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Infection and Immunity, July 2000, p. 4217-4224, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob)
Is a Lectin with Five Cys-Rich, Chitin-Binding Domains
Marta
Frisardi,1
Sudip K.
Ghosh,1
Jessica
Field,1
Katrina
Van
Dellen,1
Rick
Rogers,2
Phillips
Robbins,3 and
John
Samuelson1,*
Department of Immunology and Infectious
Diseases1 and BioMedical Imaging
Institute,2 Harvard School of Public Health, and
Department of Cell Biology, Boston University School of Dental
Medicine,3 Boston, Massachusetts
Received 31 January 2000/Returned for modification 1 March
2000/Accepted 25 March 2000
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ABSTRACT |
The infectious stage of amebae is the chitin-walled cyst, which is
resistant to stomach acids. In this study an extraordinarily abundant,
encystation-specific glycoprotein (Jacob) was identified on
two-dimensional protein gels of cyst walls purified from
Entamoeba invadens. Jacob, which was acidic and had an
apparent molecular mass of ~100 kDa, contained sugars that bound to
concanavalin A and ricin. The jacob gene encoded a 45-kDa
protein with a ladder-like series of five Cys-rich domains. These
Cys-rich domains were reminiscent of but not homologous to the Cys-rich
chitin-binding domains of insect chitinases and peritrophic matrix
proteins that surround the food bolus in the insect gut. Jacob bound
purified chitin and chitin remaining in sodium dodecyl sulfate-treated
cyst walls. Conversely, the E. histolytica plasma membrane
Gal/GalNAc lectin bound sugars of intact cyst walls and purified Jacob.
In the presence of galactose, E. invadens formed wall-less
cysts, which were quadranucleate and contained Jacob and chitinase
(another encystation-specific protein) in secretory vesicles. A
galactose lectin was found to be present on the surface of wall-less
cysts, which phagocytosed bacteria and mucin-coated beads. These
results suggest that the E. invadens cyst wall forms when
the plasma membrane galactose lectin binds sugars on Jacob, which in
turn binds chitin via its five chitin-binding domains.
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INTRODUCTION |
Entamoeba histolytica is
a luminal protozoan parasite, which is a frequent cause of dysentery
and liver abscess in persons in developing countries that cannot
prevent its fecal-oral spread (37). E. histolytica is part of a family of microaerophilic amebae, which
reside as commensals in the human colon (Entamoeba dispar and Entamoeba coli) or survive as free-living
organisms in garbage (Entamoeba moscovshii) (12).
The infectious stage of amebae is the chitin-walled, quadranucleate
cyst, which is resistant to stomach acids (2, 15). Because
it is not possible to discriminate cysts of E. histolytica
and E. dispar by microscopy, cysts identified in clinical
samples are called "E. histolytica/E. dispar"
(51).
Since E. histolytica parasites do not encyst in axenic
culture, cyst formation has been studied using the reptilian pathogen Entamoeba invadens, which also forms a quadranucleate cyst
(15). E. invadens, which is more closely related
to E. histolytica than to the human commensal E. coli, converts to cysts within 2 days when deprived of glucose
(38, 40). Amebic cyst wall proteins include 100- and 150-kDa
glycoproteins, which bind wheat germ agglutinin (WGA), and
uncharacterized antigens, which react with monoclonal anti-cyst
antibodies (7, 49). Chitin (
-1,4-linked N-acetylglucosamine [GlcNAc]) is also present in amebic
cyst walls (2). Although neither chitin synthase nor
chitinase is present in amebic trophozoites, both enzymes are expressed
by encysting parasites (3, 10, 47). Amebic chitinases, which
have catalytic domains like those of nematode, insect, and plant
chitinases, are present in hundreds of small cyst-specific secretory
vesicles (4, 11, 19, 23, 39, 46).
Amebae have a plasma membrane Gal/GalNAc lectin, which may
also be involved in encystation (8, 9). This lectin, which has been best characterized on the surface of E. histolytica
trophozoites, binds to galactose or N-acetylgalactosamine
(GalNAc) on bacteria, red blood cells and epithelial cells, or
mucin-coated beads (13, 20). The E. histolytica
Gal/GalNAc lectin is composed of a large 170-kDa subunit, which has a
transmembrane domain near its C terminus, and a small 35-kDa subunit,
which has a glycosylphosphatidylinositol anchor at its C terminus
(29, 31, 36, 44). An E. invadens gene encoding a
homologue of the Gal/GalNAc lectin small subunit has been cloned
(GenBank accession number AF016642). Since galactose but not GalNAc
inhibits the aggregation and encystation of E. invadens
parasites in vitro, it may be more accurate to refer to the E. invadens plasma membrane "galactose lectin" (8). It
has been suggested that galactose exerts its effect on aggregation and
encystation by blocking signal transduction mediated by the galactose
lectin (9). Previously, inside-out signaling by cytosolic domains of the Gal/GalNAc lectin was shown to be important for epithelial cell adherence and amebic virulence (48).
