Antibodies to the Ventral Disc Protein δ-giardin Prevent in Vitro Binding of Giardia lamblia Trophozoites.

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895
J. Parasitol., 95(4), 2009, pp. 895–899
? American Society of Parasitologists 2009
ANTIBODIES TO THE VENTRAL DISC PROTEIN ?-GIARDIN PREVENT IN VITRO BINDING
OF GIARDIA LAMBLIA TROPHOZOITES
Mark C. Jenkins, Celia N. O’Brien, Charles Murphy, Ryan Schwarz, Katarzyna Miska, Benjamin Rosenthal, and
James M. Trout
Animal Parasitic Diseases Laboratory, Agricultural Research Service, USDA, Beltsville, Maryland 20705. e-mail: mark.jenkins@ars.usda.gov
ABSTRACT:
function in mediating surface attachment. Recombinant ?-giardin antigen was expressed in Escherichia coli as a poly-histidine
fusion protein and was purified by affinity chromatography for production of antisera to ?-giardin. By immunoblotting analysis,
antisera to recombinant ?-giardin antigen recognized a 31-kDa protein on G. lamblia trophozoites. Anti-recombinant ?-giardin
was used to localize the native protein to the trophozoite ventral disk in both immunofluorescence and immunoelectron microscopy
assays. Pre-treatment of G. lamblia trophozoites with anti-?-giardin sera caused morphological changes in the parasite and
inhibited trophozoite binding to the surface of cell culture slides. Binding of antibodies to ?-giardin may provide a means of
inhibiting attachment of G. lamblia trophozoites to the intestinal epithelium and thereby prevent clinical giardiasis.
A cDNA coding for ?-giardin was cloned from Giardia lamblia trophozoites to localize the protein and to study its
Giardia lamblia, the causative agent of giardiasis, is a pro-
tozoan that infects the upper intestinal tract of many mammals,
including humans. In general, giardiasis begins by the ingestion
of water contaminated with G. lamblia cysts, which excyst in
the small intestine, releasing 4 trophozoites that attach to epi-
thelial cells lining the gut (for review, see Adam, 2001). At-
tachment is mediated by the ventral disk, an organelle that is
composed of 3 distinct structures, i.e., microtubules that are
coiled around a bare area; microribbons that protrude into the
cytoplasm; and cross-bridges that connect adjacent microtu-
bules, thereby providing structural support to the ventral disk
(Elmendorf et al., 2003; de Souza et al., 2004; Sant’Anna et
al., 2005). A ventro-lateral flange and lateral crest at the disk
perimeter may play a role in attachment of the parasite to sur-
faces (Feely, 1982; Peattie et al., 1989; Tu ˚mova ´ et al., 2007).
It has been proposed that adherence of G. lamblia trophozoites
is mediated by a suction force between the ventral disk and
epithelial cell surface (Holberton, 1974). At present, 3 major
classes of proteins, termed ?-, ?-, and ?-giardins, have been
identified as components of the ventral disk (Peattie et al., 1989;
Peattie, 1990; Nohria et al., 1992). ?-Giardins are a large class
of annexin-like molecules that are localized to the outer edges
of microribbons (Holberton et al., 1988; Bauer et al., 1999;
Wenman et al., 1993; Weiland et al., 2003, 2005), whereas ?-
giardins seem to be closely associated with microtubules (Hey-
worth et al., 1999). ?-Giardin is a 38-kDa protein that may also
be a component of microribbons (Nohria et al., 1992). A fourth
giardin, named ?-giardin, has been studied at the molecular lev-
el, but no information is available on its location in G. lamblia
(Elmendorf et al., 2001).
Maintenance of the ventral disk architecture seems to be crit-
ical for trophozoite binding. For example, disruption of micro-
tubule function and binding of microribbons by benzimadoles
prevented attachment of trophozoites to host cells (Chavez et
al., 1992; Sousa et al., 2001). In a related study, polyclonal
antibodies against G. lamblia surface proteins prevented attach-
ment of trophozoites to host cells and glass tissue culture sur-
faces (Inge et al., 1988; Samra et al., 1991). Although the an-
tigen target of this serum was unknown, cytoskeletal proteins,
such as ?- and ?-giardins, are known to be highly immunogenic
Received 29 August 2008; revised 19 November 2008; accepted 20
November 2008.
DOI: 10.1645/GE-1851.1
molecules (Roxstromlindquist, 2006). The purpose of the pres-
ent study was to express recombinant G. lamblia ?-giardin pro-
tein, produce antisera specific for recombinant ?-giardin, use
this antisera to localize the protein in G. lamblia trophozoites,
and test its effect on in vitro attachment of trophozoites.
