Proc. Natl. Acad. Sci. USAVol. 93, pp. 7628-7633, July 1996Cell Biology

Cholesterol starvation induces differentiation of the intestinalparasite Giardia lamblia

(encystation/primitive eukaryote/protozoan/membrane fluidity/bile salts)

HUGO D. LUJAIN*, MICHAEL R. MOWATr, LINDA G. BYRD, AND THEODORE E. NASHLaboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425

Communicated by Louis H. Miller, National Institutes of Health, Bethesda, MD, April 2, 1996 (received for review February 26, 1996)

ABSTRACT Giardia lamblia, like most human intestinalparasitic protozoa, sustains fundamental morphological andbiochemical changes to survive outside the small intestine ofits mammalian host by differentiating into an infective cyst.However, the stimulus that triggers this differentiation re-mains totally undefined. In this work, we demonstrate theinduction of cyst formation in vitro when trophozoites arestarved for cholesterol. Expression of cyst wall proteins wasdetected within encystation-specific secretory vesicles 90 minafter the cells were placed in lipoprotein-deficient TYI-S-33medium. Four cloned lines derived from two independentGiardia isolates were tested, and all formed cysts similarly.Addition of cholesterol, low density or very low density li-poproteins to the lipoprotein-deficient culture medium, in-hibited the expression of cyst wall proteins, the generation ofencystation-specific vesicles, and cyst wall biogenesis. Incontrast, high density lipoproteins, phospholipids, bile salts,or fatty acids had little or no effect. These results indicate thatcholesterol starvation is necessary and sufficient for thestimulation of Giardia encystation in vitro and, likely, in theintestine of mammalian hosts.

rigid wall that protects the cell in stressful environments (6, 12,13).The predilection of Giardia for the mid-jejunum (4, 14-16)

suggests that trophozoites require a high concentration ofnutrients at a relatively stable pH. Like most eukaryotic cells(17), membrane biogenesis in Giardia requires cholesterol (18,19). Because Giardia is unable to synthesize cholesterol (18),it must therefore obtain this compound from the milieu of theupper small intestine, which is particularly rich in biliary anddietary cholesterol (20, 21). In vitro, trophozoites are routinelycultured in a complex TYI-S-33 medium containing 10%serum (22), but cholesterol and phosphatidylcholine can fulfillthe serum lipid requirement of Giardia when solubilized inlipoproteins, bile salt mixtures, or cyclodextrins (23, 24).

Because the trophozoite may suffer nutrient deprivation asit traverses the intestine of the host, we decided to investigatethe effects of lipid starvation on this parasite. Here wedemonstrate, by morphological, immunological, and biochem-ical criteria, that cholesterol deprivation is necessary andsufficient to induce the differentiation of Giardia trophozoitesto cysts.

Giardiasis, the most common waterborne disease caused by anenteric parasite in humans, is produced by the flagellatedprotozoan Giardia lamblia (1). The Giardia life cycle presentstwo morphologically distinct forms, trophozoites and cysts.The disease is caused by the trophozoite forms and frequentlypresents as acute or chronic diarrhea (1). Trophozoites colo-nize the upper small intestine of the mammalian host where,by unknown mechanisms, they may differentiate into cysts,which are passed with the feces. Transmission occurs throughthe ingestion of Giardia cysts, usually from fecally contami-nated food or water or interpersonal contact (2).

Besides its medical importance, Giardia is considered anexcellent system to study the evolution of fundamental cellularprocesses since it belongs to the earliest branch of the eukary-otic lineage (3). Thus, encystation may represent a primitiveadaptive response developed by eukaryotes to survive harmfulconditions. Although encystation has been reproduced in vitro,the molecular basis for its induction remains undefined. Sev-eral conditions are known to trigger this encystation in vitro.For instance, an increase in the concentration and/or thesource of bile or bile salts in the culture medium and a shift inpH from 7 to 7.8 have been implicated in the induction ofencystation; however, encystation also has been achievedunder conditions of bile starvation (4-6). Once encystation isinitiated, cyst wall components are rapidly synthesized andsorted to characteristic secretory granules, the encystation-specific secretory vesicles (ESVs) that transport the cyst wallcomponents to the plasma membrane for release to the cellexterior (7-11). Encystation concludes in the production of a

