoutside the host, the cysts can withstand adverse conditionsfor prolonged periods and are thus critical in maintaining thelife cycle of the parasite. The relatively thick cyst wall isbelieved to contribute to the resistance of the cyst toextreme environmental conditions. Although the process ofexcystation can readily be reproduced in vitro (7), it has notbeen possible to induce cultured G. lamblia trophozoites toencyst. Prevention of encystation would provide an effectivemeans of interrupting the life cycle of the parasite andcontrolling the spread of the disease. Knowledge of themechanism of cyst formation is therefore essential in design-ing strategies directed towards the prevention of encysta-tion. Information regarding the chemical composition of thecyst wall is a fundamental prerequisite of understanding theprocess of encystation. However, although ultrastructuralfeatures of the cyst wall have been described (21), virtuallynothing is known of its chemical composition.As a prelude to understanding the mechanism of encysta-
tion, we have analyzed the sugar composition of Giardiacyst walls with the aid of sugar-binding proteins andglycosidases of known specificity. We show here that chitinis a major constituent of the cyst walls of G. lamblia and G.muris, as determined by studies of lectin binding andglycosidase digestion of partially purified cysts observed bylight and electron microscopy.
* Corresponding author.
MATERIALS AND METHODS
G. lamblia cysts. G. lamblia cysts were partially purifiedfrom feces of infected patients from the Tufts-New EnglandMedical Center and Children's Hospital Medical Center,Boston, Mass., by a modification of the method of Sheffieldand Bjorvatn (21). Fecal specimens (10 g) were emulsified inwater and filtered through several layers of gauze, and thefiltrate was centrifuged at 400 x g for 5 min. The sediment wassuspended in 5 ml of water, layered on a discontinuousdensity gradient consisting of 5 ml each of 1.5, 1.0, 0.75, and0.5 M sucrose, and centrifuged at 1,000 x g for 30 min at 4°C.Cysts were collected from the water-0.5 M sucrose and the0.5 M-0.75 M sucrose interfaces. The cyst suspension wasdiluted 10-fold with water and centrifuged at 400 x g for 5 min.The supernatant was discarded, and the pellet was washedthree times with 0.02 M phosphate buffer (pH 7.2) with 0.15M sodium chloride (PBS). Cysts were suspended in PBS toa final concentration of 106 cysts per ml and used within 24h.
G. muris cysts. G. muris cysts were isolated from feces ofinfected CF-1 mice by a modification of the method ofRoberts-Thomnson et al. (20). Stools from six CF-1 mice,infected 7 to 10 days earlier with 2,000 cysts of G. muris,were collected for 2 h, broken up in tap water, and filteredthrough several layers of gauze. Filtrate (3 ml) was layeredon 2.5 ml of 1 M sucrose and centrifuged at 400 x g for 15min at 4°C. Cysts were collected from the sucrose-waterinterface, diluted 10-fold with water, and centrifuged at 600x g for 5 min at 4°C; the pellet was suspended in 6 ml ofwater and recentrifuged on 1 M sucrose. Cysts at theinterface were diluted 10-fold with water and centrifuged at600 x g for 10 min, and the pellet was washed three times inPBS. Cysts were suspended to a final concentration of 106cysts per ml and used within 24 h.
FITC-lectin binding studies. All fluorescein isothiocyanate(FITC)-lectins were purchased from Sigma Chemical Co.,except for tomato lectin (TOL), which was purified fromtomatoes bought at local supermarkets as described previ-
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ously (16). For fluorescein conjugation, TOL was dissolvedin a borate-bicarbonate buffer (pH 8.9) at 8 mg/ml, FITC wasadded to a final concentration of 50 ,uglml, and the mixturewas allowed to react for 20 h at 4°C in the dark. Theconjugated lectin was separated from unreacted FITC on aBio-Gel P-10 column (10 by 1 cm) equilibrated with PBS andstored at 4°C with 0.02% sodium azide as preservative. Theconjugated lectin (1.8 mol of FITC per mol of lectin) had anagglutinating activity indistinguishable from that of the un-conjugated hemagglutinin.