In this study, two-dimensional protein gels identified an abundant
E. invadens cyst wall glycoprotein, which was called Jacob because it contained a ladder-like series of Cys-rich, chitin-binding domains. Jacob also contained sugars, which were recognized by the
galactose lectin on encysting E. invadens, such that
wall-less cysts formed in the presence of excess galactose.
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MATERIALS AND METHODS |
Preparations of cysts and trophozoites.
The IP-1 strain of
E. invadens was grown at 25°C in axenic culture in TYI-SS
medium (15). E. invadens encystation was induced by placing parasites for 48 h in low-glucose (LG) medium, which has reduced osmolarity, glucose, and serum levels with respect to
TYI-SS medium (38). Cysts walls were identified by staining with 2 µg of Calcofluor per ml, which binds to chitin and emits a
blue fluorescence when excited with UV light (5). Chitin and
other carbohydrates in cyst walls were also stained with 50 µg each
of fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA),
FITC-ricin, or tetramethylrhodamine isothiocyanate-conjugated (TRITC)-WGA per ml for 60 min at room temperature in phosphate-buffered saline (PBS) and washed four times. Cyst nuclei were identified by
permeabilizing cysts with 0.1% sodium dodecyl sulfate (SDS) and
staining with 1 µM Sytox green.
In an attempt to inhibit cyst wall formation, E. invadens
parasites were placed in LG medium containing 100 mM lactose,
galactose, GalNAc, or mannose and incubated for 2 to 4 days at room
temperature. Wall-less cysts, which were formed in the presence of
galactose, were motile, had four nuclei, and contained Jacob and
chitinase in secretory vesicles (see below) but lacked a chitin wall.
Because a rabbit anti-E. histolytica Gal/GalNAc lectin
antibody did not bind to E. invadens trophozoites, the
E. invadens galactose lectin was indirectly detected on
trophozoites and wall-less cysts by incubating them with green
fluorescent protein-labeled bacteria with or without galactose
(45). Wall-less cysts were also incubated in the absence of
galactose with mucin-coated beads or uncoated beads (negative control)
(20).
Two-dimensional SDS-PAGE of purified cyst walls.
To purify
cyst walls, we separated cysts from adherent trophozoites by decanting
unchilled flasks. Residual trophozoites were lysed by five short pulses
with a sonicator. Cysts were concentrated by centrifugation and
resuspended in PBS plus 100 µM E-64 to inhibit amebic cysteine
proteases. The cysts were broken by extensive sonication (
50 pulses),
and cyst walls were separated from cytosol, membranes, nuclei, and
intact cysts by centrifugation through two 60% sucrose cushions
(2). Cyst wall preparations, which were checked by
phase-contrast microscopy, fluorescence microscopy with Calcofluor, and
transmission electron microscopy (TEM), contained less than one intact
cyst per 100 walls and no trophozoites.
Purified cyst walls were boiled in 1% SDS-5%
-mercaptoethanol
(2-ME), and the supernatant of a microcentrifuge centrifugation
was
mixed with lysis buffer (540 mg of urea per ml, 2% Triton
X-100, 2%
2-ME, 2% ampholines 3 to 10, 100 µg of E-64 per ml)
and
electrophoresed on two-dimensional gels (
35). Precast gels
(Pharmacia Biotech AB, Uppsala, Sweden) contained amphollytes
from pH 3 to pH 10 in the first (isoelectric-focusing) dimension
and a gradient
of acrylamide from 10 to 20% in the second (SDS-polyacrylamide
gel
electrophoresis [PAGE]) dimension. The gels were stained with
Coomassie blue or silver (to identify less abundant cyst wall
proteins). The most abundant cyst wall glycoprotein (Jacob), which
was
acidic and was 100 kDa, was excised from a Coomassie blue-stained
gel
and digested with trypsin. Tryptic peptides were separated
by
high-pressure liquid chromatography, and N-terminal sequences
were
obtained by Edman degradation (
32). Alternatively,
two-dimensional
protein gels of cyst wall proteins were transferred to
polyvinylidene
difluoride (PVDF) filters, and Jacob, identified with
Ponceau-S.,
was excised for N-terminal sequencing. Some PVDF membranes
were
blocked with powdered milk and treated with anti-Jacob antibodies
or the
E. histolytica Gal/GalNAc lectin (as described
below).