MATERIALS AND METHODS
Giardia lamblia
Giardia lamblia (WB strain) trophozoites (assemblage A) were ob-
tained from the American Type Culture Collection (ATCC 30957, Ma-
nassas, Virginia) and cultured in modified TYI-S-33 media (Miller et
al., 1988) in 15-ml sterile polypropylene tubes at 37 C. Giardia lamblia
were grown in continuous culture by inoculating 12 ml of new media
every 2–3 days, with approximately 5 ? 105trophozoites from a viable
culture.
Identification and real-time reverse transcription-polymerase
chain reaction (RT-PCR) analysis of G. lamblia ?-giardin cDNA
cloning and expression of G. lamblia ?-giardin cDNA
A cDNA clone was identified in subtracted G. lamblia trophozoite
libraries by searching the nr database using BLAST-X and found to be
nearly identical (98–99%) to G. lamblia ?-giardin DNA sequences in
GenBank (accessions AAK32143.1, XP001707448.1, XP001707449.1).
Oligonucleotide primers (Gl?G-forward (F), 5?-AGGAGTCCTTTGG-
CGCCTTTATTG-3? and Gl?G-reverse (R), 5?-ATTCATGTCGGTGG-
CATGCTTGAG-3?) directed to the ?-giardin cDNA sequence were used
in real-time RT-PCR with a 3000XP real-time PCR machine (Strata-
gene, La Jolla, California) to compare the relative abundance of ?-
giardin mRNA between G. lamblia cysts and trophozoites. In brief, 1
ng of total RNA was subjected to real-time RT-PCR using 1 pmol of
Gl?G-forward and -reverse primers, and the Superscript III one-step RT-
PCR system (Invitrogen, Carlsbad, California) in a 25-?l reaction vol-
ume. RT-PCR consisted of reverse transcription at 47 C for 1 hr, de-
naturation at 94 C for 1 min, followed by 35 cycles of 94 C for 30 sec,
59 C for 30 sec, 72 C for 1 min, and a final extension at 72 C for 5
min. In addition, RT-PCR reactions were performed using primers di-
rected to G. lamblia glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) to control for slight differences in RNA levels between cysts
and trophozoites. Reaction conditions were identical to those described
for ?-giardin RT-PCR described above, except using GlGAPDH-F (5?-
AGCTTACCGGTATGGCCTTTCGT-3?) and GlGAPDH-R (5?-TTGT-
CGTACCAGGCAACCAGCTTA-3?) primers. All reactions were run in
triplicate and were further analyzed by polyacrylamide gel electropho-
resis to ensure that the real-time RT-PCR signal was generated from the
expected size amplification product (?-giardin, 118 base pairs [bp];
GAPDH, 267 bp). Relative expression of ?-giardin mRNA in tropho-
zoites and cysts were calculated after normalizing the data to the G.
lamblia GAPDH signal using Q-gene software (Mueller et al., 2002).

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896THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 4, AUGUST 2009
Cloning and expression of G. lamblia ?-giardin cDNA
Plasmid DNA containing ?-giardin cDNA sequence was prepared and
digested with EcoRI to release the ?-giardin coding region, which was
inserted into EcoRI-digested pET28c (Novagen, Madison, Wisconsin)
using DNA ligase and reaction conditions recommended by the manu-
facturer (New England Biolabs, Ipswich, Massachusetts). The ligation
mixtures were used to transform Escherichia coli DH5 cells according
to standard procedures (Hanahan, 1983). Recombinant ?-giardin clones
were identified by restriction digestion of plasmids derived from the E.
coli transformation. Maintenance of the reading frame between the clon-
ing vector and ?-giardin cDNA was confirmed by DNA sequencing.
Recombinant clones were used to transform E. coli Rosetta 2 cells
(Novagen) for high-level protein expression. Cultures of E. coli Rosetta
2 cells harboring pET28-?-giardin were grown at 30 C in Luria-Bertani
broth containing 50 ?g/ml kanamycin and 50 ?g/ml chloramphenicol
until an optical density600? 1.0. Induction of recombinant ?-giardin
was accomplished by growth of cultures at 30 C for 4 hr in the presence
of 1 mM isopropylthiocyanate (Sigma, St. Louis, Missouri).
Analysis of recombinant and native G. lamblia ?-giardin protein
Escherichia coli expressing G. lamblia ?-giardin protein were har-
vested by centrifugation at 3,000 g for 10 min. The cell pellets were
extracted with native binding buffer (Invitrogen) containing phenyl-
methylsulfonyl fluoride (PMSF) protease inhibitor (Sigma), frozen-
thawed 2 times between a dry ice-ethanol bath and a 37 C water bath,
and sonicated twice for 15 sec each, with incubation on wet ice for 1
min between sonications. The protein extracts were treated with 1
U/ml RNase and DNase for 30 min at room temperature and pelleted
by centrifugation at 5,000 g for 30 min. The insoluble pellet was ex-
tracted by resuspension in denaturing binding buffer (Invitrogen) for 30
min at room temperature on a rocker. The extracts were pelleted by
centrifugation at 5,000 g for 30 min, and the supernatant was subjected
to nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography to pu-
rify recombinant ?-giardin protein using procedures recommended by
the manufacturer (QIAGEN, Valencia, California).