MATERIALS AND METHODSMaterials. Cyclodextrins, bile acids, bile salts, human serum,

human low density (LDL, d = 1.019-1.063 g/ml), high density(HDL, d = 1.12-1.21 g/ml) and very low density (VLDL, d <1.006 g/ml) lipoproteins, bovine bile, porcine bile extract, andlipoprotein-deficient human serum were obtained from Sigma.Sterols and other lipids were from either Fluka or Sigma.22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-23,24-bisnor-5-cholen-3f3-ol (NBD-cholesterol) was from MolecularProbes. Adult bovine serum was from Biofluids (Rockville,MD). Lipoprotein-deficient bovine serum and total serumlipoproteins were obtained as described (25).Organisms and Cultures. Giardia lamblia trophozoites

[(WB isolate: clone 1267 (26), clone A6 (26), clone C6 (27); GSisolate, clone H7-1 (28)], were cultured axenically at 37°C inDiamond's medium TYI-S-33 containing 10% adult bovineserum (22). Only trophozoite populations with surface anti-genic homogeneity greater than 98% by indirect immunoflu-orescence were used in these studies. For cell counting, themedium and nonadherent trophozoites were discarded andreplaced with the same volume of fresh medium; then, tubeswere chilled, inverted 10 times, and the trophozoites countedwith a Coulter Model ZB1 electronic counter. To initiateencystation, cells were cultured to confluence in TYI-S-33medium (22) in 8-ml glass tubes for 72 h, typically yielding a

Abbreviations: CWP, cyst wall protein; LDL, low density lipoprotein;LPDS, lipoprotein-deficient serum; LPDM, lipoprotein-deficient me-dium; GDH, glutamate dehydrogenase.*To whom reprint requests should be addressed at: Laboratory ofParasitic Diseases, National Institute of Allergy and InfectiousDiseases, National Institutes of. Health, Building 4, Room 126,Bethesda, MD 20892-0425. e-mail: HLujan@pop.niaid.nih.gov.

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VSP + CWP cwP

U..FIG. 1. Encystation of G. lamblia in LPDM. Immunofluorescence

image of a field of Giardia WB/1267 trophozoites cultured for 24 h inLPDM. Cells were labeled simultaneously with fluorescein-conjugatedanti-1267 variant-specific surface protein (left) and rhodamine-conjugated anti-CWP1 (right) mAbs. Superimposition of the left andright panels is shown in the center. Trophozoites containing encysta-tion-specific vesicles, visualized by staining with anti-CWP1, areencysting.

cell density of approximately 1.1 x 106 cells/ml (initial inoc-ulum varied among clones due to differences in growth rates:WB/C6, 5,000 cells/ml; WB/1267 and WB/A6, 500 cells/ml;GS/H7-1, 20,000 cells/ml). Then, the medium was discarded,and the tube containing a confluent monolayer of trophozoitesrefilled with lipoprotein-deficient medium (LPDM) contain-ing human or bovine sera as described in Results.

Effects of Lipid Repletion on Encystation of G. kamblia inTYI-S-33 Medium. The media from monolayer cultures weredecanted and replaced with fresh medium containing lipopro-tein-deficient serum (LPDS), with or without lipid additions.At 24 h, the number of water-resistant cysts from the super-natant media was determined. Water-resistant cysts free oftrophozoites were obtained as described (16), and their via-bility was determined by fluorescein diacetate/propidiumiodine staining (6, 29).

Lipids were solubilized in either ethanol (0.01%) or di-methyl sulfoxide (DMSO) (0.05%) before their addition to theculture medium. Only solvent was added to control cultures,which were always carried out in parallel. Under these condi-tions, any effect due to the solvents was excluded.Immunofluorescence Microscopy. Immunofluorescence mi-

croscopy was performed essentially as described (9, 10). TheWB/1267, GS/H7, and WB/A6 variant-specific surface pro-teins were detected with the mAbs SC1, G10-4, and 6E7,respectively (26, 28). Cyst-specific antigens were detected withthe mAb 5-3C (10, 30) or 7D2 (11).

Analysis of Cholesterol Uptake by Trophozoites. To studythe uptake of fluorescent-labeled cholesterol by trophozoites,

1110 11X>-

IN IX)90 X0}-

In D ' 1 l«il 1T _ ll1T ilu~~~~~~~~~~~~~~~~~6

TI T5 IlIT

cholesterol was incorporated into purified LDL as reported(23).