Other PVDF membranes were also incubated with biotinylated
ConA,
ricin, WGA, or
Sambucus nigra agglutinin (all 4 µg/ml in PBS),
washed, and developed with avidin conjugated to
alkaline
phosphatase.
Cloning of the E. invadens jacob gene.
A segment
of the E. invadens jacob gene was isolated from DNA of
E. invadens IP-1 using PCR and degenerate primers to
N-terminal sequences of tryptic Jacob peptides (24). A
degenerate sense primer,
CA(AG)TA(CT)TT(CT)GA(AG)TG(CT)(AT)(CG)(AT)AA(CT)AC, was to
QYFECSNT, while an antisense primer,
AC(AG)TA(AG)TA(CT)TG(AG)AA(AG)TC(AG)TG, was to HDFQYYV. The
jacob PCR product, which was 488 bp long, was cloned in TA
vector and sequenced by dideoxy methods. The jacob PCR
product was used to identify jacob gDNA clones from an
E. invadens IP-1 strain DNA gDNA library (38).
Like other E. invadens genes, the E. invadens
jacob coding sequence contained no introns and had a 51% A+T
content in the third position (11, 19, 38). The E. invadens Jacob protein was compared with proteins in the GenBank
and EST databases and with products of unfinished microbial genomes by
using BLAST (1). N-terminal signal sequences were predicted
and potential transmembrane segments identified using well-established
algorithms (16, 34).
Production of anti-Jacob antibodies, Western blots, and indirect
immunofluorescence microscopy.
E. invadens Jacob was excised
from 10 two-dimensional protein gels, mixed with complete Freund's
adjuvant, and injected into rabbits. The rabbits were boosted at 3 and
6 weeks with Jacob from five gels each in incomplete Freund's
adjuvant. Western blots of two-dimensional gels of cyst wall proteins
were incubated for 60 min at 25°C with anti-Jacob serum diluted
1:1,000 in PBS. Filters were washed and incubated in anti-rabbit
antibodies conjugated to peroxidase (1:2,000 dilution), which was
detected using chemiluminescent reagents.
To localize Jacob on the surface of
E. invadens cysts,
parasites were fixed with 2% paraformaldehyde for 10 min at 4°C,
washed
in PBS, and immunostained for 60 min at 37°C with rabbit
anti-Jacob
sera, diluted 1:100 in PBS containing 1 mg of bovine serum
albumin
per ml. To localize Jacob within secretory vesicles of
encysting
E. invadens parasites, amebae were permeabilized
by incubation
with 0.1% Triton X-100 for 5 min at room temperature and
then
immunostained with rabbit anti-Jacob, which was also diluted
1:100.
The organisms were washed four times and immunodecorated for 60
min with a Texas red-conjugated goat anti-rabbit antiserum. As
a
negative control, parasites were stained with preimmune rabbit
serum.
As a positive control, encysting parasites were stained
with TRITC-WGA,
which binds chitin. Alternatively, parasites were
incubated with a
rabbit anti-
E. invadens chitinase antibody, which
was
previously made to a multiantigenic peptide containing chitinase
repeats (
19). Nuclei were stained with Sytox green, and
parasites
were observed with a Leica NT-TCS confocal microscope fitted
with
argon and krypton lasers. Three-dimensional reconstructions were
made from a series of optical sections, which were made at 0.5-
to
1-µm
intervals.
TEM and immuno-EM of encysting parasites.
Cysts and purified
cyst walls were fixed for 10 min in 1% paraformaldehyde, postfixed in
1% osmium tetroxide, stained en block with uranyl acetate, dehydrated,
and embedded in Epon. Sections, which were 60 nm thick, were stained on
the grid with uranyl acetate and lead citrate. To visualize Jacob on
the surface of cysts or wall-less cysts, encysting parasites were fixed
in paraformaldehyde, incubated with anti-Jacob antibodies as described
for fluorescence microscopy, and then incubated with
Staphylococcus protein A, which had been conjugated to
10-nm-diameter gold particles. A negative control included preimmune
rabbit serum. Parasites were washed and prepared for TEM as described
above. Alternatively, parasites were fixed in 2% paraformaldehyde,
infiltrated with 2.3 M sucrose, and frozen. Ultrathin sections were
incubated with the anti-Jacob serum and protein A-gold complex. These
parasites were prepared for TEM in the absence of osmium tetroxide, so
that the membranes remained unstained.
Methods to demonstrate binding of Jacob to chitin.