Giardia lamblia trophozoites were obtained by placing growing cul-
tures on ice for 5 min followed by centrifugation for 5 min at 2,000 g.
The pelleted trophozoites were washed once with incomplete media,
suspended in protein extraction buffer (10 mM Tris-HCl, pH 7.3, and
1 mM MgCl2) containing PMSF, and extracted by freeze-thawing, son-
ication, and treatment with RNase and DNase as described above.
Recombinant and native G. lamblia protein were treated with sample
buffer (Laemmeli, 1970) with and without the reducing agent 2-mer-
captoethanol, heated for 1 min at 95 C, and fractionated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, followed by trans-
blotting to Immobilon membrane (Millipore, Billerica, Massachusetts)
in a semi-dry transblotter apparatus (Bio-Rad, Hercules, California).
After transfer, the membranes were treated with phosphate-buffered sa-
line (PBS) containing 2% nonfat dry milk (PBS-NFDM) to block non-
specific immunoglobulin binding in subsequent steps. After blocking,
the membranes were incubated with anti-recombinant ?-giardin sera or
preimmune sera for 2–4 hr at room temperature on a laboratory shaker,
followed by 2-hr incubation with biotinylated goat-anti-rabbit immu-
noglobulin (Ig) G (1:1,000 dilution; Sigma), and 1 hr with avidin-per-
oxidase (1:5,000 dilution; Sigma). All antibodies were diluted in PBS
containing 0.05% Tween 20 (PBS-TW) and removed after each step by
3 washes with PBS-TW. Binding of ?-giardin antibodies was assessed
by a final incubation with the peroxidase substrate 0.5 mg/ml 4-chloro-
1-napthol (Sigma) and 0.015% H2O2(Sigma) in PBS.
Preparation of antisera against recombinant ?-giardin
Ni-NTA-purified recombinant ?-giardin was mixed with ImmunoMax
SR adjuvant (Repros Therapeutics, The Woodlands, Texas) and used to
immunize 2 rabbits (New Zealand White, Covance, Denver, Pennsyl-
vania) by subcutaneous injection. Pre-immunization sera were collected
from both rabbits, and in immunofluorescence assay (below) they were
found to be devoid of antibodies to G. lamblia trophozoites. Primary
and booster immunizations consisted of 25 ?g of Ni-NTA-purified re-
combinant ?-giardin in a total volume of 500 ?l, and they were admin-
istered 1 mo apart. Rabbits were killed by exsanguination, and blood
was processed for serum following protocols approved by the BARC
Animal Care and Use Committee. Preliminary studies showed that anti-
?-giardin titers were similar in both rabbits; thus, sera were pooled for
use in all assays described below.
Immunofluorescence staining (IF) of G. lamblia trophozoites
Giardia lamblia trophozoites were harvested from cell culture as de-
scribed above, suspended to 106parasites/ml in PBS, pipetted onto in-
dividual wells of multi-well glass slides (104trophozoites/well; Erie
Scientific Co., Portsmouth, New Hampshire), and allowed to air dry.
After drying, the wells were fixed for 5 min with methanol, then gently
rinsed with PBS. After fixation, the wells were treated with PBS-NFDM
for 30 min at room temperature in a humidified chamber, gently rinsed
with PBS, air-dried, and then incubated for 2 hr at room temperature
with a 1:1,000 dilution of rabbit anti-G. lamblia ?-giardin sera or control
sera (pre-immune sera or antisera to a non-G. lamblia polyHis recom-
binant protein). The wells were gently rinsed 3 times with PBS, allowed
to air dry, and then incubated for 1 hr at room temperature with a 1:
100 dilution of fluorescein isothiocyanate-anti-rabbit IgG (Sigma).
Again, the wells were gently rinsed 3 times with PBS, allowed to air
dry, overlaid with several drops of Vectashield mounting medium (Vec-
tor Laboratories, Burlingame, California) followed by a coverslip, and
then examined using epifluorescence microscopy.