Lipid Extraction and Cholesterol Analysis. Giardia tropho-zoites cultured for different periods in either normal growthmedium or LPDM were harvested by centrifugation and thecholesterol extracted twice from cells and culture mediaaccording to Slayback et al. (31). Cholesterol contents weredetermined using a Sigma test kit with cholesterol as standard(32).Other Methods. Nucleic acids were extracted and analyzed

by standard methods as described (10, 33). Determination ofthe steady-state mRNA levels of cyst wall proteins (CWPs) andthe housekeeping enzyme glutamate dehydrogenase (GDH)(34) was performed by slot-blot analysis in a Schleicher &Schuell blotting apparatus as indicated by the manufacturer.32P-end-labeled antisense oligonucleotides oMM103 (G. lam-blia CWP1 nt 272-253, GenBank accession no. U09330) andGDH9B (G. lamblia glutamate dehydrogenase nt 1080-1061,GenBank accession no. M84604) were used in RNA hybrid-ization studies.

RESULTSLipoprotein Depletion Induces Giardia Encystation in Vitro.

Initial experiments showed that cyst-like forms appeared in theculture supernatant when trophozoites (WB/1267 clone) weremaintained in LPDM, pH 7.0. Immunofluorescence analysis ofthe cyst-like forms revealed surface reactivity with the CWP-specific mAbs (result not shown). Subsequent analysis of cellscultured for 24 h in LPDM showed the presence of encystation-specific vesicles that contain CWP1, evidenced by immuno-fluorescence using a CWP1-specific mAb (Fig. 1). In contrast,control trophozoites grown in normal growth medium showedno evidence of CWP expression (results not shown).

Since the lipoprotein content of the medium appeared toinfluence differentiation, we determined whether lower con-centrations of LPDS facilitate Giardia encystation. Fig. 2Ashows that the number of encysting trophozoites and water-resistant cysts increased when LPDS concentration was low-ered from 10% to 5%. LPDS concentrations lower than 5%adversely affected encystation, suggesting that optimal con-centrations of serum components other than lipoproteins areessential for the parasite (35).Because others have reported that the pH of the medium is

important for encystation (36), we also analyzed the effect ofpH in the LPDM. Fig. 2B shows that encystation occurred

I181

2.5 5.0 7.5

Serum concentration (%)6.6 7.0 7.4 7.X 0 3 6 9 12 15 1X

pH Time (h)

FIG. 2. Optimal conditions for Giardia encystation in LPDM. Giardia trophozoites (clone WB/1267) were cultured in LPDM under the followingconditions: (A) in the presence of different concentrations of LPDS, at pH 7.0, for 24 h; (B) in the presence of 5% LPDS, at different pHs, for24 h; and (C) in the presence of 5% LPDS, at pH 7.8, for different periods. Cells were harvested and the percentage of nonencysting trophozoites(El), encysting trophozoites (L), and cysts (m) determined as described in Materials and Methods. Values are the mean of three independentexperiments performed in duplicate ± SD.

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Table 1. Effects of lipid depletion on Giardia in vitro

Addition toserum-freeTYI-S-33 Total cysts Viable cysts,medium Clone x 106/ml %

None WB/1267 0.02 ± 0.0 0 ± 0WB/C6 0.01 0.0 0 + 0WB/A6 0.00 0.0 0 + 0GS/H7 0.00 0.0 0 + 0

5% lipoprotein- WB/1267 1.60 + 0.10 36 ± 3deficient WB/C6 1.42 + 0.06 56 ± 7human serum WB/A6 0.87 + 0.01 33 + 4

GS/H7 0.45 + 0.09 46 ± 85% lipoprotein- WB/1267 1.92 + 0.03 42 ± 5

deficient WB/C6 1.65 + 0.01 61 ± 8bovine serum WB/A6 0.94 + 0.02 31 + 6

GS/H7 0.69 + 0.10 47 ± 7

within a wide pH range, but that encystation was optimal at pH7.8.At optimal conditions of serum concentration and pH (5%

