INFECTION AND IMMUNITY, June 1996, p. 2151–2157
Copyright ? 1996, American Society for Microbiology
Vol. 64, No. 6
Inhibition of Giardia lamblia Excystation by Antibodies against
Cyst Walls and by Wheat Germ Agglutinin
TZE-CHIANG MENG,1MICHAEL L. HETSKO,2AND FRANCES D. GILLIN2,3*
Division of Infectious Diseases, Department of Medicine,1Department of Pathology,2and Center for Molecular
Genetics,3University of California at San Diego Medical Center, San Diego, California 92103-8416
Received 6 December 1995/Returned for modification 9 January 1996/Accepted 8 March 1996
Although excystation is crucial to the initiation of infection by Giardia lamblia, little is known about the
regulation of this important process. We have been able to reliably induce excystation in vitro by mimicking
cyst passage through the stomach and upper small intestine by the exposure of in vitro-derived cysts to an
acidic, reducing environment (stage I) followed by protease treatment at a slightly alkaline pH (stage II).
Preexposure of cysts to polyclonal rabbit antiserum against purified cyst walls (PCWs) or to wheat germ
agglutinin (WGA) inhibited excystation by >90%. Adsorption of either ligand with PCWs eliminated inhibition,
demonstrating specificity for cyst wall epitopes. Inhibition by WGA was reversed by either chitotriose or sialic
acid, while inhibition by polyclonal antibodies against PCWs (anti-PCW) was reversed only by sialic acid,
which also inhibited binding of both ligands to intact cysts and to cyst wall antigens in immunoblots. Binding
of anti-PCW did not affect acidification of cyst cytoplasm during stage I. Exposure of cysts to anti-PCW and
WGA prior to, but not after, stage II was sufficient to inhibit excystation, and inhibition could be partially
reversed by increasing the protease concentration during stage II. A 7- to 10-fold higher proportion of WGA-
and anti-PCW-treated cysts than control cysts remained intact after stage II. Our results suggest that these
ligands, which bind cyst wall epitopes, inhibit excystation, most likely by interfering with proteolysis of cyst wall
glycoproteins during stage II.
Giardia lamblia is a major cause of waterborne gastrointes-
tinal disease in the United States and of malnutrition from
malabsorption worldwide (35). The trophozoite form that col-
onizes the human intestinal tract is capable of differentiating
into a resistant cyst in response to appropriate stimuli (1).
Cysts are absolutely required for survival outside the host, as
well as for initiating new infections (28). Ingested cysts un-
dergo excystation in the upper small intestine of the new host,
releasing the disease-causing trophozoites (1). In prior studies
(4), we completed the life cycle of G. lamblia in vitro, demon-
strating for the first time that excystation of in vitro-derived
cysts, like that of fecal cysts (3, 29), could be accomplished by
mimicking passage through the acid-filled stomach and into
the protease-rich upper small intestine. Since this critical dif-
ferentiation process is very poorly understood, we have begun
to dissect the mechanism(s) of excystation.
The cyst wall must be sufficiently impervious to protect the
parasite within from the rigors of both the external environ-
ment and passage through the stomach but must also permit
response to the external signals triggering excystation. Purified
cyst wall structures (PCWs), which morphologically resemble
the wall of intact cysts, are produced by boiling cysts in sodium
dodecyl sulfate (SDS) (11). Chemical analyses suggest the pri-
mary structural component of PCWs is a polymer of galac-
tosamine (GalN) and/or N-acetylgalactosamine (GalNAc)
(11). Under reducing conditions, several proteins and glyco-
proteins, some containing N-acetylglucosamine (GlcNAc) (21,
25), are released from PCWs (26). Recently genes encoding
two leucine-rich cyst wall proteins have been identified (14,
In our prior studies on expression and transport of cyst wall
antigens, we identified two groups of cyst antigens (10, 24, 26).