Immunoelectron microscopic (IEM) staining of G. lamblia
trophozoites
Giardia lamblia trophozoites were harvested from cell culture and pel-
leted by centrifugation for 2 min at 5,000 g. The trophozoite pellet was
briefly mixed and then suspended in 100 ?l of fixative consisting of 3%
paraformaldehyde in 0.1 M cacodylate buffer. After a 5-min fixation, the
trophozoites were transferred to a 1.5-ml microcentrifuge tube, pelleted
by centrifugation for 5 min at 5,000 g, gently washed twice with caco-
dylate buffer, and then briefly mixed to form a dispersed pellet in the
bottom of the tube. The trophozoite mixture was dehydrated in a graded
ethanol series, infiltrated overnight with LR White hard-grade acrylic
resin (London Resin Company, London, U.K.), and cured at 55 C for 24
hr. Thin sections (90 ?m thickness) were obtained using a Diatome dia-
mond knife on a Reichert/AO Ultracut microtome and collected on 200-
mesh Formvar-coated nickel grids. The grids were floated for 5 min with
the tissue section facing down on drops of PBS containing 0.1 M glycine
and 1% bovine serum albumin, followed by 5 min on drops containing
PBS-TW-NFDM. Grids were incubated tissue-side down for 2 hr at room
temperature on drops of PBS-TW containing a 1:1,000 dilution of rabbit
anti-G. lamblia ?-giardin sera or control sera (pre-immune sera or antisera
to a non-G. lamblia polyHis recombinant protein). The grids were rinsed
3 times with PBS-TW, incubated for 1 hr at room temperature on PBS
containing a 1:100 dilution of gold-labeled anti-rabbit IgG (Sigma);
washed 2 times with PBS-TW, once with PBS, and once with H2O; air-
dried; stained with 5% uranyl acetate for 30 min; and examined with a
Hitachi H7000 electron microscope.
In vitro testing of anti-G. lamblia ?-giardin sera against G. lamblia
trophozoites
Giardia lamblia trophozoites were harvested from culture by trans-
ferring to 15-ml polypropylene tubes (Falcon 2059, Falcon; BD Bio-
sciences Discovery Labware, Bedford, Massachusetts) and suspended
to 5 ? 105trophozoites/ml in complete culture medium containing 1:
100 dilution of anti-G. lamblia ?-giardin sera or control sera (antisera
to a non-G. lamblia polyHis recombinant protein). The trophozoites
were mixed on an orbital rocker for 1 hr at room temperature and then
aliquoted to 4-well Lab-Tek Chamber Slides (Nalge Nunc International,
Naperville, Illinois). The culture slides were placed in a stationary 37
C incubator to allow for attachment of G. lamblia trophozoites to the
slide surface. A sample of trophozoites were also adhered to glass mi-
croscope slides using Cytopro 7620 cytocentrifuge (Wescor, Logan,
Utah) by centrifugation for 1 min at 1,000 RPM. Adherent trophozoites
were fixed for 5 min with methanol, and then stained for 10 min with
200 ?l of Ladd Multiple Stain (Ladd Research Industries, Burlington,
Vermont). After staining, the slides were washed with PBS, and the
adherent tropozoites were overlaid with a drop of mounting medium
(50% glycerol in PBS) and a coverslip. After 4 hr, individual wells of
the chamber slides were gently washed 3 times with PBS, and the at-
tached trophozoites were treated with methanol and Ladd Multiple Stain
as described above. After staining, the slides were overlaid with mount-

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JENKINS ET AL.—ANTIBODIES TO TROPHOZOITE ?-GIARDIN897
FIGURE 1.
plification products from real-time reverse transcription-polymerase
chain reaction of RNA from Giardia lamblia cysts (C) or trophozoites
(T) using ?-giardin-specific primers (?G) or glyceraldehyde-3-phosphate
dehydrogenase-specific primers.
Analysis by polyacrylamide gel electrophoresis of am-
FIGURE 3.
ic (B–C) staining of Giardia lamblia trophozoites using antisera specific
for recombinant-? giardin protein. VD, ventral disk. Bar ? 500 ?m.
Immunofluorescence (A) and immunoelectron microscop-
FIGURE 2.
protein or recombinant (R)-? giardin protein fractionated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis using antisera spe-
cific for recombinant-? giardin protein. ?2ME, native and recombinant
proteins treated with sample buffer in the absence of 2-mercaptoethanol;
?2ME, native and recombinant proteins treated with sample buffer in
the presence of 2-mercaptoethanol; Mr, molecular size markers (kilo-
daltons).
Immunoblotting analysis of native (N) Giardia lamblia
FIGURE 4.
with control antisera (A) or with antisera specific for recombinant-?
giardin protein (B) as observed by phase contrast microscopy at ?400
magnification.