To obtain
soluble forms of Jacob, E. invadens parasites were encysted
for 24 h and cytosolic extracts were made by sonicating parasites
in the presence of E-64. These extracts were incubated with SDS-treated
cyst walls, chitin beads, or GlcNAc beads, which were washed and then
incubated with rabbit anti-Jacob antibodies and immunodecorated as
described above. Negative controls included cytosolic extracts from
E. invadens trophozoites, nonimmune sera, and Sepharose or
agarose beads. In addition, extracts of E. histolytica bound
to chitin beads were stained with antibodies to alcohol dehydrogenase 1 or Ariel (negative controls) (22, 28). Amebic proteins
binding to chitin beads were also eluted with 1% SDS-5% 2-ME and run
on one-dimensional SDS-PAGE. Gels were transferred to nitrocellulose
filters and incubated with anti-Jacob antibodies, which were detected
with chemiluminescent reagents. A positive control included cyst wall
proteins, which were run in a parallel lane.
Methods to demonstrate binding of the Gal/GalNAc lectin to
Jacob.
Binding of the E. histolytica Gal/GalNAc lectin
to cyst walls and to Jacob was demonstrated in three ways. First,
E. histolytica HM-1 parasites were stained with a
nonspecific cytosolic stain, CFDA SE (Molecular Probes, Eugene, Oreg.)
and incubated for 3 h at 37°C with intact E. invadens
cysts, which were stained with Calcofluor. Phagocytosis of cysts by
trophozoites was determined by fluorescence microscopy. Negative
controls included trophozoites incubated with cyst walls after
treatment with SDS to remove Jacob and other glycoproteins, as well as
incubation of trophozoites with cyst walls in the presence of excess
galactose. Second, E. invadens cysts were incubated with 5 µg of purified Gal/GalNAc lectin from HM-1 trophozoites per ml
(36). Cysts were washed, and bound lectin was detected with
a 1:500 dilution of rabbit anti-lectin antibodies (29),
which were immunodecorated as described for anti-Jacob antibodies.
Negative controls included replacement with SDS-treated cyst walls,
addition of excess galactose to Gal/GalNAc lectin, omission of the
lectin, or incubation with nonimmune rabbit serum. Third, a Western
blot of a two-dimensional gel of Jacob was incubated with the
Gal/GalNAc lectin, washed, and incubated with anti-lectin antibodies,
which were detected with chemiluminescent reagents. A negative control
was included in which the Gal/GalNAc lectin was omitted.
Nucleotide sequence accession number.
The nucleotide and
predicted amino acid sequence of the jacob gene have been
submitted to GenBank with accession number AF175527.
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RESULTS |
The most abundant cyst wall protein is an acidic, 100-kDa
glycoprotein (Jacob).
The walls of E. invadens cysts
were electron dense and had a uniform thickness of ~100 nm (Fig.
1A). Electron-dense material was also
present in secretory vesicles and along the plasma membrane. Purified
cyst walls, which were prepared on sucrose gradients after sonication
of cysts, closely resembled the walls of intact cysts (Fig. 1B). After
the cyst walls were boiled in SDS and 2-ME, only fibrils, which were
less tightly bound to each other, remained (Fig. 1C). These fibrils are
presumably made of chitin, because they stained with Calcofluor (data
not shown) (2).
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FIG. 1.
Transmission electron micrographs of E. invadens cyst walls. (A) Intact E. invadens cysts have
an electron-dense wall, which overlies secretory vacuoles and electron
densities along the plasma membrane. (B) Cyst walls purified by two
sucrose gradients contain electron-dense material between chitin
fibrils. (C) Chitin fibrils and little other electron-dense material
remain in cyst walls after boiling in SDS. Bar, 200 nm. Magnification,
×4,500.
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More than a dozen cyst wall proteins (Fig.
2A),
which were absent from trophozoites (Fig.
2B), were identified by
two-dimensional
SDS-PAGE (
35). Some of these cyst wall
proteins formed multiple
spots, which were the same size but had
different isolectric points.
The most abundant cyst wall-specific
protein (Jacob) was acidic
and had an apparent molecular mass of ~100
kDa. Jacob and other
amebic cyst wall proteins were glycoproteins,
which bound ConA
(Fig.
2C). Jacob also bound ricin (Fig.
2D) and WGA
(data not
shown) but did not bind
S. nigra agglutinin.
Rabbit antibodies
to purified Jacob bound to the 100-kDa spot and to
higher- and
lower-molecular-mass spots (Fig.
2E), which had the same
isoelectric
point as Jacob but were less abundant. Fluorescence
microscopy
was used to confirm the Western blot findings. ConA (Fig.
3A)
and ricin (Fig.
3B) bound much more
extensively to the surface
of cysts than to the surface of
trophozoites, while anti-Jacob
antibodies bound to cysts but not to
trophozoites (Fig.