Group I antigens consist of several broad protein bands, ?26
to 46 kDa, that are expressed beginning within ?3 h of encys-
tation and are each recognized by the monoclonal antibodies
(MAbs) GCSA-1 and 8C5 (5, 26, 34). Immunoelectron micros-
copy demonstrated that these epitopes localize to the interior
of the encystation-specific secretory vesicles and to the cyst
wall (16, 27) and also appear to be recognized by polyclonal
antibodies against PCWs (anti-PCW). Group II antigens con-
sist of at least four glycoprotein bands, ?66, 85, 120, and 140
kDa, that localize to the cyst wall and are recognized by wheat
germ agglutinin (WGA) (24, 25), which is specific for (GlcNAc)n
or sialic acid, as well by anti-PCW serum. In previous investi-
gations of host immune responses, we found that many pa-
tients with giardiasis have serum and/or mucosal antibodies
which preferentially recognize these WGA-binding cyst wall
These findings prompted us to initiate the first studies of the
mechanism of excystation of G. lamblia in vitro by examining
whether antibodies and lectins that bind to the outside of the
cyst wall could interfere with this differentiation process.
MATERIALS AND METHODS
Materials. Unless otherwise specified, reagents were obtained from Sigma
Chemical Co. (St. Louis, Mo.). Bicarbonate was purchased from Fisher Scientific
Co. (Pittsburgh, Pa.). Adult bovine serum was purchased from HyClone (Logan,
Cultivation of parasites. G. lamblia trophozoites (strain WB, ATCC 30957,
clone C6) were routinely cultivated at 37?C in Diamond TYI-S-33 medium
containing 10% adult bovine serum (7), pH 7.1, supplemented with bovine bile
(12) (0.5 mg/ml) but without added iron, vitamins, or antibiotics. Trophozoites
were subcultured twice weekly.
Preparation of cysts in vitro. Cultures grown to late-log phase were chilled and
trophozoites were enumerated in a hemacytometer. Trophozoites (3,000/ml,
final concentration) were added to freshly prepared pre-encystation medium (4):
TYI-S-33 medium containing 10% adult bovine serum, pH 7.1, without bile.
After 72 h, the attached monolayer (?60% confluent) was refed with encystation
medium: TYI-S-33 medium containing 10% adult bovine serum, adjusted to pH
7.8 with 1 M NaOH, and supplemented with lactic acid (hemi-calcium salt, 5
* Corresponding author. Mailing address: UCSD Medical Center,
Department of Pathology, 214 Dickinson St., CTF-403C, San Diego,
CA 92103-8416. Phone: (619) 543-6146. Fax: (619) 543-6614. Elec-
tronic mail address: firstname.lastname@example.org.
mM), porcine bile (0.25 mg/ml), piperacillin (500 ?g/ml; Lederle Laboratories,
Carolina, P.R.), and amikacin (125 ?g/ml). After approximately 66 h, the me-
dium containing the nonattached population, including the cysts, was removed,
and the cells were harvested by centrifugation at 2,200 ? g for 5 min at room
temperature. Trophozoites and incomplete cysts were lysed by incubation in
double-distilled water for 20 min at room temperature. After several low speed
(1,140 ? g) washes at 4?C, water-resistant cysts were stored in double-distilled
water at 4?C overnight, prior to excystation.
Excystation of in vitro-derived cysts. In vitro-derived cysts were enumerated
on the day of excystation in a hemacytometer chamber, and viability was deter-
mined by diluting cysts in an equal volume of 0.4% trypan blue (Gibco-BRL,
Gaithersville, Md.). The percentage of type I cysts, which are smooth, slightly
refractile, oval bodies with the organelles of fecal cysts and are more likely to be
viable (8), was determined by differential interference contrast microscopy. Un-
less otherwise specified, excystation was induced by a modification of the two-
step method of Rice and Schaefer (4, 29). Stage I solution was freshly made by
mixing 6.8 ml of Hanks buffered salt solution (pH 6.8) containing L-cysteine (57
mM) and reduced glutathione (32.5 mM) with 6.8 ml of 0.1 M NaHCO3and
11.36 ml of water and then adjusting the pH to 4.0 with HCl. Cysts (1.5 ? 106
total, unless otherwise specified) were pelleted at 8,300 ? g at 4?C, resuspended
in 550 ?l of prewarmed stage I solution, and incubated for 20 min at 37?C. Cysts
were then pelleted and resuspended in 1 ml of prewarmed stage II solution
(Tyrodes buffered salt solution [pH adjusted to 8.0 with 7.5% NaHCO3] with
1-mg/ml bovine trypsin type II) for 1 h at 37?C. Cysts were then pelleted at 8,300
? g and resuspended in 100 ?l of cultivation medium for 30 min at 37?C (stage
III). The emerged motile trophozoites were enumerated by a hemacytometer.
The percent excystation was calculated as the sum of the motile trophozoites and
partially emerged trophozoites divided by the initial number of viable type I cysts.