Morphology of Giardia lamblia trophozoites after treatment
ing medium and a cover slip. Attached trophozoites were enumerated
by counting total attached trophozoites in at least 5 random length-wise
scans of each culture well. Average counts were compared between
treatments for statistical differences using a two-way Student’s t test in
the GraphPad InStat software program (GraphPad Software Inc., San
Diego, CA). Adherent and attached trophozoites were examined by
phase contrast microscopy using a Zeiss Axioskop 2 microscope, and
images were exposed using the AXIOVs v4.6.3.0 software (Zeiss, Got-
tingen, Germany).
RESULTS
Comparison of ?-giardin mRNA levels between G. lamblia
cysts and trophozoites
Real-time RT-PCR analysis revealed a 16-fold greater level
of ?-giardin mRNA signal in trophozoites compared with cysts.
GAPDH mRNA signals were nearly identical in real-time RT-
PCR analysis of trophozoites. Analysis of ?-giardin or GAPDH
real-time RT-PCR products by acrylamide gel electrophoresis
confirmed that the amplicons were of expected size (Fig. 1).
Analysis of recombinant and native G. lamblia ?-giardin
protein
Recombinant G. lamblia ?-giardin protein was highly ex-
pressed in E. coli, representing nearly 50% of total denaturing
soluble protein at concentration of approximately 5 ?g/ml cul-
ture medium (data not shown). Antisera specific for recombi-
nant G. lamblia ?-giardin protein recognized a 36-kDa recom-
binant protein and a 31-kDa native G. lamblia trophozoite pro-
tein by immunoblotting assay (Fig. 2). The Mrdifferences be-
tween recombinant and native ?-giardin proteins are because of
the short polyHis fusion peptide at the amino terminus of the
?-giardin coding sequence. Inclusion of 2-mercaptoethanol had
no effect on the apparent Mrof both recombinant and native G.
lamblia ?-giardin protein (Fig. 2).
Immunofluorescence (IF) and IEM staining of G. lamblia
trophozoites with anti-recombinant ?-giardin sera
In the IF assay, antibodies to recombinant ?-giardin protein
recognized an antigen associated with the G. lamblia tropho-
zoite ventral disk (Fig. 3A). Varying degrees of staining inten-
sity were observed, with an occasional trophozoite exhibiting
negligible staining of the ventral disk (Fig. 3A). IEM staining
of G. lamblia trophozoites with anti-recombinant ?-giardin sera
localized the protein to microribbons of the ventral disk (Figs.
3B, C). Close observation of numerous IEM images revealed
an even distribution across the entire ventral disk.
Effect of anti-recombinant ?-giardin sera on in vitro
attachment of G. lamblia trophozoites
Binding of anti-recombinant ?-giardin sera had a noticeable
effect on the morphology of G. lamblia trophozoites (Fig. 4B).
Although a few control antisera-treated trophozoites displayed
an irregular appearance (Fig. 4A), a much larger proportion of
G. lamblia treated with anti-recombinant ?-giardin exhibited a
distorted morphology (Fig. 4B). Many trophozoites were mis-
shapen, and all were noticeably less active than those treated
with control sera. In addition, anti-recombinant ?-giardin sera

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898THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 4, AUGUST 2009
TABLE I. Comparison of numbers of Giardia lamblia trophozoites
bound to surface of glass culture slides after treatment with either anti-
recombinant ?-giardin or control sera.
Study
No.
Serum
treatment
Average no.
trophozoites/scan*
Percent
inhibition**
1 Control
Immune
Control
Immune
Control
Immune
231 ? 58
127 ? 29
79 ? 23
11 ? 3
263 ? 41
104 ? 18
45.0
2
86.1
3
60.5
* Values are an average of 5 random vertical scans of well surface.
** No. trophozoites control wells – no. trophozoites immune wells/no. trophozo-
ites control wells
affected the binding of trophozoites to the culture well surface.
In all 3 studies, a significant decrease (P ? 0.05) was observed
in the number of bound G. lamblia trophozoites that were treat-
ed with anti-?-giardin sera compared to those treated with con-
trol sera (Table I). Although the absolute number of bound tro-
phozoites varied between studies, trophozoites attachment after
anti-?-giardin sera treatment was consistently lower (45.0–
81.1% inhibition) than attachment observed in control serum
(Table I).