3C).
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FIG. 2.
Two-dimensional gels of E. invadens
cyst wall proteins. (A and B) Silver stains show that Jacob, which is
by far the most abundant cyst wall protein (arrow in panel A), is
absent from trophozoites (B). (C and D) Western blots show that ConA
(C) and ricin (D), visualized with acid phosphatase, bind to Jacob and
numerous other cyst wall glycoproteins. (E) In contrast, anti-Jacob
antibodies, visualized by chemiluminescence, bind to Jacob and larger
and smaller proteins with the same charge. Purified E. histolytica Gal/GalNAc lectin, which was detected with anti-lectin
antibodies, binds to Jacob excised from a Ponceau-stained
two-dimensional gel (inset in panel E). A negative control, in which
the Gal/GalNAc lectin was omitted, did not bind the anti-Gal/GalNAc
antibodies (data not shown).
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FIG. 3.
Fluorescence micrographs of E. invadens cysts
and trophozoites. (A and B) FITC-ConA (arrowheads in panel A) and
FITC-ricin (B) stained the surface of cysts (c) much more intensely
than the surface of trophozoites (t). (C) Similarly, anti-Jacob
antibodies, made to the native Jacob protein, bound to the surface of
cysts but not to trophozoites. (D) The E. histolytica
Gal/GalNAc lectin, which was detected with anti-lectin antibodies, also
bound to E. invadens cyst walls but not to the surface of
trophozoites.
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Jacob contains five Cys-rich domains, separated by acidic
spacers.
N-terminal sequences of tryptic peptides of Jacob were
used to design degenerative oligonucleotide primers to obtain a segment of the E. invadens jacob gene by PCR (24). The
jacob PCR product was in turn used to obtain the entire
coding region of the jacob gene of E. invadens,
which predicted a 405-amino-acid protein (Fig.
4). The formula weight of Jacob
(Mr 45,122) was less than half the apparent
molecular mass (100 kDa) of Jacob on two-dimensional protein gels (Fig.
2A). A signal sequence (MLSDILFGIAAA), which was identified by
sequencing the N terminus of undigested Jacob, was cleaved at a site
predicted by the -3,-1 rule as applied to eubacteria rather than
eukaryotes (34). The predicted E. invadens Jacob
also contained N-terminal sequences of peptides obtained from tryptic
digests of Jacob. The amino acid composition of the predicted protein,
which was rich in acidic (16%), basic (15%), and polar (43%) amino
acids, matched that of Jacob excised from the gel.
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FIG. 4.
Primary structure of E. invadens Jacob, in
single-letter code. An asterisk marks the stop codon. The signal
sequence, proven by N-terminal sequencing of intact Jacob, is
underlined twice, while the N-terminal sequences of Jacob tryptic
peptides are each underlined once. PCR primers for cloning the E. invadens jacob gene were made to QYFECSNT and HDFQYYV. A possible
site of Asn-linked glycosylation (NDT) is marked with a wavy underline.
Conserved Cys residues in putative chitin-binding domains, which are
aligned, are marked in bold.
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Jacob had one site of possible N-linked glycosylation (Asn33) and
numerous Ser and Thr residues for possible O-linked glycosylation
(
21) (Fig.
4). Jacob did not have any predicted
transmembrane
segments (
16). Instead, it was composed of
five similar domains,
each of which contained six Cys residues spaced
7, 12, 9, 5, and
12 amino acids apart. Chitin-binding domains of
peritrophins and
fungal, nematode, and insect chitinases have
mirror-image spacing
of Cys residues, which are 12, 5, 9, 12, and 7 amino acids apart
(all ±1) (
17,
23,
39,
46). Jacob repeat
domains also contained
conserved aromatic amino acids (Tyr, Phe, and
Trp), which may
be involved in binding sugars, as described for WGA
(
52). Between
the Jacob repeat domains were acidic domains,
which were similar
in their composition but not their sequence to
repeats in amebic
chitinases, Ser-rich
E. histolytica
proteins, or Ariel surface
protein (
11,
28,
41). There were
no proteins homologous
to amebic Jacob in GenBank, EST, or unfinished
microbial genome
databases, although BLAST with Jacob identified the
large subunit
of the
E. histolytica Gal/GalNAc lectin and
Giardia lamblia variable
surface proteins, which are also
Cys rich (
1,
29,
33,
44).
Jacob, which is released from multiple loci to the surface of
encysting parasites, is a chitin-binding lectin.