Antibodies against cyst wall antigens. PCWs were obtained by boiling water-
resistant in vitro-derived cysts in 1% SDS for 10 to 15 min (11). New Zealand
White rabbits were immunized intravenously with 106PCWs without adjuvant,
twice weekly for five immunizations (26). Complement was inactivated by incu-
bation of serum for 1 h at 56?C prior to use. MAb GCSA-1 was a gift from H.
Ward (34) and MAb 8C5 was a gift from G. Faubert (5).
Inhibition of excystation. Unless otherwise stated, in vitro-derived cysts were
preincubated prior to stage I in 0.1 to 0.4 ml of diluted antiserum (10% in water)
or lectin diluted in lectin solution (73 mM NaCl, 100 mM MgCl2, 100 mM CaCl2)
for 1 h at 4?C. Preincubation in water, preimmune serum, or lectin solution,
respectively, was used as a control to calculate percent inhibition. After deter-
mining reliable inhibitory concentrations, specificity of the inhibition was deter-
mined by adsorption and sugar competition. Reduced PCWs were produced by
boiling in vitro-derived cysts in 1% SDS for 5 min in the presence of 70 mM
dithiothreitol. Anti-PCW serum (10%) was adsorbed against PCWs or reduced
PCWs (final concentration, 5 ? 107/ml) overnight at 4?C on a rocker. Initial
experiments indicated that overnight incubation of WGA resulted in loss of
inhibitory activity; therefore, WGA (10 ?g/ml) was incubated with PCWs or
reduced PCWs for 1 h at 4?C immediately prior to preincubation with the cysts.
Competition for binding was evaluated by adding the sugar to diluted antibody
or lectin before the addition of cysts. Unbound antibodies, lectin, or sugar was
removed by pelleting the cysts at 8,300 ? g for 5 min at 4?C. Stability of binding
was evaluated by preincubating cysts with anti-PCW serum or WGA, removing
unbound antibody or WGA, and then incubating the treated cysts with sialic acid
or chitotriose. The effect of the timing of exposure was evaluated by varying the
stage at which the cysts were preincubated with anti-PCW serum or WGA.
The efficiency of excystation varied among experiments, as would be expected
of a complex differentiation process. Therefore, the mean inhibition by various
ligands was normalized to the respective control for each experiment. All exper-
iments were repeated at least twice. P values were calculated by paired two-tailed
t test. Data shown are means ? standard deviations for at least two independent
experiments. To calculate the mean reversal of inhibition by competing sugars,
the results of each experiment were normalized to the maximal inhibition by
antibody or WGA for that experiment. P values were calculated by paired
two-tailed t test with the normalized comparison value set at 100% for control
excystation or 0% for maximal inhibition.
Measurement of cyst cytosol pH. Cysts were resuspended to 107/ml and incu-
bated with intracellular pH indicator 2?,7?-bis-(2-carboxyethyl)-5 (and-6)-car-
boxyfluorescein-acetoxymethyl ester (BCECF-AM) (5 mM; Molecular Probes,
Eugene, Oreg.) (30) in labeling buffer (pH 6.9; 10 mM N-2-hydroxyethylpipera-
zine-N?-2-ethanesulfonic acid [HEPES], 130 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 10 mM glucose) at 37?C for 30 min. Excess label was removed by washing
three times with labeling buffer, and then cysts were preincubated with antibody
(10%) as described above. After washing the cysts three times with water, 100 ?l
of cyst suspension (1.6 ? 107to 2.0 ? 107/ml) was added to 1.9 ml of the target
pH buffer (pH[out]). Emission at 535 nm was determined with excitation at 505
and 439 nm by using a Perkin-Elmer LS3 fluorescence spectrophotometer. In-
ternal pH (pH[in]) was determined by interpolation from a standard curve of the
535 nm emission ratio, with excitation at 505 and 439 nm, of labeled cysts in high
potassium pH buffers [pH range 4.0 to 7.8; 125 mM KCl, 1.2 mM KH2PO4, 1 mM
MgSO4, 1.2 mM CaCl2; buffered with 10 mM sodium acetate for pH 4.0 and 5.0;
10 mM piperazine-N,N?-bis(2-ethanesulfonic acid) (PIPES) for pH 6.0 and 6.4;
or 10 mM HEPES for pH 6.8, 7.2, and 7.8] in the presence of the potassium
ionophore nigericin (10 mM; Molecular Probes), which equalizes pH[in]and
Preparation of immunoblots. Cyst antigens for replicate immunoblots were
prepared by scaling up excystations 5- to 10-fold, maintaining the ratio of cyst
number to stage I and stage II solution volumes as previously described. Cells
were washed three times in water in the presence of 1 mM phenylmethylsulfonyl
fluoride and 1 mM trans-epoxysuccinyl-L-leucyl(4-guanidino)-butane (E64).