DISCUSSION
The present study demonstrates that ?-giardin is a component
of the G. lamblia trophozoite ventral disk. Similar to other giar-
dins, native ?-giardin (31 kDa) is between 29 and 38 kDa and
seems to be associated with microribbons that, together with
microtubules and bridges, make up the ventral disk cytoskele-
ton. The ?-giardin cDNA shares no sequence similarity to ?-
or ?-giardins, which are different from one another. Although
the overall sequence similarity is low (?20%), ?-giardin shares
conserved amino acid motifs with ?-giardin, suggesting that
they belong to the same protein family. Regardless, it remains
unclear how the 4 classes of giardins interact to provide struc-
ture to the ventral disk. Antibodies to purified native ?32-kDa
protein (Crossley and Holberton, 1983, 1985; Crossley et al.,
1986) or to recombinant protein produced by molecular cloning
(Holberton et al., 1988; Peattie et al., 1989; Alonso and Peattie,
1992) have shown that ?-giardins are a large class of proteins
that are related to annexins (Morgan and Fernandez, 1995; Wei-
land et al., 2005). ?-Giardins are found in variety of G. lamblia
cytoskeletal components, such as flagella, ventral disk, and the
plasma membrane (Alonso and Peattie, 1992; Wenman et al.,
1993; Bauer et al., 1999; Weiland et al., 2003, 2005; Vahrmann
et al., 2008). In particular, ?-1 giardin has been localized to the
outer edges of microribbons of the ventral disk (Peattie et al.,
1989). Other microribbon proteins, such as ?-giardin (29 kDa)
and ?-giardin (38 kDa), were identified in studies of 29–38-
kDa proteins of G. lamblia trophozoites (Crossley and Holber-
ton, 1983; Crossley et al., 1986) or by molecular cloning of the
respective genes for these proteins (Baker et al., 1988; Holber-
ton et al., 1988; Aggarwal and Nash, 1989; Nohria et al., 1992).
Results in the present study indicate that ?-giardin (Elmen-
dorf et al., 2001) is also a microribbon protein that may be
involved in attachment of G. lamblia trophozoites. The cyto-
skeletal locale of ?-giardin is consistent with its predicted struc-
ture. For example, motif searching using PFAM (http://
pfam.sanger.ac.uk or http://ebi.ac.uk/interpro) and Conserved
Domains (http://www.ncbi.nlm.nih.gov/structure/cdd) programs
revealed that ?-giardin shares homology with the striated fiber-
assemblin (SFA)/?-giardin family (E value ? 2.00e?03). SFAs
are acidic 33-kDa proteins that represent a major component of
striated microtubule-associated fibers.
In this study, binding of the ventral disc using antibodies to
?-giardin appeared to have an effect on morphology and motil-
ity of trophozoites. Examination of G. lamblia trophozoites im-
mediately after the 1 hr treatment with control or immune serum
revealed a high percentage of distorted trophozoites in those
incubated with anti-?-giardin antibodies (Fig. 4B). In addition,
G. lamblia trophozoites treated with anti-?-giardin sera were
noticeably less mobile than trophozoites treated with control
sera. While control trophozoites exhibited a classical rapid,
tumbling movement, virtually all of those parasites bound with
anti-?-giardin antibodies were immobile. Subsequent binding
assays revealed an effect of anti-?-giardin serum on trophozoite
attachment to glass surface. Although it is clear that in our
assay most trophozoites in both control and immune treatments
did not bind to glass culture surface (the maximum number
observable in a single scan would equal 2,000 trophozoites),
there was a consistent and highly significant inhibition of G.
lamblia attachment in the presence of anti-?-giardin sera. Our
data indicates that binding of ?-giardin antibodies to the ventral
disc affects the ability of trophozoites to bind/or remain at-
tached to inanimate surfaces. This phenomenon is not without
precedent. Antibodies to whole G. lamblia blocked in vitro
binding of trophozoites to enterocytes (Inge et al., 1988) and
glass culture surfaces (Samra et al., 1991). How binding of a
cytoskeletal protein by antibodies prevents attachment of G.
lamblia trophozoites is unknown. It is possible that binding of
cytoskeletal elements either directly interferes with trophozoite
binding or hinders the flexibility of the ventral disk, thereby
preventing parasite attachment to gut epithelial cells. The mor-
phological change in trophozoites that had been treated with
anti-recombinant ?-giardin sera may indicate that G. lamblia
attachment is at least partly dependent on morphology. Binding
to cultured cells or glass surfaces is probably multi-factorial
because a role for lectins in attachment of trophozoites has also
been observed (Inge et al., 1988; Sousa et al., 2001). Whether
post-translational moieties on giardins play a role in G. lamblia
binding to surfaces or host cells remains unknown. Many giar-
dins, such as ?2-, ?-, and ?-giardin seem to have sites for po-
tential N-glycosylation and O-glycosylation, but it is unknown
whether lectins that have been shown to interfere with tropho-
zoite attachment (Inge et al., 1988; Sousa et al., 2001) are bind-
ing to giardins or to other surface proteins. The ability of an-
tibodies that react with whole G. lamblia trophozoites or with
specific giardin molecules to inhibit attachment of the parasite
in vitro may indicate a possible therapy against giardiasis. In-
deed, it seems that a humoral immune response, in particular
secretory IgA, is necessary for development of immunity
against G. lamblia (Faubert, 2000; Gillin and Eckmann, 2002;
Langford et al., 2002; Eckman 2003) and that a strong response
to cytoskeletal proteins is observed in natural infections
(Roxstr m-Lindquist et al., 2006).
