Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine

Molecular and Biochemical Parasitology, 32 (1989) 121-132

121

Elsevier MBP 01065

Giardia cyst wall-specific carbohydrate: evidence for the presence of

galactosamine Edward L. Jarroll 1, Paul Manning 1, Donald G. Lindmark 1, James R. Coggins 2 and S t a n l e y L. E r l a n d s e n 3 1Department of Biology, Cleveland State University, Cleveland, OH, U.S.A.; 2Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, U.S.A.; and 3Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN, U.S.A. (Received 25 February 1988; accepted 4 August 1988)

Gas chromatographic (GC), mass spectrometric (MS), lectin binding and enzymatic analyses of the carbohydrates from Giardia cyst walls, intact cysts and trophozoites were performed to investigate the carbohydrate composition of Giardia cyst walls and to test the hypothesis that the Giardia cyst wall is composed largely of chitin. Galactosamine, verified by MS, was present in Giardia cyst walls and intact cysts (ca. 47 nmol 10 -6 cysts). Since not even trace amounts of it were detected in trophozoites by either GC or lectin binding, galactosamine is hypothesized to be a cyst wall-specific amino hexose. Based on the putative binding affinity of Phaseolus lirnensis lectin, galactosamine may be present in cyst walls as N-acetylgalactosamine. Neither glucosamine nor sialic acid were detected in as much as 11 mg dry weight of cysts, cyst walls, or trophozoites. Glucose, the most abundant carbohydrate, and ribose were detected in Giardia cysts and trophozoites. Galactose (ca. 10 nmol 10 -6 cysts) was detected in cysts but not in trophozoites. The lack of detectable levels of (1) glucosamine in cyst wall hydrolysates, (2) cyst staining by Calcofluor M2R, (3) endogenous chitinase activity and (4) N-acetylglucosamine when cysts served as a substrate for exogenous chitinase suggests that the Giardia cyst wall is not composed largely of chitin as previously reported. 13-N-Acetylgalactosaminidase, EC 3.2.1.32, activity was detected in cysts and trophozoites and represents the first carbohydrate splitting hydrolase detected in Giardia. Key words: Giardia; Protozoan cyst wall; Carbohydrate; Amino hexose; Galactosamine; 13-N-Acetylgalactosaminidase

Introduction Parasitic protozoa in the genus Giardia exist in two morphologically distinct forms: a trophozoite and a cyst. The cyst form of this flagellate is responsible for the transmission of the parasite from one host to another, but compared to the trophoCorrespondence address: E.L. Jarroll, Department of Biology, Cleveland State University, 1983 E. 24th Street, Cleveland, OH 44115, U.S.A. Abbreviations: LBA, Phaseolus limensis lectin; GalNAc, Nacetylgalactosamine; GalNHz, galactosamine; GlcNAc, Nacetylglucosamine; GIcNH2, glucosamine; SA, N-acetylneuraminic (sialic) acid; MA, muramic acid; SDS, sodium dodecyl sulfate; TEM, transmission electron microscopy; LVSEM, low voltage scanning electron microscopy; GC, gas chromatography; MS, mass spectrometry; NAGase, 13-N-acetylglucosaminidase; WGA, wheat germ agglutinin

zoite, little is known of its structure and organization. A cyst measures 6--10 ~m in length and is composed of a pair of trophozoites encased within a fibrous cyst wall which measures 0.3-0.5 Ixm in thickness [1]. In 1955, Filice [2] stated that the cyst wall of Giardia from the laboratory rat (1) did not contain chitin or cellulose since it was Feulgen negative after hydrolysis with hydrochloric acid (no details of the procedure were given), (2) did not contain a simple protein or saccharide since pepsin, trypsin, papain, animal diastase, and plant amylase had no detectable effect on it, and (3) was not composed mainly of lipids since it was not stained with Sudan IV. In 1965, Dutta [3] reported the presence of polysaccharides (acridine orange positive after sulfation) in the cyst wall of Giardia intestinalis (synonym for G. lamblia and G. duodenalis [2]) and based on other cytochem-

0166-6851/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

122 ical properties (periodic acid-Schiff positive and amylase resistant) postulated that these polysaccharides were in combination with proteins in the cyst wall. In 1985, Ward et al. [4] reported that the cyst walls of G. muris and G. lamblia were composed largely of chitin. This conclusion was based on the binding of wheat germ agglutitiin (WGA; with putative specificity for N-acetylglucosamine (GlcNAc) and sialic acid (SA)) and succinylated-WGA (sWGA, with putative specificity for GlcNAc) which were abolished after treatment of the cysts with chitinase (shown to exhibit other glycosidic and proteolytic activities [5]). Further indirect support for the possible presence of chitin in the Giardia cyst wall came from a report by Gillin et al. [6] which mentioned briefly that chitin synthetase activity had been detected in encysting Giardia trophozoites. To date, published studies on the composition of these cyst walls have been histochemical in nature. This study extends the earlier studies by employing gas chromatographic, mass spectrometric, enzyme and lectin binding analyses to investigate the carbohydrate composition of Giardia cyst walls, and to test the hypothesis that chitin is a major cyst wall component. These analyses focus on cysts of G. muris and G. lamblia from animals, and cysts of G. duodenalis generated axenically in vitro [7]. Materials and Methods