LPDS and pH 7.8), trophozoites differentiated into cysts fasterthan any other reported methods (4-7) (Fig. 2C). Expressionof CWPs and formation of encystation-specific vesicles wasobserved by immunoblotting and immunofluorescence, re-spectively, as early as 90 min after the cells were placed inLPDM (results not shown); nearly 50% of the trophozoitesbecame cysts within 18 h (Fig. 2C).We also investigated whether lipoprotein deprivation in-

duces encystation of other cloned lines of the parasite. Table1 shows a comparative study on encystation of four differentcloned lines from two independent Giardia isolates in TYI-S-33 medium containing 5% lipoprotein-deficient human orbovine sera for 24 h at pH 7.8. All four cloned lines encystedsimilarly, even the WB-derived clone A6, which has beendescribed as "encystation deficient" (37). Cysts obtained wereresistant to hypotonic lysis, and '40% appeared viable at 24 has judged by the method of exclusion of propidium iodine anduptake of fluorescein diacetate (29).

Cholesterol Repletion Inhibits Giardia Encystation in Li-poprotein-Deficient Medium. In TYI-S-33 medium, lipids arederived primarily from the lipoprotein fraction of the serumsupplement (18, 19, 23). In serum, cholesterol is mainlyassociated with lipoproteins, but a small fraction can be foundin mixed micelles along with phospholipids, lower glycerides,

and fatty acids (38). It is known that the addition of traceamount of bile or bile salts to serum-containing Giardia culturemedium stimulates trophozoite growth, most likely by enhanc-ing the transfer of lipids from the medium to the parasite (23,39, 40). Bile salts, whose concentration in serum is negligible,are transported in serum in association with albumin. Further-more, it is known that if an excess of bile salts is added abovethe amount that can be carried by albumin, unesterifiedcholesterol and some phospholipids are removed from thelipoproteins to form mixed micelles with bile salts (38), whichare preferentially taken up by the parasite (23).To further analyze the mechanisms of cholesterol consump-

tion by trophozoites, we first determined the concentration ofcholesterol in both complete and LPDM, before and duringGiardia culture. Results showed that the cholesterol contentwas -200 ,uM in normal growth medium and -10 A/M inLPDM (Fig. 3A, 0 time); trace or no cholesterol was found inserum-free TYI-S-33 medium (not shown). During trophozo-ite cultivation, cholesterol content decreases -20% at 24 h(160 ,M) in complete medium, and to undetectable values inLPDM (Fig. 3A). While the total number of cells increased, thetotal cholesterol content per cell remains constant (Fig. 3B), anindication that cholesterol is utilized by Giardia for growth andproliferation. Because the parasite requires cholesterol tomake new membranes during growth, we hypothesized thatcholesterol starvation might trigger Giardia to encyst inLPDM. To test that possibility, we supplemented the deficientmedium with cholesterol. The minimal cholesterol concentra-tion required to totally block cyst formation was dependent onthe compound used to solubilize cholesterol: 20 ,tM whensolubilized in either cyclodextrins, ethanol, or DMSO (Fig. 3A,asterisk) and 5 AM when solubilized by bile salts (not shown).This agrees with our previous report that the degree ofcholesterol uptake by trophozoites depends on the compoundused to solubilize cholesterol in the medium (23).To further investigate the specificity of the inhibition of

encystation by cholesterol, we performed comparative reple-tion studies. The number of cysts and the percentage of viablecysts were determined after 24 h culture in LPD-TYI-S-33medium (pH 7.8). Table 2 shows that serum lipoproteins(except high density lipoprotein), serum lipid mixtures, andsolubilized cholesterol were most effective in inhibiting encys-tation. Under these conditions, cyst production was totallyabolished. Cyst yields decreased and cyst viability diminishedwhen either hydroxysterols, squalene, or lanosterol wereadded. Little or no effect was observed with either sitosterol,

B

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1.50

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0 6 12 18 24

Encystation Time (h)

0 1.25

t 1.00c -,Opc_2 0.75

2U_M

S 0.50o

u 0.25- *

0.00

0 6 12 18

Encystation Time (h)

FIG. 3. Cholesterol in Giardia and culture media during growth and encystation. Trophozoites (1.1 x 106 cells/ml) were cultured in growth(O) or LPDM (El) for different times. Cells were harvested and the cholesterol content in media (A) and cells (B) determined as described. Valuesrepresent the mean of two independent experiments performed in duplicate ± SD. Line with asterisk represents the minimum concentration ofcholesterol required to totally block Giardia encystation in LPDM.