Cells were resuspended to a concentration of 3 ? 108/ml in SDS sample buffer,
with or without dithiothreitol (35 mM final concentration), and boiled for ?5
min. Proteins were separated by SDS–10% polyacrylamide gel electrophoresis
(13) and transferred to nitrocellulose (33). After blocking for 1 h, blots were
incubated in antibody diluted in gelatin buffer (25) (1:100) or WGA-horseradish
peroxidase (HRP) diluted in lectin solution (0.5 ?g/ml) for 1 h. Goat anti-rabbit
immunoglobulin G alkaline phosphatase (1:3,000) was utilized to localize the
rabbit polyclonal antibodies. To evaluate whether sugars block recognition of
specific cyst wall epitopes, anti-PCW serum (1.0%) and WGA-HRP (20 ?g/ml)
were preincubated with chitotriose or sialic acid (50 and 100 mM, respectively)
for 30 min at 4?C prior to addition to immunoblots of PCW antigens.
Immunocytochemistry. Cysts were incubated with anti-PCW serum (10%),
WGA-HRP (1:2,000), or their respective controls, with or without prior incuba-
tion of ligand with chitotriose or sialic acid (50 mM), for 1 h at 4?C. Antiserum-
treated cysts were washed three times with ice-cold phosphate-buffered saline
(PBS) and then incubated with anti-rabbit immunoglobulin G-HRP (1:500) for 1
h at 4?C. After washing, cysts were developed in substrate (0.5-mg/ml 4-chloro-
1-naphthol, 16.7% [vol/vol] methanol, and 0.015% [vol/vol] H2O2in PBS) and
observed under differential interference contrast microscopy (24).
Inhibition of excystation. Preincubation of in vitro-derived
cysts in anti-PCW serum at a concentration of 10% consis-
tently inhibited excystation ?90% (Fig. 1), while preincubation
in preimmune serum did not. To determine which of the an-
tigens recognized by anti-PCW serum were involved in inhibi-
tion, we tested MAbs GCSA-1 and 8C5, which are specific for
the group I antigens, and WGA, which is specific for the group
II antigens. Preincubation of cysts in the MAbs had no appar-
FIG. 1. Effects of anti-PCW serum and lectins on excystation. Percent excys-
tation is compared with preimmune serum or lectin solution, respectively. The
means ? one standard deviation of two experiments are shown. Cysts were
preincubated in diluted serum or lectin prior to stage I of excystation. ?, P ? 0.05,
paired two-tailed t test, compared with preimmune serum or lectin solution
controls; Con A, concanavalin A.
2152MENG ET AL.INFECT. IMMUN.
ent inhibitory effect, with maximal inhibition of ?20 and ?7%
for MAbs GCSA-1 and 8C5, respectively, at ascites concentra-
tions of 25%. WGA, on the other hand, consistently inhibited
excystation at a concentration of 10 ?g/ml (Fig. 1). This sug-
gests that the group II glycoprotein antigens, rather than the
group I antigens, are involved in the inhibition of excystation,
although the lack of effect of the MAbs may be secondary to
limited avidity or a narrow range of epitope recognition. Inhi-
bition was proportional to the concentrations of WGA (Fig. 1)
and anti-PCW (inhibition at 5% was less than 10% [data not
shown]). Preincubation of cysts in WGA or antibody did not
affect cyst viability, as determined by trypan blue exclusion
(data not shown; see also below).
As WGA recognizes oligomers of D-N-acetylglucosamine
(GlcNAc)nor N-acetylneuraminic acid, we evaluated several
other lectins to determine if inhibition of excystation was spe-
cific to certain carbohydrate epitopes. Lycopersicon esculentum
lectin, which binds (GlcNAc)3, recognized the group II anti-
gens on immunoblots (data not shown) but did not reliably
inhibit excystation. Phaseolus limensis lectin, which recognizes
GalNAc and has been reported to bind to cysts (11), failed to
inhibit excystation. Concanavalin A, which recognizes ?-D-
mannose or ?-D-glucose and binds an 85-kDa protein on im-
munoblots of cyst antigens (23), also failed to inhibit excysta-
tion (Fig. 1); however, this epitope does not appear to be
exposed on the external cyst surface. Datura stramonium lectin
(which recognizes [GalNAc]2) and Tetragonolobus purpureas
lectin (which recognizes ?-L-fucose) have not been shown to
bind to cysts and did not inhibit excystation (data not shown).