Source of chemicals. Sugar standards were purchased from Supelco (Bellefonte, PA) unless otherwise stated. Calcofluor M2R was purchased from Polysciences, Inc. (Warrington, PA). All other chemicals, including chitin, fluorescently labeled Phaseolus limensis lectin (LBA), having affinity for N-acetyl-D-galactosamine (GalNAc), chitinase, and [3-N-acetylglucosaminidase (NAGase, 3.2.1.30) were purchased from Sigma (St. Louis, MO). LBA labeled with fluorescein was also purchased from Vector Laboratories (Burlingame, CA). Giardia cultures. G. lamblia (G. duodenalis type) trophozoites (Portland 1 strain) were cultured in TYI-S-33 medium without bile at 37°C. Cells were harvested after 96 h in all experiments. G. duo-

denalis (MR4 strain) trophozoites were cultured in TYI-S-33 supplemented with bile [8]. Giardia cysts. G. lamblia cysts from the feces of infected gerbils [9] and G. muris cysts from the feces of infected CF-W mice [10] were harvested and purified by the method of Sauch [11]. Feces from the same number of uninfected gerbils and mice were harvested and purified in the same manner as those from infected animals and used as background controls in the purification procedure. Cysts of G. duodenalis (MR4) were generated in vitro by the method of Schupp et al. [7]. Cyst numbers were approximated by counting in a hemocytometer. Excystation was induced as described by Rice and Schaefer [12]. Giardia cyst walls. Cysts purified from the feces of gerbils and mice were placed in a 1% solution of sodium dodecyl sulfate (SDS), and this mixture was placed in a 100°C water bath for 2-5 min. The resulting structures were washed 3 x in distilled water and were termed cyst walls (CW). Sugar and chitin standards used in the chemical analyses of these walls were treated in the same manner prior to hydrolysis and quantitation. Staphylococcus aureus, Saccharomyces cerevisiae and Entamoeba invadens. S. aureus cells were grown for 24 h in nutrient broth at 37°C. These cells were used as a control for the detection of glucosamine (GlcNH2) and muramic acid (MA) after acid hydrolysis of peptidoglycan. S. cerevisiae were grown for 48 h on YEP (yeast extractglucose-peptone) slants at 30°C. E. invadens (ATCC 30020) trophozoites were grown in TPS1 [13] and induced to form cysts in vitro [14]. Electron microscopy. For transmission electron microscopy (TEM), approximately 2.7 x 10 7 intact and SDS-treated Giardia cysts were collected in 25 ml centrifuge tubes and fixed overnight with an excess of phosphate-buffered 4% glutaraldehyde. After washing in phosphate buffer, cysts were postfixed in phosphate buffered 1% osmium tetroxide for 2 h at 4°C. The pellet was stained en bloc using 2% aqueous uranyl acetate for 30 min; subsequent dehydration was through an ascending ethanol series. Material was infil-

123 trated in LR white resin (Polysciences, Inc., Warrington, PA). Cysts were polymerized in gelatin capsules at 60°C for 24 h. Silver to gray sections were cut and then stained with uranyl acetate and lead citrate. Sections were viewed using an Hitachi H-600 microscope operated at 75 kV in the Electron Microscopy Laboratory at the University of Wisconsin-Milwaukee. For scanning electron microscopy (SEM), intact, freshly isolated G. muris cysts and SDS extracted cyst walls were attached to glass chips with 0.1% poly-L-lysine. These chips were then placed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, dehydrated in an ascending ethanol series, and critically point dried using the CO2 method of Anderson [15] as previously described [16]. The samples were then coated with platinum using an ion beam micro sputter coater (Ion Tech, Ltd., Middlesex, U.K.) and examined at 1-1.5 kV with the Hitachi S-900 low voltage scanning electron microscope (LVSEM) in the Integrated Microscopy Resource at the University of Wisconsin (Madison, WI).

Hydrolysis, methanolysis, gas chromatography (GC) and mass spectrometry (MS). Trophozoites, intact cysts (IC), CW, S. aureus, chitin, control fecal samples, and the sugar standards, were hydrolyzed, under nitrogen, at 100°C in either 2 N HCI for 3 h [17], 6 N HC1 for 6 h [18], or subjected to methanolysis in 1.5 N methanolic HCI for 3 h at 85°C [19,20]. The digests from these treatments were applied to Dowex 1 (carbonate form) columns and the sugars were eluted with water [17]. These eluates were dried by flash evaporation, and the sugars were collected in the appropriate volume of 70% ethanol. Prior to GC analysis the ethanol was evaporated under nitrogen, and trimethylsilane derivatives of all sugars were made [21,22]. For quantitation of sugars, 0.1 ixmol of mannitol were added to each sample prior to hydrolysis or methanolysis [19]. This system of detection required an initial minimum of approximately 30 nmol of each sugar prior to acid hydrolysis and 25 nmol prior to methanolysis. Approximately 50 nmol of SA prior to methanolysis were required. GC analysis was performed on a HewlettPackard (Model 5880A) chromatograph equipped