Jacob mRNAs,
detected by reverse transcription-PCR, were abundant in extracts of
E. invadens cysts but were weakly present in extracts of
trophozoites, which inevitably contain some encysting parasites (data
not shown). Encystation-specific expression of Jacob was similar to but
more abundant than that of chitinase (11). Anti-Jacob
antibodies showed that Jacob was secreted from numerous foci onto the
surface of encysting parasites with one nucleus (Fig. 5A and
B) and continued until cysts had four
nuclei (Fig. 5C and D). Jacob was present as electron-dense material in
clumps between and on the surface of chitin fibrils (Fig. 6A and
C). Jacob was also present in numerous
large secretory vesicles in encysting parasites (Fig. 5E and 6D). These
Jacob-associated secretory vesicles were larger than those visualized
with anti-chitinase antibodies (Fig. 5F). It is possible that small
chitinase-containing vesicles are lysosomes, which are released during
excystation.
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FIG. 5.
Confocal micrographs of encysting E. invadens
stained with anti-Jacob antibodies (red in panels A through E),
anti-chitinase antibodies (red in panel F) and Sytox green (nuclear
stain in panels A through F). Jacob was present in multiple places on
the surface of encysting parasites with one nucleus (in section [A]
and three-dimensional composite [B]) and became more dense on
parasites with four nuclei (in section [C] and composite [D]).
Jacob was present in numerous relatively large secretory vesicles (in
composite [E]) that surround the nuclei of encysting parasites, which
were permeabilized before labeling. Chitinase (in composite [F]) was
present within hundreds of smaller secretory vesicles of an encysting
parasite. Bars, 5 µm.
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FIG. 6.
Immuno-EM of anti-Jacob antibodies binding to cysts and
wall-less cysts. (A) Anti-Jacob antibodies, visualized with gold
particles, were present over electron-dense material on the surface of
encysting parasites, which were stained prior to fixation. (B) In
contrast, wall-less cysts, which were made in the presence of
galactose, lacked chitin and had a thin layer of electron-dense
material that bound few anti-Jacob antibodies. (C and D) When encysting
parasites were fixed and sectioned prior to staining, anti-Jacob
antibodies bound to cyst walls and to numerous large secretory
vesicles. Bars, 200 nm. Magnification, ×4,500.
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An extract of encysting
E. invadens, which contained soluble
Jacob, was incubated with purified cyst walls that had been treated
with SDS to remove their proteins. Jacob, which was detected with
anti-Jacob antibodies, bound to chitin remaining in SDS-treated
cysts
walls (data not shown). Anti-Jacob antibodies did not bind
to
SDS-treated cyst walls, which had not been pretreated with
extracts of
encysting parasites. Jacob also bound to chitin beads
(Fig.
7). Jacob did not bind to Sepharose or
agarose beads, while
abundant
E. histolytica proteins
including alcohol dehydrogenase
1 and Ariel did not bind to either
SDS-treated cyst walls or chitin
beads (
22,
28). These
results suggest that the Cys-rich domains
of Jacob, which resemble
those of peritrophins and chitinase,
are chitin-binding domains
(
17,
23,
39,
46).
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FIG. 7.
SDS-PAGE and Western blots of Jacob binding to chitin
beads. Western blots with anti-Jacob antibodies to trophozoite proteins
(T) before ( ) and after (+) binding to chitin beads, total proteins
from encysting parasites (E) before () and after (+) binding to
chitin beads, and cyst wall proteins (C) (positive control) are shown.
Two bands labeled with anti-Jacob antibodies correspond to 100-kDa and
high-molecular-mass forms of Jacob identified on two-dimensional
protein gels (Fig. 2). Molecular mass standards are marked on the
right.
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Wall-less cysts are produced when amebae encyst in the presence of
galactose.
We confirmed here the previous observation that
galactose is able to block aggregation and encystation by amebae in
vitro (8, 9). Remarkably, galactose-treated parasites in
encysting medium, which are referred to as wall-less cysts, were
ameboid and quadranculeate, lacked Calcofluor-binding material on their surface, and were filled with numerous secretory granules containing Jacob and chitinase (Fig. 6B and 8A to
C). Quadranucleate, wall-less cysts were also formed in the presence of
lactose but not in the presence of GalNAc or mannose (data not shown).
In contrast, negative-control parasites, which were treated with
galactose in normal culture medium, were mononucleate and lacked Jacob
and chitinase (data not shown).
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FIG. 8.
Confocal micrographs of wall-less cysts, which were made
by encysting E. invadens parasites for 2 days in the
presence of 50 mM galactose. Wall-less cysts, which were permeabilized
before labeling, had four nuclei (stained with Sytox green in panels A,
B, E, and F) and contained numerous secretory vesicles stained with
anti-Jacob antibodies (red in panel A) and anti-chitinase antibodies
(red in panel B). The vast majority of wall-less cysts, which were
labeled with anti-Jacob antibodies (red in panel C), lacked chitin on
their surface, which was detected with WGA (yellow in panel C). In
contrast, many control parasites encysting in the absence of galactose
were spherical and bound WGA to their cyst walls (yellow in panel D).