I

A220 -

200 -

180 -

.160-

140 -

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Table 2. Effects of lipid repletions

Addition to lipoprotein-deficient Total cysts Viable cysts,TYI-S-33 medium X 106/ml (%) %

None 1.60 ± 0.0 (100) 37Lipoproteins and other natural lipidsLDL (100 ,ug/ml) 0.00 ± 0.0VLDL (100 ,ig/ml) 0.00 ± 0.0HDL (100 ,ug/ml) 1.62 ± 0.1 (104) 40Bovine lipids (0.01%) 0.00 ± 0.0Bovine bile (0.02% wt/vol) 0.10 ± 0.0 (6) 0

Cholesterol (0.2 mM)Cholesterol/bile salts mix (5 mM) 0.00 ± 0.0Cholesterol/cyclodextrins (0.05%) 0.00 ± 0.0Cholesterol/ethanol (0.01%) 0.00 ± 0.0

Phosphatidylcholine (0.2 mM)Phosphatidylcholine/bile salts mix (5 mM) 1.48 ± 0.6 (93) 31Phosphatidylcholine/cyclodextrins (0.05%) 1.53 ± 0.9 (96) 26Phosphatidylcholine/ethanol (0.01%) 1.53 ± 0.1 (96) 33

Conjugated bile saltsBile salts mix (5 mM) 1.17 ± 0.1 (73) 38Glycocholic acid (1.08 mM) 1.42 ± 0.0 (89) 41Glycodeoxycholic acid (0.54 mM) 1.22 ± 0.2 (76) 27Glycochenodeoxycholic acid (1.08 mM) 1.26 ± 0.0 (79) 36Taurocholic acid (0.36 mM) 1.36 ± 0.4 (85) 40Taurodeoxycholic acid (0.18 mM) 1.23 ± 0.1 (77) 38Taurochenodeoxycholic acid (0.36 mM) 1.20 ± 0.8 (75) 31

Sterols (0.2 mM)25-Hydroxycholesterol/ethanol (0.01%) 0.38 ± 0.5 (24) 257-B-Hydroxycholesterol/ethanol (0.01%) 0.88 ± 0.1 (55) 12B-Sitosterol/ethanol (0.01%) 1.21 ± 0.2 (76) 21Lanosterol/ethanol (0.01%) 0.56 ± 0.3 (35) 33Squalene 0.46 ± 0.3 (29) 14

Fatty acidsOleic acid 1.61 ± 0.2 (101) 31Palmitic acid 1.60 ± 0.1 (100) 48Myristic acid 1.47 ± 0.2 (92) 37Linoleic acid 1.60 ± 0.1 (100) 36

SolventsCyclodextrins (0.05%) 1.99 ± 0.0 (124) 46Ethanol (0.01%) 1.60 ± 0.0 (100) 32

VLDL, very low density lipoprotein; HDL, high density lipoprotein

phosphatidylcholine (Table 2), or primary bile salts (notshown). Bovine bile blocked encystation by 94%, but conju-gated bile salts, individually or in mixtures, inhibited by lessthan 25%, indicating bile constituents other than conjugatedbile salts are responsible for suppression of encystation.

Effects of Cholesterol on CWP mRNA Levels. To assess thepoint in encystation at which cholesterol and other compoundsinhibit cyst formation, the steady-state levels of CWP1 andGDH (a housekeeping gene) mRNAs were examined and thehybridization signals quantified by phosphorimage analysis.Fig. 4 shows that CWP1 mRNA levels, but not GDH mRNAlevels, are dramatically reduced (-99%) in cells cultured inLPDM in the presence of cholesterol. Taurodeoxycholate,lanosterol, and 25-hydroxycholesterol showed less inhibitoryactivity on the CWP1 mRNA levels (45%, 32%, and 68%,respectively). Consistent with the morphological results, sol-vents used to solubilize cholesterol (e.g., ethanol, Fig. 4), orphosphatidylcholine, primary bile salts, or fatty acids had noeffect on the level of CWP1 mRNA (results not shown).Neither cell viability, flagellar movement, nor trophozoiteattachment was altered by these treatments.To examine the mechanism of the partial inhibition of

encystation observed with conjugated bile salts (Table 2 andFig. 4), cells were cultured for 6 h in LPDM (to allow theconsumption of cholesterol). Then, either cholesterol or tau-rodeoxycholic acid was added to the culture, trophozoites wereharvested after 0, 3, and 6 h, and the transcripts analyzed by

slot-blot analysis. Fig. 5 shows that the levels of CWP1 mRNAwere down-regulated when cholesterol was added to encystingcells. In contrast, CWP1 mRNA levels increased with time inthe control without addition of cholesterol. Because taurode-oxycholate showed no effect on- encysting trophozoites in theabsence of cholesterol, these results indicate that the partialinhibition of encystation by conjugated bile salts is likely due

addition to LPDM GDH CWP

cholesterol _

25,hydroxycholesterol _

taurodeoxycholate _

lanosterol _

ethanol ^ .