with a flame ionization detector and a fused silica capillary column (Hewlett-Packard SE-54). The hydrogen carrier gas flow rate was 2.1 ml min -1 and the nitrogen make-up gas flow rate was 28.1 ml min -1. For the separation of amino sugars after acid hydrolysis the column temperature was 160°C for 20 min, increased at 10°C min -1 for 2 min and remained at 180°C for 10 min. For the separation of neutral sugars after methanolysis, the column temperature was 140°C for 0.5 min, increased at 0.5°C min -1 to 180°C, and then maintained at 180°C for 20 min. GC and MS analyses were performed on a Finnigan TSQ 45 GC/MS/MS/DS operated in the MS mode. The GC/MS was equipped with a HewlettPackard capillary column (5% phenylmethyl silicone), and the temperature was ramped linearly from 170°C to 250°C in 30 min (2.67°C min-~). Sugar identification was based on the comparison of both spectra and retention time to authentic samples.

Lectin staining. Cysts of G. muris and G. duodenalis (isolated from beavers) were isolated from feces using the sucrose gradient method of Sheffield and Bjorvatn [23] and attached to slides using 0.1% poly-L-lysine at room temperature for 45-60 min prior to staining. The unfixed cysts, mounted on slides, were treated with 0.1% bovine serum albumin in phosphate buffered saline (PBS) at pH 7.4 for 10 min. Excess fluid was wicked off the slide and the cysts were stained with 50 Ixg m1-1 of LBA in PBS for 30 min. The slides were rinsed in PBS and mounted in an antifade medium containing 0.1% paraphenylenediamine m1-1 of glycerol, diluted with PBS (9:1, v/v). The slides were then examined with an Olympus epifluorescence microscope and photographed on Ektachrome film ( A S A 400, daylight). Controls for the lectin staining included the absorption of the LBA with either 100 mM GalNAc or GlcNAc for 24 h prior to staining. Calcofluor staining. Glutaraldehyde fixed and unfixed cysts of both Giardia species, cysts of E. invadens, and S. cerevisiae were subjected to staining with Calcofluor M2R (stains 13-1,4 and 13-1,3 linked polysaccharides such as chitin and cellulose) according to the method of Arroyo-Begov-

124 ich et al. [18]. Slides were examined with a Nikon epifluorescence microscope. E n z y m e assays. Chitinase (EC 3.2.1.14) activity in 108 intact G. muris cysts, G. muris cysts which had been induced to excyst [12], and G. lamblia trophozoites was assayed by the colorimetric method of Roberts and Cabib [5]. Chitinase activity was assayed in the presence and absence of exogenous 13-N-acetylglucosaminidase (EC 3.2.1.30, N A G a s e ) . Chitinase from Streptomyces griseus (Sigma) and Serratia marcescens (Sigma) was used as controls. All chitinase assays included Triton X-100 and were p e r f o r m e d at 25°C and 37°C for incubation times of up to 72 h. In addition, 108 intact G. muris cysts were used to replace the chitin in assays for which purified chitinase was used. Chitin from crab shells (Sigma) served as a known substrate. The limit of detection of G l c N A c in the chitinase assays was 0.3 nmol [24]. 13-N-Acetylgalactosaminidase (EC 3.2.1.32)

activity was assayed in G. lamblia trophozoites and G. muris cysts using the colorimetric method described by Mtiller [25]. These assays include Triton X-100 and were p e r f o r m e d at 30°C for 30 rain at p H 5. Enzyme units are defined as the amount of enzyme necessary to degrade 1 ixmol of substrate min -a under the assay conditions stated. The limit of detection in this assay was 0.1 m U (mg protein) -~. Results Electron microscopy. Fig. 1 depicts an intact cyst of G. muris showing a typical internal morphology, peritrophic space, an intact cyst m e m b r a n e , and the fibrous CW. The SDS treatment of intact cysts yields intact cyst walls lacking the SDS-soluble trophozoites and cyst m e m b r a n e as demonstrated in Fig. 2. Only a small amount of SDS-insoluble debris remained within these walls. Cysts of G. lamblia from gerbils gave similar results (data not shown). High resolution studies of the

Fig. 1. G. muris untreated control cyst with encysted trophozoite (12 000×). Trophozoite with two nuclei (Nu) and flagellar axonemes (A) near the anterior end of the cell. The peritrophic space (P) is enclosed by the electron-dense inner membrane (arrowhead) and the fibrous outer cyst wall (C). Scale bar indicates 1.0 p.lxm. Fig. 2. G. muris cysts after SDS treatment (8000×). Only scattered debris remains as remnants of the encysted trophozoites (asterisk), and the inner cyst membrane has been destroyed. C marks the junction of three cyst walls. Scale bar indicates 1.0 ~m. Greater than 90% of the cysts examined exhibited this pattern after SDS-treatment.