(i) Unfixed cysts. A total of i05 washed G. lamblia or G.muris cysts were incubated with 10 ±g of FITC-labeledlectins in 100 ,u1 of PBS at 23°C for 30 min. The cysts werethen washed twice in PBS, suspended in 100 R1 of PBS, andexamined with a Zeiss ICM microscope with phase-contrastand fluorescence optics. Fluorescence was arbitrarily gradedas - (no apparent fluorescence), + (weak fluorescence), or+ + (strong fluorescence). The inhibition of FITC-lectinbinding to cysts was studied by mixing the FITC-lectin withthe following specific sugar inhibitors at room temperaturefor 30 min and then incubating the mixture with the cysts asdescribed above: 100 mM N-acetyl-D-glucosamine (GIcNAc)and 1 mM N,N',N",N"'-tetraacetylchitotetraose for wheatgerm agglutinin (WGA) and TOL; 100 mM N-acetyl-D-galactosamine (GalNAc) for phytohemagglutinin, soybeanagglutinin (SBA), and Helix pomatia agglutinin; 100 mMD-mannose for concanavalin A and Lens culinaris agglutinin;and 100 mM D-galactose (D-Gal) for peanut agglutinin.
(ii) Fixed cysts. A 25-pI volume of suspension containing106 cysts per ml in PBS was placed on a glass slide, air dried,and fixed in methanol for 10 min at 23°C. Slides were washedin PBS for 10 min and then incubated with 25 ,ul of FITC-lectin (WGA, 50 ,ug/ml; TOL, 20 ,ug/ml; other lectins, 100,ug/ml) in PBS for 30 min at 23°C. The slides were thenwashed in PBS, mounted in PBS containing glycerol (10%[vol/vol]), and observed under the microscope. Inhibitionstudies were performed by preincubating the FITC-lectinwith its specific sugar inhibitor as described above. Fluores-cence was graded as for the unfixed cysts.
Cytochemical visualization of lectin binding. Lectin bindingto Giardia cysts was also visualized by cytochemical stain-ing by the avidin-biotin-peroxidase technique (2). A total of2.5 x 104 washed cysts were placed on a glass slide, airdried, and fixed in methanol as described above for fixedcysts. Endogenous peroxidase activity was blocked by incu-bating the cysts in 2% hydrogen peroxide (i.e., by activationof the endogenous enzyme in the absence of the chromogensubstrate). The fixed tissue was incubated with 100 ,ug offiltered mouse liver powder (Cappel Laboratories) per ml inPBS for 10 min (to reduce nonspecific binding), washed withPBS, and then incubated for 30 min at 230C with thefollowing biotinylated lectins: WGA, 50 ,ug/ml; succinylatedWGA (S-WGA), 20 p.g/ml; concanavalin A, 10 ,ug/ml; peanutagglutinin, 10 ,ug/ml; Ricinus communis agglutinin I, 10,ug/ml; Dolichos biflorus agglutinin, 10 ,ug/ml; SBA, 10p.g/ml; Ulex europaeus agglutinin I, 10 ,ug/ml; and Bandeireasimplicifolia agglutinin, 10 ,ug/ml (all from Vector Laborato-ries, Inc.). Mouse liver powder (100 ,ug per slide) was thenadded, and the slides were incubated for another 30 min andwashed three times for 5 min each time with PBS. Slideswere then incubated with avidin-biotin-peroxidase complex(Vector) as described previously (2). Avidin binds to thebiotinylated lectin as well as to the biotin-labeled peroxidaseto form a stable complex (2). Peroxidase was then visualizedby incubation with PBS containing the chromogen substrate3,3'-diaminobenzidine tetrahydrochloride (Sigma) and hy-
drogen peroxide, which gives a dark-brown reaction prod-uct. Finally, the slides were washed in tap water for 5 min,counterstained with methyl green, and dehydrated, andcover slips were attached with Permount. Positive stainingwas demonstrated by the dark-brown reaction product.
