From the New England Eye Center and Departments of
§ Ophthalmology, ¶ Physiology, and
Biochemistry,
Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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Acanthamoeba keratitis is a
vision-threatening corneal infection. The mannose-binding protein of
Acanthamoeba is thought to mediate adhesion of parasites to
host cells. We characterized the amoeba lectin with respect to its
carbohydrate binding properties and the role in amoeba-induced
cytopathic effect (CPE). Sugar inhibition assays revealed that the
amoeba lectin has the highest affinity for -Man and Man(
1-3)Man
units. In vitro cytopathic assays indicated that
mannose-based saccharides which inhibit amoeba adhesion to corneal
epithelial cells were also potent inhibitors of amoeba-induced CPE.
Another major finding was that
N-acetyl-D-glucosamine (GlcNAc) which does not
inhibit adhesion of amoeba to host cells is also an inhibitor of
amoeba-induced CPE. The Acanthamoebae are thought to
produce CPE by secreting cytotoxic proteinases. By zymography,
one metalloproteinase and three serine proteinases were detected in the
conditioned media obtained after incubating amoebae with the host
cells. The addition of free
-Man and GlcNAc to the co-culture media
inhibited the secretion of the metalloproteinase and serine
proteinases, respectively. In summary, we have shown that the
lectin-mediated adhesion of the Acanthamoeba to host cells
is a prerequisite for the amoeba-induced cytolysis of target cells and have implicated a contact-dependent
metalloproteinase in the cytopathogenic mechanisms of
Acanthamoeba.
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INTRODUCTION |
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Acanthamoeba keratitis is a painful, vision-threatening
corneal infection. It is caused by parasites of the genus
Acanthamoeba (1, 2). The mechanism by which
Acanthamoebae produce keratitis has not been fully
elucidated. Contact lens wear is thought to be the leading risk factor.
However, the disease also occurs, if less frequently, in non-contact
lens wearers. It is believed that minor corneal surface injury caused
by contact lens wear or other noxious agents and exposure to
contaminated solutions, including lens care products and tap water, are
two major factors in the pathogenesis of the keratitis. The adhesion of
the parasite to the host cells is thought to be a critical first step
in the pathogenesis of infection (3-5). Studies aimed at the
characterization of the molecular mechanisms by which amoebae adhere to
corneal epithelium have shown that: (i) the adhesion of
Acanthamoebae to corneal buttons in organ culture and to
monolayer cultures of corneal epithelium can be inhibited by
methyl--D-mannopyranoside (
-Man) but not by several
other monosaccharides (6, 7), and (ii) a mannose-binding protein is
present on the surface membranes of Acanthamoeba (8).
In vitro, Acanthamoeba parasites have been shown to produce
a cytopathic effect (CPE)1 on
a variety of cell types (9-11) including rabbit and human corneal
stromal and epithelial cells (5, 12, 13). The parasite's ability to
cause cytolysis and necrosis of host tissues is clearly a key component
of Acanthamoeba infection but our understanding of how this
is achieved is still rudimentary. In the present study, we show that
the Acanthamoeba lectin binds
-Man and
1-3-D-mannobiose with highest affinity and that the
lectin-mediated adhesion of the amoeba to host cells is a prerequisite
for the amoeba-induced cytolysis of target cells.
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EXPERIMENTAL PROCEDURES |
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Characterization of the Carbohydrate-binding Specificity of Acanthamoeba Lectin
An Acanthamoeba strain derived from an infected human
cornea (MEEI 0184; Acanthamoeba castellanii based on
morphological characteristics) was used throughout this study. The
parasites were axenically cultured in a proteose peptone/yeast
extract/glucose medium (14). To characterize the sugar binding
properties of the amoeba lectin a solid-phase assay was used (8).
