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: email@example.com.
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Editor: J. M. Mansfield
VOL. 64, 1996 INHIBITION OF GIARDIA EXCYSTATION2157