125

Fig. 3. Low voltage scanning electron micrographs of G. muris cysts. (A) Untreated control, 13500x; and (B) SDS treated, 15 000x. Scale bar indicates 1 Ism.

surface of intact, freshly isolated cysts using LVSEM clearly demonstrated the filamentous component of the wall (Fig. 3A). Examination of the SDS-treated cyst (i.e., CW) by LVSEM also revealed a similar filamentous pattern (Fig. 3B). Sugar analysis. Fig. 4 and Table I present the re-

suits of representative GC analyses of carbohydrates from Giardia and S. aureus hydrolyzed in 2 N HCI at 100°C for 3 h. Galactosamine (GalNH2) was the only amino sugar detected in hydrolysates of cysts from either Giardia species (Fig. 4A-C, GalNH2 peaks: 1A, 1B, 1C). The mass spectra (not shown) and retention times of the GalNH 2 peaks in Giardia matched those of authentic samples. The quantity of GalNH2 (ca. 47 nmol 10 -6 cysts) was not significantly different among G. muris IC and CW, G. lamblia CW, and G. duodenalis (MR4) IC cultivated in vitro. The only determination for G. lamblia IC from gerbils that was quantitated gave a level of GalNH2

which was just above the lower 95% confidence interval for the mean GalNH 2 from G. muris CW. GalNH2 constituted ca. 30% of the dry weight of G. muris CW, and ca. 12% of the dry weight of G. lamblia CW. GalNH2 was below the level of detection in G. lamblia trophozoite hydrolysates (Fig. 4D). GIcNH 2 and SA were not detected in either Giardia CW, IC or trophozoite hydrolysates of up to 11 mg dry weight, but GIcNH2 (not GalNH2) was detected in the serum supplement for the medium in which G. lamblia trophozoites were grown, and GIcNH 2 and MA were detected in 2 mg dry weight of S. aureus (Fig. 4E, GlcNH2 peaks: 2A, 2B, 2C; MA peaks: 5A, 5B). GIuNH2 and MA were below the level of detection in the hydrolysates of Giardia cysts purified from feces and in hydrolysates of cyst-free controls. Gas chromatograms of 6 N HCI hydrolysis and 1.5 N HC1 methanolysis revealed no additional amino sugars (data not shown).

126

3A

3A

3B

3B

~4

4

115

10

210 3B

2'5

10

1~5

20

~'s

Retention Time (min.)

8-C

D

I

~4-

_

1 ~B

15

1~

20

~s

lb

1~

20

2's

Retention Time (min.)

3A

f.,i

8- E

2A

4

5B 5A

2B

2b

2'5

3B

I~

Lectin staining. Intense staining of the cyst wall with L B A lectin is shown in Fig. 5A, but control trophozoites showed no staining (data not shown). Absorption of the L B A with 100 m M G a l N A c (Fig. 5B) to L B A staining (Fig. 5C) reduced the staining of the cyst wall to background levels. In contrast to this, absorption of L B A with 100 m M GlcNAc failed to noticeably diminish cyst wall staining with L B A (Fig. 5C). Calcofluor staining. Neither glutaraldehyde-fixed nor fresh Giardia cysts of either species stained with Calcofluor M2R. However, E. invadens cysts and S. cerevisiae gave positive staining results similar to those described by Arroyo-Begovich et al. [18] for E. invadens.

1 1o

Glucose, verified by MS, was detected in both cysts and trophozoites of Giardia. Glucose represented the sugar in highest concentration in both forms of the organism. All Giardia cysts isolated from animals contained equivalent amounts of glucose (ca. 65 nmol 10 -6 cysts). G. duodenalis cysts (MR4) and G. lamblia trophozoites, both cultured in vitro, contained significantly higher quantities of glucose (587 nmol 10 -6 cysts; 131.5 nmol 10 -6 trophozoites) than cysts from either animal host. The in vitro induced cysts (MR4) contained significantly more glucose than did the G. lamblia trophozoites. Galactose (ca. 10 nmol 10 .6 cysts) and ribose (ca. 5 nmol 10 .6 cysts) were detected by G C and verified by MS in Giardia cysts after methanolysis. Ribose (ca. 1 nmol 10 .6 cells), but not galactose, was detected in G. lamblia trophozoites after methanolysis.

2'o

~'5

E n z y m e assays. W h e n G. lamblia trophozoites, G. muris IC, and G. muris IC which had been induced to excyst were assayed for chitinase with and without exogenous N A G a s e , chitinase activ-

Retention Time (min.)