Glycosidase treatment of fixed cysts. A total of 2.5 x 104cysts per ml were fixed on glass slides in methanol asdescribed above and incubated in a moist chamber with thefollowing glycosidases in PBS: N-acetyl-p-glucosaminidasefrom Jack beans (Sigma), 2.75 mU, for 48 h at 23°C;muramidase (lysozyme-mucopeptide N-acetyl muramylhydrolase [Sigma]) from chicken egg white, 40 U, for 30 minat 23°C; neuraminidase from Vibrio cholerae (Calbiochem-Behring), 25 mU, for 30 min at 37°C; chitinase from Strep-tomyces griseus (Sigma), 75 mU, for 24 h at 230C; andpurified chitinase from Serratia marcescens (a gift fromEnrico Cabib, National Institutes of Health, Bethesda, Md.),45 mU, for 24 h at 23°C. As a control for the variousenzymes, cysts were incubated with equal volumes of PBSfor similar periods of time. Another control involved inhibi-tion of chitinase activity (by preincubating the enzyme withan equal volume of 50% purified crab shell chitin in PBS for30 mmin at room temperature) before incubation with thecysts.The slides were then washed twice with PBS and reacted
with FITC-WGA as described above for the fixed cysts orwith biotinylated WGA as described above for cytochemicalvisualization of lectin binding.
Electron microscopy studies. A total of 5 x 105 washedcysts were incubated with 50 ,ul of purified Serratia marces-cens chitinase at a concentration of 1.8 U/ml for 18 h at 23°C.The cysts were then washed twice in PBS, fixed in Trumpsfixative (1% glutaraldehyde, 4% Formalin) in PBS, andpostfixed in 2% aqueous osmium tetroxide. Specimens weredehydrated in graded ethanol, stained en bloc with 5.0%uranyl acetate, and embedded in Epon 812. Sections wereviewed with a Philips 300 electron microscope.
RESULTSSelective reaction of GlcNAc-binding lectins with cysts of G.
lamblia and G. muris. A systematic analysis of surfacecarbohydrate residues on the cyst walls of G. lamblia and G.muris was undertaken with 12 lectins of various sugarspecificities (Table 1). The most notable feature was theexclusive binding of WGA and TOL, two GlcNAc-bindinglectins (1, 18), to the cyst walls of both Giardia species, asdemonstrated by fluorescence microscopy (Fig. 1) or bycytochemistry (Fig. 2). The binding of WGA and TOL wasvia their carbohydrate binding sites, since it was completelyabolished by preincubation of the lectins with the specifichapten N,N',N",N"'-tetraacetylchitotetraose. No differ-ence in reactivity was detected between cysts of G. lambliaand G. muris or between freshly harvested and methanol-fixed cysts (data not shown). Lectins with specificity for ot-or P-D-Gal (peanut agglutinin, SBA, B. simplicifolia aggluti-nin, and R. communis agglutinin I), oa- or P-D-GalNAc(phytohemagglutinin, D. biflorus agglutinin, H. pomatiaagglutinin, and SBA), cc-D-mannose and a-D-glucose (ConAand LCA), and cc-L-fucose (UEA-I) did not react with eithercyst.
Binding of WGA and TOL to Giardia cyst walls mediated byGlcNAc residues. The carbohydrate specificity of WGAincludes terminal sialic acid as well as GlcNAc and itsp1->4-linked oligomers (1, 6). However, the binding of WGAto sialic acid and GlcNAc residues may be distinguished bysuccinylation of the lectin (17), since S-WGA binds only to
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TABLE 1. Lectin binding to G. lamblia and G. manris cyst walls
Binding assessed by:Lectin" Major sugar specificity" Fluores- Cyto-
D-Gal at or Pl1-RCA-I D-Gal a or P1-s NDBS-I D-Gal al-s NDConA D-Man aol-; D-Glc axl-s1LCA D-Man al-; D-Glc aol- - NDUEA-I L-Fuc aol- ND -
" PNA, Peanut agglutinin; PHA, phytohemagglutinin; DBA, D. bfflorilsagglutinin; HPA, H. pomtia agglutinin; RCA-I. R. c(otlmmoiis agglutinin l;BS-l, B. sirnplicifolia agglutinin; ConA, concanavalin A: LCA, L. cuilinarisagglutinin; UEA-1, U. eiuropaeois agglutinin I.
b See reference 19. NeuNAc, Sialic acid; D-Man, D-mannose; D-Glc,D-glucose; L-Fuc, L-fucose.