Reversal of inhibition. Inhibition of excystation by anti-PCW
serum was overcome by increasing the concentration of cysts
(Table 1), suggesting that the inhibitory factor could be titrated
or adsorbed out. Specificity of the inhibition to antibodies
directed against cyst wall epitopes was shown by abrogation of
inhibition when anti-PCW serum (10%) was adsorbed with
PCWs (Table 1) prior to excystation. Since boiling PCWs in
reduced sample buffer released many proteins and glycopro-
teins, as determined by immunoblot, we also adsorbed anti-
PCW serum with reduced PCWs prior to excystation. Surpris-
ingly, inhibition was also abrogated by reduced PCWs (Table
1), suggesting that the ligand(s) associated with inhibition was
either strongly attached to the cyst walls or intrinsic to the cyst
wall. Not unexpectedly, inhibition by WGA was also eliminated
by adsorption with PCWs or reduced PCWs (Table 1).
WGA-mediated inhibition of excystation was efficiently
blocked by preincubation with triacetylchitotriose (GlcNAc)3
or sialic acid (10 mM) (Fig. 2), probably through competition
for WGA binding. Interestingly, the inhibitory action of anti-
PCW serum was prevented by coincubation with sialic acid but
not by triacetylchitotriose or other sugars (Fig. 2). Importantly,
lack of competition by glucuronic acid supports the specificity
for sialic acid.
Moreover, binding of anti-PCW and WGA to cysts was re-
versible. Addition of sialic acid, but not chitotriose, after cysts
were preincubated with anti-PCW reversed the inhibition of
excystation by anti-PCW serum, presumably by displacing
bound ligand; both sugars reversed inhibition by WGA (data
not shown). This also demonstrated that binding by WGA or
anti-PCW serum did not kill the cysts. The sugars alone did not
affect excystation at the same concentrations (data not shown).
Immunocytochemistry demonstrated that intact cysts were rec-
ognized by anti-PCW serum but not by preimmune serum. This
recognition was blocked by the addition of sialic acid but not by
chitotriose (Fig. 3). Interestingly, preincubation of anti-PCW
serum and WGA with sialic acid, but not chitotriose, abrogated
recognition of cyst wall epitopes on immunoblots (Fig. 4).
These results suggest that the binding epitope(s) for the inhib-
itory antibodies, like that for WGA, is a carbohydrate-contain-
ing moiety of the cyst wall.
Stage of inhibition by anti-PCW serum and WGA. In order
to further evaluate the mechanism of inhibition by anti-PCW
or WGA, we examined the effects of ligand binding on the
individual steps of in vitro excystation. Stage I of excystation
primarily mimics passage of the cyst through the acid-filled
stomach (3), while stage II primarily mimics passage of the cyst
into the protease-rich, slightly alkaline environment of the
small intestine, distal to the entry of the common bile duct
(29). To determine if antibody binding interferes with the
physiologic response to external acidification or alkalinization,
we measured internal cyst pH as a function of external pH in
the presence and absence of bound anti-PCW or preimmune
serum. While mean pH[in]decreased by 0.83 ? 0.48 pH units
when cysts were placed in stage I solution (pH 4), no differ-
ences were found between cysts preincubated in anti-PCW
serum (mean decrease, 0.90 ? 0.51 pH units) or preimmune
serum (mean decrease, 0.70 ? 0.38 pH units). Antibody-
treated and control cysts also had similar cytoplasmic pH re-
sponses to mild external alkalinization (data not shown).
We next examined whether antigens recognized by anti-
PCW serum or by WGA change during excystation and if this
was affected by the presence of bound ligand. We compared
immunoblots of cysts preincubated with preimmune serum,
anti-PCW serum, or WGA before excystation, after stage I and
after stage II. The patterns of the antigens recognized by either
anti-PCW serum or WGA were altered after both stage I and
stage II. However, no major differences were observed among
cysts pretreated with preimmune serum, anti-PCW serum, or
WGA (data not shown).