The galactose lectin was present on the surface of wall-less cysts,
which phagocytosed GFP-labeled bacteria (stained green in panel E) or
mucin-coated beads (stained red in panel F) when excess galactose was
removed. Bars, 5 µm.
|
|
Galactose was demonstrated on the surface of cysts in three ways.
First, purified
E. histolytica plasma membrane Gal/GalNAc
lectin, which was detected with a monospecific rabbit antibody
to
the
E. histolytica Gal/GalNAc lectin, bound to intact
E. invadens cysts but not to
E. invadens
trophozoites or to
E. invadens cysts
treated with SDS to
remove cyst wall glycoproteins (Fig.
3D).
The anti-lectin antibody did
not bind to cysts of negative controls
in which the Gal/GalNAc lectin
was omitted. Second, the
E. histolytica Gal/GalNAc lectin,
again detected with the anti-lectin antibody,
bound to Western blots of
Jacob (inset in Fig.
2E), while a negative
control without the
E. histolytica Gal/GalNAc lectin did not bind
Jacob. These Western
blots do not rule out the possibility that
the Gal/GalNAc lectin binds
to other cyst wall glycoproteins.
Third,
E. histolytica
trophozoites rapidly phagocytosed intact
E. invadens cysts
but not SDS-treated cysts, and phagocytosis
was inhibited by galactose
(data not
shown).
A putative
E. invadens galactose lectin was shown on the
surface of quadranucleate, wall-less cysts by two indirect methods.
First, wall-less cysts phagocytosed bacteria labeled with green
fluorescent protein (
45), and the phagocytosis was inhibited
by galactose (Fig.
8E). Second, wall-less cysts phagocytosed
mucin-coated
beads but not uncoated beads (Fig.
8F). Nonencysting
E. invadens trophozoites also phagocytosed bacteria and
mucin-coated beads
(data not shown), suggesting the galactose lectin is
constitutively
expressed on
E. invadens parasites.
| |
DISCUSSION |
A model of amebic cyst wall construction.
Our results suggest
a two-lectin model of E. invadens cyst wall construction.
First, the plasma membrane galactose lectin binds sugars on Jacob and
perhaps other encystation-specific secretory glycoproteins that are
part of the cyst wall (13, 29, 36, 44). Second, Jacob itself
is a lectin with five Cys-rich domains, which bind chitin (3, 10,
47). Wall-less cysts are formed in the presence of galactose,
because the galactose lectin no longer binds Jacob and other cyst wall
glycoproteins and Jacob no longer binds chitin.
Weaknesses of this two-lectin model include the following. The gene
encoding the large subunit of the
E. invadens galactose
lectin has not been identified. Conversely, the gene encoding
the
E. histolytica homologue of Jacob has not been identified.
Sugars on Jacob have not yet been analyzed. The possibility that
galactose blocks secretion of Jacob and chitinase cannot be ruled
out.
It is also possible that the
E. invadens galactose lectin
is
involved in signal transduction during encystation, as has
been
suggested (
9).
Examples of convergent and coincident evolution.
Jacob appears
to be a chitin-binding lectin for three reasons. First, Jacob is
localized to chitin-binding fibrils by immuno-EM. Second, Jacob binds
chitin in SDS-treated cyst walls and binds chitin beads but does not
bind to other carbohydrates such as Sepharose or agarose. Third, Jacob
contains five putative domains, each of which has six Cys residues with
conserved spacing and numerous aromatic amino acids present in
chitin-binding domains of peritrophins and fungal, nematode, and insect
chitinases (17, 23, 39, 46). Because the spacing between Cys
residues in repetitive domains of Jacob is the mirror-image of those in
lectin domains of peritrophins and chitinases, it appears that Jacob is
related to these chitin-binding proteins by a common structure rather
than a common ancestry (convergent evolution) (14).
Convergent evolution is possible, because chitin-binding domains are
short (~50 amino acids) and contain fewer conserved residues than are present in the active sites of most enzymes.