Fig. 4. Representative gas chromatograms of carbohydrates from 2 mg dry weight of Giardia and Staphylococcus hydrolyzed under nitrogen in 2 N HC1 at 100°C for 3 h: (A) intact G. muris cysts; (B) SDS-treated G. muris cysts; (C) intact, in vitro generated G. duodenalis (MR4) cysts; (D) G. lamblia trophozoites (Portland 1); (E) intact S. aureus; (F) SDS-treated G. muris cysts with superimposed glucosamine peaks (---) using mannitol as the common reference. Peak identification for

all chromatograms is as follows: et-galactosamine (GalNHz) (1A), 13-GalNH2 (1B), open chain GalNH2 (1C); a-glucosamine (GlcNHz) (2A), open chain GIcNH2 (2B), [3-GIcNHz (2C); a-glucose (3A), I~-glucose(3B); mannitol as an internal standard (4); muramic acid (5A, 5B); unknown (U). Column temperature was maintained at 160°C for 20 min, increased at 10°C min 1 for 2 rain, and remained at 180°C for 10 min.

127 TABLE I Quantitation of galactosamine (GalNH2) and glucose in cysts and trophozoites of Giardia after 2 N HCI hydrolysis Organism

No. a (dry weight)

nmolb 10-6 cells GalNH2

G. G. G. G.

muris IC muris CW muris T lamblia IC

(gerbils)

15 (2 mg) 54 (2 mg)

No. of Glucose

trials

41.4 (6.8)d 63.3 (28.3)d

75.5 (15)d 63.7 (3)d

3 3

c

c

8 (2 mg)

15.8~

57.4~

32 (2 mg) 18 (2 mg)

42.0 0.0

61.5 131.5 (3)f

1 3

37,0 (5.5)~

587.0 (11)f

2

1

G. lamblia CW

(gerbils) G. lamblia T G. duodenalis

IC(MR4)

7 (2 mg)

aNn. = the number of cysts x 106. bMean number of nmol of the sugar (-+ S.D.). CG. muris trophozoites have not been cultured in vitro, and thus are not available in sufficient quantity for examination. °Comparison of means within sugar types by Student's t-test showed that they do not differ significantly (P > 0.05). eThe single glucose value closely approximates the mean glucose values for the G. rnuris in vivo cysts. The GalNH2 single determination falls near the lower 95% confidence limit (11.17) of the G. muris mean (63.33). fStudent's t-test comparisons of these means with the others for glucose show that they differ significantly (P < 0.001). IC, intact cysts; CW, cyst wall; T, trophozoites; MR4 cysts were generated by in vitro encystment.

ity was b e l o w the d e t e c t i o n limit of 0.3 n m o l . F u r t h e r m o r e , the attempted digestion of G. m u f f s I C as a s u b s t r a t e for purified chitinase p r o d u c e d n o d e t e c t a b l e G l c N A c (0.3 n m o l limit of detection). B - N - A c e t y l g a l a c t o s a m i n i d a s e activity was exh i b i t e d by G. l a m b l i a t r o p h o z o i t e s a n d by G. m u r i s cysts. T h e specific activity of this e n z y m e

was a p p r o x i m a t e l y 10 m U (mg p r o t e i n ) -1 in trophozoites a n d cysts.

Discussion T h e data p r e s e n t e d in this p a p e r , b a s e d o n G C a n d MS analyses, constitute the first chemical investigation of the G i a r d i a C W . T h e p r e s e n c e of a

Fig. 5. Fluorescence microscopy of intact G. muffs cysts at 500x. (A) LBA lectin binding with putative specificity for N-acetyln-galactosamine. (B) LBA plus 100 mM N-acetyl-D-galactosamine. (C) LBA plus 100 mM N-acetyl-n-glucosamine. The location of the cyst wall is marked by an arrowhead. Scale bar indicates 1 ~,m.

128 significant amount of GalNH 2 (ca. 12-30% of dry weight) in the CW of two distinct species of Giardia [2] has been clearly demonstrated. Since GalNH 2 was detected chromatographically from CW and IC hydrolysates (but not in trophozoite hydrolysates), and since LBA staining of the CW was abrogated by preincubation with GalNAc but not GlcNAc, the results strongly indicate that most, if not all, of the GalNH2 is located in the CW. Based on putative LBA binding affinity, GalNH2 may be present as GalNAc. Additionally, GalNH 2 may function as a stage specific marker in this genus since it is present in the cyst, but it is below the level of detection, by either GC or lectin binding, in nonencysting trophozoites. Our data more closely agree with the presence of CW saccharides, possibly in combination with proteins as suggested by Dutta [3], the apparent absence of CW chitin suggested by Filice [2], and thus, do not agree with the presence of chitin as a major CW component [4] in Giardia. Even though low levels of chitin could be present in the Giardia CW, we were unable to detect (1) as little as 30 nmol (6.6 txg) of GlcNH 2 in as much as 11 mg dry weight of CW and IC, (2) chitinase activity even in excysting trophozoites, (3) GlcNAc when IC were used as substrate for chitinase, and (4) Calcofluor M2R staining of either fresh or fixed Giardia cysts. The lack of detectable quantities of GIcNH2 was surprising in light of recent studies which suggest its presence [4,26,27]. Hill et al. [26] reported that GlcNAc was present on living G. lamblia trophozoites since WGA agglutinated 22.9% of the trophozoites tested in a microassay. However, they were unable to detect any agglutination of the trophozoites in a standard macroagglutination assay with cell concentrations of up to 2.5 x 106 cells m1-1. Despite this, more than 90% of the cells exhibited fluorescence with fluorescein isothiocyanate (FITC)-conjugated WGA. Ward et al. also reported FITC-WGA binding to Giardia cysts [4] and trophozoites [27]. In the case of cysts, W G A binding was abrogated and the CW ultrastructure was abolished by 24 h of incubation in chitinase, but WGA binding was not affected by treatment with NAGase, neuraminidase, lysozyme, or chitinase which was preincubated with chitin. FITC-conjugated lectins for