Although there was little difference in immunoblots, differ-
ential interference contrast microscopy revealed that cysts pre-
incubated with either ligand appeared much less degraded
TABLE 1. Blockage of inhibition by increasing cyst numbers or by
adsorption with PCWs or reduced PCWs
% Excystation with indicated
No. of cysts
1 ? 106
5 ? 106
1 ? 107
Reduced PCW adsorbed
Reduced PCW adsorbed
aIncreasing cyst number during preincubation (1 ml constant volume of 10%
anti-PCW) reduced the inhibitory effect, suggesting the inhibitory factor could be
titrated out. The percent excystation of controls also appeared to decrease with
increasing cyst concentrations, possibly because of decreased efficiency of pro-
teolysis during stage II. Adsorption of anti-PCW serum (10%) or WGA (10
?g/ml) with PCWs or reduced PCWs (5 ? 107per ml of diluted serum or lectin)
prior to preincubation with cysts (4.5 ? 105per condition) reversed the inhibition
of excystation, indicating specific binding to PCW epitope(s).
bAbsolute percent excystation.
VOL. 64, 1996INHIBITION OF GIARDIA EXCYSTATION 2153
than their respective controls after stage II (Fig. 5). A greater
proportion of treated cysts retained the smooth, refractile, oval
appearance of intact cysts (WGA 7.7% ? 2.5% versus control
1.1% ? 1.5%, P ? 0.12; anti-PCW 9.3% ? 2.2% versus pre-
immune 0.6% ? 1.1%, P ? 0.04; paired two-tail t test) (Fig. 5B
and D), suggesting that degradation of the cyst wall is neces-
sary for the emergence of G. lamblia trophozoites.
To further elucidate the mechanism by which inhibition by
FIG. 2. Percent reversal of (A) WGA- or (B) anti-PCW-serum-mediated inhibition of excystation by coincubation with the indicated sugar concentration (mM).
Values are the means ? one standard deviation of at least two separate experiments with each sugar, and the reversal of inhibition is normalized to the maximal
inhibition by WGA and anti-PCW serum for the corresponding experiments. ?, P ? 0.05, paired two-tailed t test, compared with WGA (10 ?g/ml) or anti-PCW serum
FIG. 3. Immunocytochemistry of cysts treated with anti-PCW serum in the
presence or absence of sugars. (A) Preimmune serum did not bind to intact-
appearing cysts. (B) Anti-PCW serum bound to intact cysts. Binding to nonintact
cysts is also present, although some reactivity likely represents trapping of con-
jugate. (C) Preincubation of anti-PCW serum with chitotriose (100 mM) did not
prevent binding to intact-appearing cysts. (D) Preincubation of anti-PCW serum
with sialic acid (50 mM) prevented binding to intact-appearing cysts. Bar, 10 ?m.
FIG. 4. Immunoblot of reduced PCW antigens in the presence or absence of
sugars. Lanes: 1, WGA-HRP; 2, WGA-HRP preincubated with chitotriose (100
mM); 3, WGA-HRP preincubated with sialic acid (50 mM); 4, anti-PCW serum;
5, anti-PCW serum preincubated with chitotriose (100 mM); 6, anti-PCW serum
preincubated with sialic acid (50 mM). Sialic acid, but not chitotriose, eliminated
recognition of PCW antigens by both WGA and anti-PCW serum. Sialic acid did
not interfere with peroxidase or alkaline phosphatase activity.
2154 MENG ET AL.INFECT. IMMUN.
these ligands occurs, the effect of timing of exposure to anti-
PCW or WGA was examined. Inhibition of excystation by
anti-PCW was most effective when cysts were incubated be-
tween stage I and stage II. Inhibition by WGA, however, was
equally effective if cysts were incubated prior to stage I or
between stage I and stage II (Fig. 6). Immunocytochemistry
demonstrated that both anti-PCW and WGA bound during the
preincubation prior to stage I were still present on the cyst
surface after stage I (data not shown). WGA may bind more
avidly and be more resistant to dissociation in the acidic and
reducing stage I solution. WGA may also bind to nonprotein
intrinsic cyst wall epitopes. Addition of anti-PCW serum or
WGA after stage II had no effect on excystation.
Our results suggest that inhibition of excystation by anti-
PCW serum or WGA is most likely mediated by interfering
with a stimulus or process during stage II. This process may
primarily involve degradation of the cyst wall, as increasing the
concentration of trypsin during stage II partially reversed the
inhibition by WGA and anti-PCW serum (Table 2).