' Lectin binding to fixed as well as unfixed cysts was assessed by fluores-cence microscopy as described in the text. Fluorescence was arbitrarilygraded as + + (strongly positive), + (weakly positive), or - (negative). ND,Not done.
d Lectin binding to fixed cysts was assessed by cytochemistry as describedin the text. Positive staining was indicated by a dark-brown reaction product.
GlcNAc and not to sialic acid (17). S-WGA reacted with theGiardia cysts in a manner indistinguishable from that ofWGA (Table 1), strongly suggesting that the cyst WGAreceptors contain GlcNAc residues. This possibility is inaccord with the finding that extensive neuraminidase diges-tion of the cysts did not affect WGA binding (Table 2) andwith the results obtained with TOL, since oligomers of13l-s4-linked D-GlcNAC are the only saccharides known toreact with this lectin (16, 18).
Binding of WGA to cyst walls is specifically abolished bychitinase. To directly demonstrate the nature of the WGAreceptors, Giardia cysts were treated with various glyco-sidases of known sugar specificity and then assayed for their
FIG. 1. FITC-WGA binding to fixed G. muris cysts (x 1,440).
FIG. 2. Cytochemical visualization of biotinylated WGA bindingto fixed G. laImblia cyst by the avidin-biotin-peroxidase method(x 1,440).
ability to bind WGA. Chitinase digestion completely elimi-nated WGA binding to the cyst walls of the two Giardiaspecies, whereas digestion with other glycosidases did noteliminate such binding (Table 2, Fig. 3). A commerciallyavailable chitinase from Streptomyces griseuis, which maybe contaminated with other glycosidases and with proteases,gave the same results as an affinity-purified chitinase fromSerratia marcescens. In either case, the effect of chitinasewas fully prevented by mixing the enzyme with chitin beforeincubation with the cysts (Table 2). N-Acetyl-p3-glucosaminidase, which cleaves terminal 1-linked GIcNAcresidues, partially inhibited WGA binding to the cysts.Digestion of the cysts with proteases such as trypsin (25,ug/ml, 30 min at 37°C) did not affect WGA binding, a resultwhich is in accordance with the view that WGA reacts witha polysaccharide on the cysts. Taken together, these resultstherefore identify chitin as the receptor for WGA on the cystwalls of G. lamblia and G. miuris.
Digestion of the Giardia cyst wall by chitinase. Sincechitinase treatment of the cysts abolished WGA binding,electron microscopy studies were performed to visualize theultrastructural effects of the enzyme treatment. G. Iambliacysts which had been treated with PBS as a control dis-
TABLE 2. Chitinase treatment specifically abolishes WGAbinding to G. lamblia and G. mlaris cysts
Fixed cysts were treated with various glycosidases or controls as de-scribed in the text and subsequently assessed for WGA binding by fluores-cence microscopy or cytochemistry. See Table 1 for grading of fluorescence.
h ND, Not done.
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FIG. 3. Complete inhibition of biotinylated WGA binding tofixed G. lamblia cyst by chitinase digestion. The negatively stainedcyst is indicated by an asterisk (x 1,440).
played an ultrastructure similar to that described by Sheffieldand Bjorvatn (21). The cyst wall contained thin fibrilsinterspersed with fine granular material (Fig. 4). Examina-tion of chitinase-treated cysts revealed a striking alterationof the cyst wall. The outer limiting margin of the wallcompletely disappeared, leaving a sparse amount of granularmaterial in a disorganized array (Fig. 5). This finding wastypical for the majority of chitinase-treated cysts.