Briefly, wells of microtiter plates were coated with bovine serum
albumin--D-mannopyranosylphenylisothiocyanate (Man-BSA
(Sigma), 15-25 mol of
-D-mannopyranoside/mol of
albumin, 0.03 µg/ml, 50 µl/well in 0.1 M sodium
carbonate buffer, pH 9.6, 4 °C, overnight); nonspecific binding was
blocked with 1% BSA in phosphate-buffered saline (1 h, room
temperature), a 50-µl aliquot of 35S-labeled
Acanthamoebae (5 × 106 cells/ml in
phosphate-buffered saline; 1-2 cpm/parasite, >95% trophozoites) was
added to each well, and the plates were incubated for 2 h at room
temperature. After the cells were rinsed with phosphate-buffered saline
to remove unbound Acanthamoebae, 0.1 ml of 2% SDS was added
to each well, and the radioactivity in aliquots of solubilized material
was determined in a scintillation counter. To characterize the
specificity of the amoeba lectin, a range of saccharides were tested
for their ability to inhibit the adhesion of amoebae to Man-BSA (see
Table I). The 25 saccharides tested were purchased from Sigma, V-Labs
Inc. (Covington, LA), and Pfanstiehl Laboratories Inc. (Waukegan, IL),
and included
1-3-D-mannobiose,
1-6-D-mannobiose,
1-3,
1-6-D-mannotriose, and
1-3,
1-6,
1-3,
1-6-D-mannopentose (Table I).
Serial dilutions of sugars were tested to calculate the concentration
required for 50% inhibition of the binding.
To determine whether any other lectin, besides the mannose-binding protein is present on the amoeba surface, the solid-phase assays were performed using microtiter wells coated with a number of neoglycoproteins including N-acetyl-D-glucosamine-BSA(GlcNAc-BSA), N-acetyl-galactosamine-BSA, fucose-BSA, and galactose-BSA. These neoglycoproteins contained 15-30 mol of sugar/mol of albumin.
Characterization of the Role of the Mannose-binding Protein in the Cytopathogenic Mechanisms of Acanthamoeba
To characterize the role of carbohydrate-mediated host-parasite interactions in the cytopathogenic mechanisms of Acanthamoeba, in vitro cytopathic assays were performed using cell cultures of corneal epithelium as target cells.
Immortalization of Rabbit Corneal Epithelial Cells--
Since
primary cultures of corneal epithelium have limited life span and can
only be passaged 2 to 3 times without significant alteration in cell
morphology, corneal epithelial cells with extended life span were
produced. For this, the primary corneal epithelial cell cultures
prepared as described earlier (15, 16) were infected with SV40 large T
antigen cDNA (17). Retroviruses were used to transduce both the
3'-phosphotransferase gene conferring G418 antibiotic resistance and
the SV40 large T antigen cDNA into the primary cultures. For
infection, irradiated producer cells (CRIP-SV40, 1 × 106 cells/100-mm dish) were added to confluent primary
cultures of corneal epithelium. Two to three days after infection, the
transformed cells were selected by adding G418 (500 µg/ml) into the
culture medium. To promote the growth of the selected cells, initially they were co-cultured with irradiated fibroblast cells. After 10-12
days, when the cultures reached 30-40% confluence, G418 as well as
fibroblasts were withdrawn. As of this stage, cells were cultured in
Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) containing fetal
bovine serum (15%), dimethyl sulfoxide (0.5%), gentamicin (40 µg/ml), epidermal growth factor (10 ng/ml), insulin (5 µg/ml), and
cholera toxin (0.1 µg/ml). When these cells reached over 50%
confluence, they were passaged with a split ratio of 1:2. Upon
phase-contrast microscopy, no fibroblastic cells could be detected
subsequent to the first passage. The entire cell population was
epithelioid. The cultures reacted positively with mAb AE5 which reacts
with keratin K3, a marker for corneal epithelial cells. This further
established the epithelial origin of the immortalized cells.