The giardial cyst wall has previously been viewed primarily
as an inert protective structure. Although biochemical analyses
have yielded insights into cyst wall carbohydrate composition
(11, 15) and demonstrated the presence of a variety of proteins
(34) and glycoproteins (21, 25, 27), little is known about the
interactions of the giardial cyst wall with the host milieu. Our
ability to produce high-quality G. lamblia cysts in vitro allows
us to avoid the problems associated with fecal cysts, particu-
larly the unavailability of high-quality clinical specimens on a
regular basis, difficulties in purifying sufficient cysts from stool
contaminants, biologic hazards of other enteric pathogens in
clinical specimens, and extreme variation in biologic excysta-
tion efficiency (0 to ?90%) (31). We demonstrate here that
ligands that specifically bind to the wall of in vitro-derived cysts
FIG. 5. Appearance of cysts by differential interference contrast microscopy after excystation (stage III). Cysts appeared more degraded when they were
preincubated in lectin solution (A) or preimmune serum (10%) (C) than when they were preincubated in WGA (10 ?g/ml) (B) or anti-PCW serum (10%) (D)
suggesting that these ligands have interfered with the breakdown of the cyst wall during excystation. Ten times more of the cysts preincubated in anti-PCW serum
retained the smooth, refractile, oval morphology of intact cysts than did cysts preincubated in preimmune serum. Solid arrows, intact cysts; open arrows, motile excysting
trophozoites. Bar, 10 ?m.
FIG. 6. Effect of time of addition of anti-PCW or WGA on excystation.
Percent excystation is compared with matched preimmune serum or buffer con-
trols. Cysts were incubated in serum (10%) or WGA (10 ?g/ml) for 1 h at 4?C at
the stage indicated. SI, stage I; SII, stage II.
VOL. 64, 1996 INHIBITION OF GIARDIA EXCYSTATION2155
efficiently inhibit excystation. Our results suggest that the in-
hibition may be mediated by binding to cyst wall carbohydrate
epitopes, possibly in cyst wall glycoproteins.
Excystation is a complex process which occurs in response to
relatively rapid and extreme changes in temperature and pH,
as well as exposure to intestinal proteases. Upon ingestion by
a new host, cysts pass from a cold, hypotonic environment with
a pH of ?5 to 6 in water to the warm stomach with a pH of ?2
to 4, and then to the small intestine with a pH of ?7 to 8 (6).
Bingham and Meyer first showed that exposure to acid was the
key stimulus to excystation of fecal cysts in vitro (3). Subse-
quently, Rice and Schaefer demonstrated that the efficiency of
excystation of fecal cysts in vitro was increased by exposure to
a reducing environment and proteases (29), and we extended
those findings to in vitro-derived cysts (4). There is little un-
derstanding, however, of the transduction of these external
environmental signals across the cyst wall. Moreover, the phys-
iologic cellular responses of mainly dormant cysts (22) to these
changes have not been investigated previously.
We found that cyst cytoplasmic pH responded to changes in
ambient pH, but that cytoplasmic pH was maintained closer to
neutral than the external pH (pH[out]). Anti-PCW serum did
not appear to alter the overall change in pH[in]of cysts in
response to changes in pH[out], indicating that inhibition of
excystation by this ligand does not involve interference with the
cytosolic pH response.
As excystation involves exposure to proteases, another pos-
sible mechanism for inhibition by anti-PCW serum and WGA
is that bound ligand might prevent the digestion of an exposed
protein(s) on the cyst surface that is required for signaling or
for the emergence of the parasite. While there were clear
changes in the pattern of proteins and glycoproteins recog-
nized by anti-PCW serum and WGA after both stage I and
stage II, we observed only subtle differences among cysts pre-
treated with anti-PCW serum, WGA, or controls in immuno-
blots after exposure to proteases. The surface-exposed por-
tions of key cyst wall antigens that need to be cleaved or
altered for excystation may be small. Therefore, inhibition of
this alteration by bound antibody or WGA might not be de-
tectable. Another possibility is that the (hypothetical) alter-
ation is too subtle to be detected by immunoblots or that the
targets are not extractable from the cysts under the conditions
utilized for preparing immunoblots or reduced PCWs.