DISCUSSIONThis study demonstrates for the first time that the Giardia
cyst wall is composed largely of chitin. This conclusion isbased on the finding that the cyst walls of both G. lamblia
I
V
and G. muris were destroyed in a specific manner by theenzyme chitinase, as revealed by ultrastructural studies (Fig.5). The presence of chitin on the cysts was also demon-strated by lectin binding studies. Only those lectins withspecificity for D-GlcNAc residues, namely, WGA, S-WGA,and TOL, were capable of reacting with the cysts, as shownby fluorescence microscopy and cytochemistry (Fig. 1 and2). WGA is known to recognize sialyl and GlcNAc residuesin glycoconjugates in solution or on cell membranes (1, 6),but the succinyl derivative of the lectin, due to its negativecharge, reacts only with GlcNAc residues (17). The identicalreaction of the native and succinyl lectins with the cysts andthe lack of an effect of preincubation of the cysts withneuraminidase are therefore in accord with the concept thatchitin is the structure recognized by WGA. This conclusionis also supported by the results with TOL, since this lectin isspecific for ,1l->4-linked polymers of D-GIcNAc (16, 18). Itis of interest that WGA was found to react selectively withtrophozoites of G. lamblia (11); however, the relationshipbetween the WGA receptors on the trophozoites, whichremain unknown, and the chitin we demonstrated on thecysts of the same parasite species needs to be clarified.
Chitin is a linear polysaccharide composed of chains of,1-*4-linked GIcNAc units. It is a major structural compo-nent of yeasts, fungi, insects, and crustacea (9, 13). Itspresence has also been reported in a number of ciliates (8), inthe egg envelope of helminths (12), and recently in the cystwall of Entamoeba invadens, an intestinal parasite of snakes(3, 4). Chitin chains associate by the formation of very stronghydrogen bonds between the >N-H groups of one chainand the C=O groups of the adjacent chain, which accountsfor the extreme insolubility of chitin in most solvents. Thepresence of chitin in the cyst wall of protozoa such asEntamoeba and Giardia species may thus explain the resist-ance of cysts to harsh environmental conditions.
Chitin is synthesized from the substrate UDP-GlcNAc by
I
ew
\ 7PM
V V
....-,y. . \Ka
FIG. 4. Electron micrograph of G. lamblia cyst. The cyst wall (CW) is distinctly demarcated. The plasma membrane (PM), peripheralvacuoles (V), and microtubules of the suction disc (MT) are also seen (x52,400).
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Cw
PM
*''~V' V
.,0'sR.,V
FIG. 5. Electron micrograph of G. lamblia cyst treated with chitinase. The outer margin of the cyst wall (CW) has disappeared, leavinga sparse amount of granular material (x52,400). See Fig. 4 legend for definitions of abbreviations.
the action of chitin synthetase (9). Since chitin is not presentin mammals, including humans, it is an ideal target forstrategies directed towards preventing its synthesis. Such astrategy has been successfully employed in the use of chitinsynthesis inhibitors such as Polyoxin and Dibenzfluron asinsecticides (14). Recently, Avron et al. have shown thatboth Polyoxin and Nikkomycin, which are structural ana-logues of UDP-GlcNAc, inhibit the formation of E. invadenscysts in vitro and also prevent the incorporation of radiola-beled [3H]GlcNAc into cysts (5).
Since chitin appears to be a major structural component ofGiardia cyst walls, it is possible that chitin synthesis may bea crucial step in the encystation process of this parasite. Theconcept should be investigated by determining whetherchitin synthesis inhibitors are effective in preventing cystformation in vivo. If so, it would represent a new approachin the study of the mechanisms of Giardia cyst formation atthe molecular level and potentially in the control of trans-mission of the disease by preventing the encystation processin vivo.
ACKNOWLEDGMENTS
We thank E. Cabib for kindly supplying us with purified chitinase,Vibha Goyal for technical assistance, and Wanda Chin-Coker forsecretarial help.
This work was supported by a grant from the Rockefeller Foun-dation.
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