GalNAc (i.e., phytohemagglutinin (PHA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA)) failed to react with Giardia cysts. In the case of the trophozoites, FITC-WGA binding, observed in 80-85% of the cells, was abrogated by NAGase, but not by chitinase. To confirm these findings, the Ward group [27] incubated one set of trophozoites in PBS and another set in 2.5 mU of NAGase for 48 h at room temperature. To assess the effect of this treatment, both sets were labeled with 125I-WGA in a solution of PBS with bovine serum albumin. These investigators reported that after such treatment a reduction (ca. 50%), not abrogation, of binding resulted. Neither in the study by Hill et al. [26] nor in the studies by Ward et al. [4,27] were the lectin binding results confirmed by means other than by treatment of Giardia cysts and trophozoites with enzymes of undetermined purity which may, as in the case of chitinase, be contaminated with other hydrolases including proteolytic enzymes [5]. These results, and those of other lectin binding studies, without their confirmation by more definitive chemical analysis should be interpreted cautiously especially since: (1) Ward et al. [4] were unable to detect lectin binding to cysts with three lectins (PHA, DBA, HPA) having affinity for various forms of GalNAc (possibly a configuration incompatibility), when clearly by GC and MS analyses of CW hydrolysates we have shown that approximately 30% of its dry weight is composed of GaINH 2 (potentially as GalNAc) and (2) Monsigny et al. [28] reported that the agglutination of horse red blood cells by 20 ~g m1-1 WGA can, for example, be inhibited by GlcNAc (10 raM), GalNAc (200 mM), nitrophenyl-tx-GalNAc (3 mM), nitrophenyl-[3-GalNAc (1 mM), [~-GalNAc]4 bovine serum albumin (0.012 mM), and N-acetyl-Lphenylalanine (10 raM). The results of Monsigny et al. [28] call into question the practice of using preincubation of lectins with a large excess of a single known inhibiting substance as a control for specificity when, in fact, the only parameter being tested may be the lectin's affinity for that particular inhibitor and not its ability to bind other compounds in the absence of the inhibitor. It is, of course, impossible to completely rule out the presence of GlcNAc on Giardia cysts or tropho-

129 zoites since it could exist below the level of detection by our methods. However, in the case of cysts, if GlcNAc (or chitin) were present in hydrolysates at a level below our detection limit that would imply that GlcNAc (chitin) constitutes less than 0.27% of 2 mg dry weight of CW (less than 0.05% of the 11 mg dry weight of CW, IC or trophozoites). Furthermore, if a small amount of GlcNAc were present in both the CW and the trophozoite, either free or in combination with chitin, it would not constitute a stage specific sugar. Gillin et al. [6] reported detecting chitin synthetase activity in encysting Giardia trophozoites. This enzyme activity was measured by the incorporation of 3H-GIcNAc into an unidentified trichloroacetic acid (TCA)-precipitable material which was digested by chitinase. However, purified chitinase has been shown to have proteolytic as well as other glycolytic activities [5] associated with it. The exact chemical nature of the TCAprecipitate was not determined, and the specificity of the synthetase for GIcNAc was not shown. Incorporation of GlcNAc into the TCA precipitate was inhibited by Polyoxin-D and Nikkomycin which are reported to inhibit chitin synthetase in other organisms [29]. Despite the fact that inhibitors of chitin synthetase in other systems prevented labeled GlcNAc incorporation into an unidentified TCA precipitate, it does not confirm that those inhibitors are acting on the same system in Giardia. Since these inhibitors are structural analogs of GlcNAc, they may also inhibit incorporation of GIcNAc (and possibly GalNAc) into products other than chitin. In many cell systems, the formation of GalNAc occurs through the epimerization of GlcNAc [30], which means that if UDP-GalNAc 4' epimerase, the enzyme responsible for this, exists in Giardia, then the radioactivity incorporated may have represented not the incorporation of GlcNAc into chitin, but, instead, the incorporation of GalNAc into a TCAprecipitable protein. Our results suggest that the presence of chitin synthetase activity in Giardia requires further evaluation. While it is not possible to rule out glucose as a CW carbohydrate based on the findings presented here, the fact that it was detected in trophozoites, IC, and CW suggests that it may be