FIG. 4. Expression of CWP1 mRNA during culture in LPDM.Giardia trophozoites were cultured in LPDM for 24 h in the presenceof either 200 ,uM of cholesterol, lanosterol, 25-hydroxycholesterol, ortaurodeoxycholate, or 0.01% ethanol (solvent control). Total mRNAwas extracted and applied to nylon membrane filters (2,ug per slot) andhybridized with 32P-labeled oligonucleotides for the housekeepingenzyme GDH or CWP1, as described.

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50time after additon h)probe 1 2 3 addition

GDH 1 1CWP I I I none

GDH I

CWP Igg cholesterol

GDH I I ltaurodeoxycholate

CWP j I I

FIG. 5. Kinetics of CWP mRNA accumulation in the presence ofcholesterol. Giardia trophozoites were cultured in LPDM for 6 h, thenexposed to 200 jLM of cholesterol or taurodeoxycholate for 1, 2, or 3 h.GDH and CWP1 mRNA levels were analyzed for each time point asdescribed in Fig. 4.

to their ability to enhance cholesterol uptake and not due toa direct effect on the trophozoites. Similar results were ob-served when CWP2 transcripts were analyzed (not shown).High Concentrations of Bile Salts Inhibit Cholesterol Up-

take by Giardia. Several reports have suggested that highconcentrations of bile salts are responsible for Giardia encys-tation (4-6). Reiner et al. (37) reported that the absence of cystantigens and reduced secretory vesicle formation in the WB-derived clone A6 during encystation in vitro correlate with adeficiency of bile salt uptake by this subline. Nevertheless,reports by others (41), as well as the work we present here,clearly establish that encystation is possible in the absence ofbile. To analyze whether the concentration of bile salts playsa role in encystation, the uptake of fluorescent cholesterol bytrophozoites was measured in the presence of increasingconcentrations of taurodeoxycholate. As shown in Fig. 6, traceamounts of bile salts added to the culture medium enhancecholesterol uptake; however, concentrations above the criticalmicelle concentration blocked transfer of labeled cholesterolto the parasite. These results indicate that the concentration ofbile salt molecules in solution and their physical state (e.g.,monomers or micelles, ref. 42) are important for cholesteroluptake by Giardia.

DISCUSSIONLiving cells can respond to changes in the extracellular envi-ronment by modulating metabolic functions. For example,many microorganisms respond to stresses such as nutrientdeprivation by initiating a process of differentiation to aquiescent and environmentally stable form (43-45). Giardia,like most eukaryotic cells, requires cholesterol for membranebiogenesis, but is unable to synthesize cholesterol and phos-pholipids de novo (18). In this work, we present compellingevidence that cholesterol starvation is necessary and sufficientfor Giardia trophozoites to differentiate into environmentallyresistant cysts.We also demonstrated a secondary role for bile salts in

Giardia encystation. Bile salts are steroids that aid in thedegradation, solubilization, and absorption of lipids from theintestinal lumen (20, 21, 46-48). The primary bile salts aresynthesized from cholesterol by the liver, conjugated to eitherglycine or taurine, and stored in the gallbladder (49, 50).During digestion, these compounds are released into theproximal duodenum and are later reabsorbed by an activetransport system in the distal ileum (21, 46, 49, 50). Previousreports suggested that bile is essential for Giardia encystation

'0 40

11c1

i 230

0

_ =

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- l \

OTI I I °-0_?~~~~I

0 1 2 3 4 5 6 7taurodeoxycholate(mM)

FIG. 6. Analysis of fluorescent cholesterol transfer from the cul-ture medium to Giardia trophozoites. NBD-cholesterol was incorpo-rated into LDL as described and incubated with trophozoites for 5 minat 37°C in the presence of increasing concentrations of taurodeoxy-cholate. Subsequently, the percentage of fluorescent probe transferredto the cells was determined. Values represent the mean ± SD of threeindependent experiments performed in duplicate.