Our results support the hypothesis that both anti-PCW se-
rum and WGA interfere with stage II stimuli, possibly by
binding to sialic acid-containing glycoproteins, which may in-
clude the group II antigens. Free sialic acid may prevent the
inhibition of excystation by occupying the binding site of the
antibody, resulting in steric hindrance or conformational
The most striking difference we observed between cysts pre-
treated with anti-PCW serum or WGA and the controls was
the preservation of the morphologic appearance of many cysts
after stage II. This suggests that the binding of anti-PCW or
WGA prevented the normal breakdown of the G. lamblia cyst
wall during excystation. Mechanisms by which bound ligand
could interfere with excystation without affecting overall diges-
tion of the antigens recognized by either ligand include inhi-
bition of proteolysis, via steric hindrance, of adjacent cyst wall
proteins; cross-linkage of cyst wall epitopes, preventing cyst
wall conformational changes that allow release of emerging
parasites; and interference with signaling across the cyst wall
that may activate endogenous proteases or glycanases that
digest the cyst wall. The ability of increased trypsin concentra-
tions to partially reverse the inhibition supports a direct effect
of bound ligand on cyst wall proteolysis; lack of complete
restoration of excystation may be secondary to the inability of
trypsin to remove the interfering ligand completely, preventing
adequate access to the cyst wall surface.
Cyst wall antigens appear to be much more conserved (25)
than the variable surface proteins that form the predominant
surface antigen of trophozoites (2, 9, 19). Our original assump-
tion had been that antibodies directed against cysts would not
affect excystation because they would have to act on the exter-
nal surface and because of the speed at which excystation
occurs (?1 h). However, our present results suggest that cyst
wall antigens might be protective immunogens, since blocking
excystation could prevent infection.
Since as few as 10 viable cysts can establish infection (20,
28), inhibition of excystation would need to be complete or
near complete in order to be effective. In addition, binding by
endogenous mucosal antibodies would not occur until passage
of the cysts into the upper small intestine and therefore would
be competing with ongoing excystation. The presence of mul-
tiple proteases in the small intestine may also diminish the
effectiveness of this inhibition. Although the relevant inhibi-
tory antibody concentration present in our diluted anti-PCW
serum (10%) is likely to be higher than any achievable con-
centration of mucosal antibodies in vivo, the ‘‘inoculum’’ is also
much greater (1 ? 106to 5 ? 106cysts) than would be expected
in a naturally acquired infection. Binding of anti-PCW anti-
body also appears to be relatively stable, as demonstrated by its
continued inhibitory effect and its presence on the cyst surface
after exposure to the reducing and acidic conditions of stage I
solution. Vaccination of mothers may therefore be of benefit to
breast-fed infants, as maternal secretory antibodies (17, 25)
directed against the cyst wall might reduce the likelihood of
acquiring infection. Finally, cysts coated with mucosal anti-cyst
wall antibodies that are shed into drinking water supplies may
be less infective, resulting in decreased community transmis-
sion in areas of poor sanitation.
These studies demonstrate the importance of cyst wall
epitopes in excystation, provide the first insights into the phys-
iologic responses of giardial cysts to specific environmental
stimuli, and raise the possibility that antibodies directed
against the cyst wall might interfere with the transmission of
giardiasis. Further research is required to determine the exact
mechanism(s) of inhibition.
This work was supported by PHS grants AI24285, AI19863,
HD29275, and DK35108 from the National Institutes of Health.
We thank S. Pandol for his assistance with the intracellular pH
experiments; G. Faubert and H. Ward for their gifts of monoclonal
antibodies; A. Manzi, R. Chammas, and A. Varki for their advice and
critical reading of the manuscript; and D. S. Reiner for technical
TABLE 2. Partial reversal of inhibition of excystation by increasing
% Control excystation at trypsin concn (mg/ml)
WGA (10 ?g/ml)
17.6 ? 6.5
6.7 ? 5.8
64.3 ? 0.8b
17.3 ? 9.3
47.2 ? 5.0b
38.6 ? 2.3b
aCysts were preincubated for 1 h at 4?C with ligand or respective control and
excysted in the usual manner, except for varying the trypsin concentration during
stage II of excystation. Data are expressed as percent control excystation and are
the means (? 1 SD) of two experiments.
bP ? 0.05, two-tailed paired t test, compared with 1-mg/ml trypsin.
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Editor: J. M. Mansfield
VOL. 64, 1996 INHIBITION OF GIARDIA EXCYSTATION2157