present primarily as glycogen which remains within the walls after SDS treatment. Dutta [3] reported the presence of PAS-positive, amylase labile carbohydrates in Giardia trophozoites while the carbohydrate in the cyst wall was PAS-positive, amylase fast. The exact localization of glucose in these cysts must await our further analysis of CW components. The amount of glucose in Giardia encysting in vitro is significantly higher than that in cysts from animals or that from nonencysting, growing trophozoites. It is interesting to speculate that this could be due to increased accumulation of glycogen within trophozoites preparing to encyst. However, the excess glucose also could have come from trophozoites which were lysed to separate them from in vitro generated cysts. The lower amount of glucose in cysts from animals might be due to sustained glycolytic activity for a longer time than those from encysting cultures, thus reducing endogenous glycogen reserves. Further study will be required to elucidate which of these possibilities is correct in the context of cyst physiology. On the basis of the chemical analyses presented here, it is not possible to establish the structural relationship of the carbohydrates detected in these crude CW preparations. It is conceivable that GalNH 2 could exist alone or in combination with other carbohydrates as a polysaccharide, a glycoprotein, proteoglycan, or a glycolipid. Lindmark [31] was unable to detect 13-N-acetylglucosaminidase (EC 3.2.1.30), 13-galactosidase (EC 3.2.1.23), ~-glucuronidase (EC 3.2.1.31), Ot-D-glucosidase (EC 3.2.1.20), 13-D-glucosidase (EC 3.2.1.21), or f3-o-xylosidase (EC 3.2.1.37) in Giardia trophozoites. Thus, the observation that cysts and trophozoites of Giardia exhibit 13-Nacetylgalactosaminidase activity, but lack detectable chitinase activity, is of interest because it is the first report of a carbohydrate splitting hydrolase in Giardia. Its presence in the battery of Giardia hydrolases coupled with the occurrence of GalNH2 in the CW is being investigated as part of the mechanism used by trophozoites for excystation and encystment. A method for removing SDS-soluble trophozoite material from Giardia cysts has been de-

130

scribed. This method leaves the remaining CW microscopically unaltered, and provides a means for preparing samples enriched for CW components. While SDS-soluble proteins possibly associated with the CW would be removed by this treatment, available microscopic evidence suggests that the fibrillar nature of the CW is unchanged by the SDS treatment. Additionally, statistical analysis confirms that the GalNH 2 composition of G. muris IC and SDS-generated CWs is not significantly different. We have also demonstrated that the carbohydrate composition of the G. duodenalis type cysts generated in vitro is similar to that of cysts generated in vivo. This information coupled with the observations of Schupp et al. [7] that cysts generated in vitro are viable and infective for mice and gerbils suggests that a suitable substitute for animal derived cysts may have been found. Metabolic studies on cysts

generated in vitro compared to those generated in vivo would aid in confirming this observation.

Acknowledgements We wish to thank Dr. Julius Kerkay, CSU Chemistry Department, for the use of the gas chromatograph, and Mr. Rodolfo Bongiovanni for his technical assistance with that instrument. We wish to acknowledge Dr. Roger Binkley, CSU Chemistry Department, for his help and advice in this study, and Dr. David Hehemann, CSU Chemistry Department, for his help with and the use of the mass spectrometer. We thank Lee Ann Sherlock for her excellent technical assistance. This research was supported in part by grants from the Thrasher Research Fund (#2799-5), Ohio Board of Regents Academic and Research Challenge Programs, and the Minnesota Medical Foundation.

References 1 Feely, D.E., Erlandsen, S.L. and Chase, D.G. (1984) Structure of the trophozoite and cyst. In: Giardia and Giardiasis (Erlandsen, S,L. and Meyer, E,A., eds.), pp. 3-31, Plenum Press, New York. 2 Filice, F.P. (1952) Studies on the cytology and life history of Giardia from the laboratory rat. Univ. Calif. Publ. Zool. 57, 53-143. 3 Dutta, G.P. (1965) Demonstration of neutral polysaccharides with fluorescence microscopy using acridine orange. Nature 205,712. 4 Ward, H.D., Alroy, J., Lev, B., Keusch, G. and Pereira, M. (1985) Identification of chitin as a structural component of Giardia cysts. Infect. Immun. 49,629-634. 5 Roberts, R. and Cabib, E. (1982) Serratia marcescens chitinase: one step purification and use for the determination of chitin. Anal. Biochem. 127,402-412. 6 Gillin, F.D., Reiner, D.S., Gault, M.J., Douglas, H., Wunderlich, A. and Sauch, J.F. (1987) Encystation and expression of cyst antigens by Giardia lamblia in vitro. Science 235, 1040-1043. 7 Schupp, D.G., Januschka, M., Sherlock, L., Erlandsen, S., Meyer, E., Bemrick, W. and Stibbs, H. (1988) Production of viable Giardia cysts in vitro: determination by fluorogenic dye staining, excystation, and animal infectivity in the mouse and Mongolian gerbil. Gastroenterology 95, 1-10. 8 Keister, D.B. (1983) Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans. R. Soc. Trop. Med. Hyg. 77,487-488. 9 Belosevic, M., Faubert, G.M., MacLean, J.D., Law, C. and Croll, N.A. (1983) Giardia lamblia infections in Mon-