(4-6); however, these studies failed to clearly demonstrate therole of bile salts in the differentiation of this parasite. Wepropose that the effects of bile salts on encystation are directlyrelated to the uptake of cholesterol by the trophozoites. Ourconclusions are supported by the following observations: (i)Giardia can grow in the absence of bile salts (22) indicating thatthese compounds are not essential for the trophozoites; (ii)trace amounts of bile salts stimulate growth of Giardia becausethey solubilize lipids and increase their availability to the cells(23, 40); (iii) paradoxically, high concentrations of bile saltsinhibit uptake and therefore favor encystation (this work).Additionally, some studies have shown that the jejunum, wherethe bile concentration is highest, is the primary site of colo-nization (14, 15) and not encystation of Giardia trophozoites.Based on our results, we propose the following model for

encystation in vivo: Trophozoites colonize the mammalianmid-jejunum of mammals, an environment rich in dietary(400-700 mg/day) and biliary (750-1200 mg/day) cholesterol(21, 38). Cholesterol absorption occurs almost completely inthe jejunum, whereas bile acid absorption occurs in the lowerileum (21, 37, 49, 50). Bacterial populations in the lower smallintestine cause profound chemical changes to the cholesterolmolecules, thereby decreasing the amount of cholesterol avail-able for parasite growth. As trophozoites travel down theintestine, they confront an environment increasingly devoid ofcholesterol, and it is this cholesterol starvation that triggersparasite differentiation. Gillon et al. (42) reported that ingerbils, the greatest trophozoite numbers were found in themid-jejunum, whereas relatively few organisms were found inthe duodenum (where the pH is acid) or in the ileum (wherethe biliary lipid concentration is lower) (49, 50). Studies in thesuckling mouse model (4) and in the gerbil model (29) showedthat cysts are found primarily in the lower two-thirds of thesmall intestine and in the colon, where most of the dietary andbiliary lipids have been absorbed or modified (49, 50).

Precisely how cholesterol deprivation induces encystation isnot obvious, but there are two possible mechanisms we canpropose for future studies: (i) cholesterol-induced changes inplasma membrane fluidity of trophozoites, and (ii) cholesterol-mediated regulation of transcription. The former model dic-tates that the physical state of the plasma membrane can beinfluenced by the cholesterol concentration. Changes in mem-

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brane cholesterol content are inversely related to membranelipid fluidity (51, 52). That cholesterol plays an important rolein membrane function is evidenced by the fact that changes incholesterol levels or the cholesterol-phospholipid ratio dra-matically influence many membrane-related activities, such aspermeability and enzyme and receptor function (53-56). De-pletion of cholesterol in the Giardia environment might in-crease the fluidity of trophozoites' plasma membrane, therebymodulating the activity of membrane-associated proteins,which, directly or indirectly, may trigger a cascade of signaltransduction mechanisms that culminate with encystation-specific gene expression.On the other hand, the cellular concentration of cholesterol

is maintained in mammalian cells within a narrow range bycomplex feedback mechanisms that control sterol synthesisand uptake by means of the LDL receptor. High intracellularconcentrations of cholesterol suppress the transcription ofgenes for several cholesterol biosynthetic enzymes and theLDL receptor (17, 57-59). In Giardia, the intracellular con-centration of cholesterol may be directly dependent on theexternal concentration of this compound. The high levels ofcholesterol available to the parasite in either the serum- andbile-rich culture medium or the upper small intestine mightrepress the transcription of CWP genes, but when the envi-ronmental cholesterol concentration reaches a critical lowlevel, transcription of the genes needed for encystation may beactivated.

1. Adam, R. D. (1991) Microbiol. Rev. 55, 706-732.2. Craun, G. F. (1990) in Giardiasis, ed. Meyer, E. A. (Elsevier,

Amsterdam), pp. 267-293.3. Sogin, M. L., Gunderson, J. H., Elwood, H. J., Alonso, R. A. &

Peattie, D. A. (1989) Science 243, 75-77.4. Gillin, F. D., Reiner, D. S., Gault, M. J., Douglas, H., Das, S.,

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