?o

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JENKINS ET AL.—ANTIBODIES TO TROPHOZOITE ?-GIARDIN899
LITERATURE CITED
ADAM, R. D. 2001. Biology of Giardia lamblia. Clinical Microbiology
Reviews 14: 447–475.
AGGARWAL, A., R. D. ADAM, AND T. E. NASH. 1989. Characterization
of a 29.4-kilodalton structural protein of Giardia lamblia and lo-
calization to the ventral disk. Infection and Immunity 57: 1305–
1310.
ALONSO, R. A., AND D. A. PEATTIE. 1992. Nucleotide sequence of a
second alpha giardin gene and molecular analysis of the alpha giar-
din genes and transcripts in Giardia lamblia. Molecular and Bio-
chemical Parasitology 50: 95–104.
BAKER, D. A., D. V. HOLBERTON, AND J. MARSHALL. 1988. Sequence of
a giardin subunit cDNA from Giardia lamblia. Nucleic Acids Re-
search 16: 7177.
BAUER, B., S. ENGELBRECHT, T. BAKKER-GRUNWALD, AND H. SCHOLZE.
1999. Functional identification of alpha 1-giardin as an annexin of
Giardia lamblia. FEMS Microbiology Letters 173: 147–153.
CHAVEZ, B., R. CEDILLO-RIVERA, AND A. MARTIZNEZ-PALOMO. 1992.
Giardia lamblia: Ultra-structural study of the in vitro effect of
benzimidazoles. Journal of Protozoology 39: 510–515.
CROSSLEY, R., AND D. V. HOLBERTON. 1983. Characterization of proteins
from the cytoskeleton of Giardia lamblia. Journal of Cell Science
59: 81–103.
———, AND ———. 1985. Assembly of 2.5 ?m filaments from giardin,
a protein associated with cytoskeletal microtubules in Giardia.
Journal of Cell Science 78: 205–231.
———, J. MARSHALL, J. T. CLARK, AND D. V. HOLBERTON. 1986. Im-
munocytochemical differentiation of microtubules in the cytoskel-
eton of Giardia lamblia using monoclonal antibodies to alpha-tu-
bulin and polyclonal antibodies to associated low molecular weight
proteins. Journal of Cell Science 80: 233–252.
DE SOUZA, W., A. LANFREDI-RANGEL, AND L. CAMPANATI. 2004. Contri-
bution of microscopy to a better knowledge of the biology of Giar-
dia lamblia. Microscopy and Microanalysis 10: 513–527.
ECKMANN, L. 2003. Mucosal defences against Giardia. Parasite Immu-
nology 25: 259–270.
ELMENDORF, H. G., S. C. DAWSON, AND J. M. MCCAFFERY. 2003. The
cytoskeleton of Giardia lamblia. International Journal for Parasi-
tology 33: 3–28.
———, S. M. SINGER, J. PIERCE, J. COWAN, AND T. E. NASH. 2001.
Initiator and upstream elements in the alpha2-tubulin promoter of
Giardia lamblia. Molecular and Biochemical Parasitology 113:
157–169.
FAUBERT, G. 2000. Immune response to Giardia duodenalis. Clinical
Microbiology Reviews 13: 35–54.
FEELY, D. E., J. V. SCHOLLMEYER, AND S. L. ERLANDSEN. 1982. Giardia
spp.: Distribution of contractile proteins in the attachment organ-
elle. Experimental Parasitology 53: 145–154.
GILLIN, F. D., AND L. ECKMANN. 2002. Central importance of immuno-
globulin A in host defense against Giardia spp. Infection and Im-
munity 70: 11–18.
HANAHAN, D. 1983. Studies on the transformation of Escherichia coli
with plasmids. Journal of Molecular Biology 166: 557–580.
HEYWORTH, M. F., J. D. FOELL, AND T. W. SELL. 1999. Giardia muris:
Evidence for a beta-giardin homologue. Experimental Parasitology
91: 284–287.
HOLBERTON, D., 1974. Attachment of Giardia-a hydrodynamic model
based on flagellar activity. Journal of Experimental Biology 60:
207–221.
———, D. A. BAKER, AND J. MARSHALL. 1988. Segmented alpha-helical
coiled-coil structure of the protein giardin from the Giardia cyto-
skeleton. Journal of Molecular Biology 204: 789–795.