10

11

12

13

14

15

16 17

18

19

golian gerbils: an animal model. J. Infect. Dis. 147, 222-226. Roberts-Thomson, I., Stevens, D., Mahmoud, A. and Warren, K. (1976) Giardiasis in the mouse: an animal model. Gastroenterology 71, 57-61. Sauch, J.F. (1984) Purification of Giardia muris cysts by velocity sedimentation. Appl. Environ. Microbiol, 50, 1434-1438. Rice, E. and Schaefer, F. (1981) Improved in vitro excystation procedure for Giardia lamblia cysts. J. Clin. Microbiol. 14, 709-710. Diamond, L. (1968) Techniques of axenic cultivation of Entamoeba histolytica Schaudinn 1903 and E. histolyticalike amoebae. J. Parasitol. 54, 1047-1056. Rengpien, S. and Bailey, G. (1975) Differentiation of Entamoeba: a new medium and optimal conditions for axenic encystation of E. invadens. J. Parasitol. 61, 24-30. Anderson, T.F. (1951) Techniques for the preservation of three dimensional structure in preparing specimens for the electron microscope. Trans. NY Acad. Sci. 13, 130-134. Erlandsen, S. and Bemrick, W. (1987) SEM evidence for a new species, Giardia psittaci. J. Parasitol, 73,623--629. Levvy, G., Hay, J., Conchie, J. and Strachan, I. (1970) The determination of the individual neutral and amino sugars in carbohydrates. Biochim. Biophys. Acta 222,333-338. Arroyo-Begovich, A., Carabez-Trejo, A. and Ruis-Herrera, J. (1980) Identification of the structural component in the cyst wall of Entamoeba invadens. J, Parasitol. 66, 735-741. Bhatti, T., Chambers, R. and Clamp, J. (1970) The gas chromatographic properties of biologically important N-

131

20

21

22

23 24

25

acetylglucosamine derivatives, monosaccharides, disaccharides, trisaccharides, tetrasaccharides and pentasaccharides. Biochim. Biophys. Acta 222, 339-347. Chambers, R. and Clamp, J. (1971) An assessment of methanolysis and other factors used in the analysis of carbohydrate-containing materials. Biochem. J. 125, 1009-1018. Sweeley, C., Bentley, R., Makita, M. and Wells, W. (1963) Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. J. Am. Chem. Soc. 85, 2497-2507. Sweeley, C., Wells, W. and Bentley, R. (1966) Gas chromatography of carbohydrates. Methods Enzymol. 8, 95-109. Sheffield, H. and Bjorvatn, B. (1977) Ultrastructure of the cyst of Giardia lamblia. Am. J. Trop. Med. Hyg. 26, 23-30. Reissig, J., Strominger, J. and Leloir, L. (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J. Biol. Chem. 217, 959-966. Mfiller, M. (1973) Biochemical cytology of trichomonad flagellates. I. Subcellular fractionation of hydrolases, dehydrogenases, and catalase in Tritrichomonas foetus. J. Cell Biol. 57, 453-474.

26 Hill, D., Hewlett, E. and Pearson, R. (1981) Lectin binding by Giardia lamblia. Infect. Immun. 34, 733-738. 27 Ward, H., Alroy, J., Lev, B., Keusch, G. and Pereira, M. (1988) Biology of Giardia lamblia. Detection of N-acetylD-glucosamine as the only surface saccharide moiety and identification of two distinct subsets of trophozoites by lectin binding. J. Exp. Med. 167, 73-88. 28 Monsigny, M., Roche, A., Sene, C., Maget-Dana, R. and Delmontte, F. (1980) Sugar-lectin interactions: How does wheat germ agglutination bind sialoglycoconjugates? Eur. J. Biochem. 104, 147-153. 29 Gooday, G., Woodman, J., Casson, E. and Browne, C. (1985) Effect of nikkomycin on chitin spine formation in the diatom Thalassiosirafluviatilis, and observations on its peptide uptake. FEMS Microbiol. Lett. 28,335-340. 30 Schachter, H. and Roseman, S. (1980) Mammalian glycosyltransferases: their role in the synthesis and function of complex carbohydrates and glycolipids. In: The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W.J., ed.), pp. 85-160, Academic Press, New York. 31 Lindmark, D.G. (1988) Giardia lamblia: localization of hydrolase activities in lysosome-like organelles of trophozoites. Exp. Parasitol. 65, 141-147.