The plasma membrane Gal/GalNAc lectin, which is implicated here in
binding sugars on Jacob and cyst wall formation, is an
important
vaccine candidate on the surface of
E. histolytica (
29,
44). The Gal/GalNAc lectin is also an important virulence factor,
because the amebic lectin binds sugars on host epithelial cells
and red
blood cells (
13,
36,
37), as well as on bacteria,
which are
the major energy source for parasites in the distal
colon
(
20). Although there is no fossil record to prove or
disprove
our assertion, it is likely that cyst wall formation is
ancient
and was present among free-living ancestors of amebae. Further
invasion of host tissue is a reproductive dead end for amebae,
which
are spread by chitin-walled cysts (
37). It seems likely,
then, that the Gal/GalNAc lectin has been selected for its involvement
in cyst wall formation and bacterial killing and that its involvement
in host cell killing is an example of coincident evolution
(
20).
Similar arguments have been made for coincident
evolution of other
amebic virulence factors including amebapores,
cysteine proteinases,
p21
racA, and vacuolar
ATPase (
20,
25). Cysteine proteinases may also
be important
for cyst wall destruction during amebic excystation,
as has been shown
for excystation of
Giardia (
50).
Discrepancies with previous results.
There are at least two
ways in which our results are different from what might have been
expected from the literature on amebae. First, it is likely that
wall-less cysts, which are formed in the presence of galactose, did
not miss the signal for encystation, as recently suggested
(9), because they are quadranucleate and produce
encystation-specific Jacob and chitinase in abundance (11).
Indeed, wall-less cysts resemble quadranucleate ameboid forms, which
appear when parasites excyst in vitro (our unpublished observations).
It is possible that the quadranucleate wall-less cysts were missed in
recent studies of galactose-induced inhibition of encystation, because
the assay for cysts involved putting parasites in water and treating
them with detergent, which would lyse the wall-less cysts (8,
9). Second, although amebic lipophosphoglycans contain galactose,
these galactose residues do not appear to be accessible on the surface
of E. invadens or E. histolytica (42). E. histolytica trophozoites, which have the Gal/GalNAc
lectin on their surface, do not adhere to and phagocytose each other (29, 36, 43). E. invadens and E. histolytica parasites fail to bind ricin by fluorescence
microscopy, and both parasites are resistant to high concentrations of
ricin (our unpublished data). In contrast, Jacob, which is the first
amebic glycoprotein identified that contains Gal or GalNAc (29,
43), is accessible on the surface of encysting parasites, so that
ricin binds to E. invadens cysts and E. histolytica trophozoites phagocytose cysts in a
galactose-inhibitable manner.
The E. invadens cyst wall is more like the insect
peritrophic matrix than the walls of Giardia cysts or
fungi.
Like the peritrophic matrix that lines the food bolus in
the insect gut, the E. invadens cyst wall is composed of
chitin and a lectin (peritrophin and Jacob, respectively), which has
five putative chitin-binding domains (17, 39). The E. invadens cyst walls and peritrophic membranes have a similar
thickness and appearance by TEM, and strong detergents are necessary to disrupt both structures. In contrast, the Giardia cyst wall
contains polymers of GalNAc rather than GlcNAc, and the abundant
Leu-rich proteins of the Giardia cyst walls (CWP1 and CWP2)
show no similarity in structure to Jacob or peritrophins (27,
30). Like Giardia, the amebic cyst wall is synthesized
simultaneously from numerous loci across the surface of parasites,
while cyst walls of budding yeast or elongating fungal hyphae are
secreted from particular loci (6, 18, 26). While chitin is a
primary structural component of the amebic cyst wall and peritrophic
matrices, it is often used to shape fungal walls, which have a complex
architecture and composition (2, 5, 6). Future studies will
attempt to identify other cyst wall proteins and to determine whether anti-Jacob antibodies may be used to discriminate cysts of E. histolytica and E. dispar (49, 51).
| |
ACKNOWLEDGMENTS |
This work was supported in part by a National Institute of
Allergy and Infectious Diseases Supplement to Promote Reentry into Biomedical and Behavioral Research Careers (M.F.) and by National Institutes of Health grants AI-33492 (J.S.), GM-31318 (P.R.), and
HL-330099 and HL-43510 to R.R.
We acknowledge the expert technical support of Jean Lai of the Harvard
School of Public Health for confocal microscopy and image analysis and
Maria Ericsson of the Harvard Medical School for TEM and immuno-EM.
Thanks to William Lane of the Microchemistry Facility at the Biological
Laboratories of Harvard University for sequencing N-terminal peptides.
Thanks to of Daniel Eichinger of New York University Medical School for
the E. invadens IP-1 strain DNA gDNA library. Thanks to
Barbara Mann and Bill Petri of the University of Virginia Medical
School for purified E. histolytica Gal/GalNAc lectin and
rabbit anti-lectin antibodies.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. Phone: (617) 432-4670. Fax:
(617) 738-4914. E-mail: jsamuels{at}hsph.harvard.edu.
Editor:
W. A. Petri Jr.
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Abstract
Introduction