INGE, P. M., C. M. EDSON, AND M. J. FARTHING. 1988. Attachment of
Giardia lamblia to rat intestinal epithelial cells. Gut 29: 795–801.
LAEMMELI, U. K. 1970. Cleavage of structural proteins during the as-
sembly of the head of bacteriophage T4. Nature 227: 680–685.
LANGFORD, T. D., M. P. HOUSLEY, M. BOES, J. CHEN, M. F. KAGNOFF, F.
D. GILLIN, AND L. ECKMANN. 2002. Central importance of immu-
noglobulin A in host defense against Giardia spp. Infection and
Immunity 70: 11–18.
MILLER, R. L., A. L. WANG, AND C. C. WANG. 1988. Identification of
Giardia lamblia isolates susceptible and resistant to infection by
the double-stranded RNA virus. Experimental Parasitology 66:
118–123.
MORGAN, R. O., AND M. P. FERNANDEZ. 1995. Molecular phylogeny of
annexins and identification of a primitive homologue in Giardia
lamblia. Molecular Biology and Evolution 12: 967–979.
MUELLER, P. Y., H. JANOVJAK, A. R. MISEREZ, AND Z. DOBBIE. 2002.
Processing of gene expression data generated by quantitative real-
time RT-PCR. Biotechniques 32: 1372–1379.
NOHRIA, A., R. A. ALONSO, AND D. A. PEATTIE. 1992. Identification and
characterization of gamma-giardin and the gamma-giardin gene
from Giardia lamblia. Molecular and Biochemical Parasitology 56:
27–37.
PEATTIE, D. A. 1990. The giardins of Giardia lamblia: Genes and pro-
teins with promise. Parasitology Today 6: 52–56.
———, R. A. ALONSO, A. HEIN, AND J. P. CAULFIELD. 1989. Ultrastruc-
tural localization of giardins to the edges of disk microribbons of
Giarida lamblia and the nucleotide and deduced protein sequence
of alpha giardin. Journal of Cell Biology 109: 2323–2335.
ROXSTR M-LINDQUIST, K., D. PALM, D. REINER, E. RINGQVIST, AND S.
G. SVA¨RD. 2006. Giardia immunity—An update. Trends in Para-
sitology 22: 26–31.
SAMRA, H. K., N. K. GANGULY, AND R. C. MAHAJAN. 1991. Human milk
containing specific secretory IgA inhibits binding of Giardia lam-
blia to nylon and glass surfaces. Journal of Diarrhoeal Diseases
Research 9: 100–103.
SANT’ANNA, C., L. CAMPANATI, C. GADELHA, D. LOURENC ¸O, L. LABATI-
TERRA, J. BITTENCOURT-SILVESTRE, M. BENCHIMOL, N. L. CUNHA-E-
SILVA, AND W. DE SOUZA. 2005. Improvement on the visualization
of cytoskeletal structures of protozoan parasites using high-reso-
lution field emission scanning electron microscopy (FESEM). His-
tochemistry and Cell Biology 124: 87–95.
SOUSA, M. C., C. A. GONC ¸ALVES, V. A. BAIROS, AND J. POIARES-DA-
SILVA. 2001. Adherence of Giardia lamblia trophozoites to Int-407
human intestinal cells. Clinical and Diagnostic Laboratory Immu-
nology 8: 258–265.
TU˚MOVA´, P., J. KULDA, AND E. NOHY´NKOVA´. 2007. Cell division of Giar-
dia intestinalis: Assembly and disassembly of the adhesive disc,
and the cytokinesis. Cell Motility and the Cytoskeleton 64: 288–
298.
VAHRMANN, A., M. SARIC´, H. SCHOLZE, AND I. KOEBSCH. 2008. ?14-
Giardin (annexin E1) is associated with tubulin in trophozoites of
Giardia lamblia and forms local slubs in the flagella. Parasitology
Research 102: 321–326.
WEILAND, M. E., J. E. PALM, W. J. GRIFFITHS, J. M. MCCAFFERY, AND
S. G. SVA¨RD. 2003. Characterisation of alpha-1 giardin: An im-
munodominant Giardia lamblia annexin with glycosaminoglycan-
binding activity. International Journal for Parasitology 33: 1341–
1351.
———, A. G. MCARTHUR, H. G. MORRISON, M. L. SOGIN, AND S. G.
SVA¨RD. 2005. Annexin-like alpha giardins: A new cytoskeletal gene
family in Giardia lamblia. International Journal for Parasitology
35: 617–626.
WENMAN, W. M., R. U. MEUSER, Q. NYUGEN, R. T. KILANI, K. EL-
SHEWY, AND R. SHERBURNE. 1993. Characterization of an immu-
nodominant Giardia lamblia protein antigen related to alpha giar-
din. Parasitology Research 79: 587–592.
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