Cytopathic Assay-- To evaluate the Acanthamoeba-induced CPE on corneal epithelial cells, the parasites (>95% trophozoites) were rinsed three times in a serum-free medium supplemented with 0.4% BSA (18) and aliquots of the parasite suspension (0.2 × 105 to 1 × 106 parasites/ml; 300 µl/well for 24-well plates; 1 ml/well for 6-well plates) were added to wells of confluent cultures of epithelium which had been rinsed and preincubated in the serum-free medium for 2 h. The plates were then incubated at 37 °C in a CO2 incubator and were periodically examined under a phase-contrast microscope for the presence of cell-free plaques in the monolayer for up to 28 h. Control wells contained the target cells in the medium without the parasites. To evaluate the effect of saccharides on Acanthamoeba-induced CPE, the assays were performed in the presence of sugars (0.03-100 mM). To estimate the relative inhibitory potency of each sugar, the CPE assays were terminated when, based on observations under a phase-contrast microscope, the culture wells incubated with the amoeba in the absence of saccharides exhibited approximately 50% destruction of the monolayer (10-14 h). At the end of the incubation period, the cultures were stained with Giemsa (Diff-Quik, Dade Diagnostic Inc., Aguada, PR). Approximate cell density in each well was estimated by scanning the stained plates in a calibrated, computer-assisted Bio-Image Scanner (Mllipore, Ann Arbor, MI). CPE was expressed as percentage of the optical density by the following formula.
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(Eq. 1) |
Measurement of Acanthamoeba-induced Target Cell Lysis
Cell lysis was quantified by measuring 51Cr release
from prelabeled cells. For radiolabeling, confluent monolayer cultures
of corneal epithelium in 24-well plates were incubated in cell culture medium containing Na51CrO4 (460 mCi/mg,
12 µCi/ml, NEN Life Science Products) for 18-20 h. At the end of the
labeling period, the cells were extensively rinsed and were then
incubated with Acanthamoeba (1 × 106
parasites/ml) in the presence or absence of saccharides (50 mM -Man or GlcNAc). The cultures were periodically
examined under a phase-contrast microscope for CPE. When approximately
50-70% cell loss occurred in the monolayers, aliquots of cell-free
conditioned media from each well were analyzed for specific
51Cr release. To determine specific 51Cr
release values, counts/min released in control wells incubated in media
alone without the parasites were deducted from the counts/min released
in the test wells. Percent specific release was calculated using a
formula: E/T × 100, where E is
counts/min released in test wells minus the counts/min released in
control wells, and T is the total counts/min (counts/min
cells + counts/min supernatant in control wells). Counts/min of cells
was determined by incubation of the control wells with 0.5% Triton
X-100. Conditioned media from each well was also analyzed for lactate
dehydrogenase release using an lactate dehydrogenase assay kit
purchased from Sigma. In each case, there were four wells/group. The
same procedure was used to determine whether ACM possesses cytolytic
activity.
Analysis of Proteinases by Zymography
To determine whether the mannose-mediated host-parasite
interactions influence the levels of secretory proteinases, conditioned media obtained by incubating the amoebae with primary cultures of
corneal epithelium in the presence and absence of saccharides (-Man
and GlcNAc, 50 mM, 6-8 h) were analyzed by zymography. The
zymography was performed using separating gels containing 10%
acrylamide and 2 mg/ml gelatin, essentially as described by Kleiner and
Stetler-Stevenson (19). Samples to be analyzed were diluted in the
electrophoresis sample buffer (20) without mercaptoethanol and were
then applied to the gels. After electrophoresis, the proteinases
separated on gels were renatured by incubating the gels in 2.5% Triton
X-100 in 50 mM Tris-HCl, pH 7.5, to remove SDS. Following
this, the gels were incubated for 18 h in a developing buffer (50 mM Tris-HCl, pH 7.5, containing 10 mM
CaCl2) and were then stained with Coomassie Brilliant Blue.
Areas of digestion were visualized as nonstaining regions of the gel.
In some experiments, samples were pretreated with phenylmethylsulfonyl
fluoride (PMSF, 1 mM), an inhibitor of serine proteinases
(21), for 1 h prior to electrophoresis, or 1,10-phenanthroline (1 mM), an inhibitor of metalloproteinases (22), was added to
the developing buffer.
To determine whether GlcNAc and/or -Man influence the levels of
secretory proteinases of Acanthamoeba by contact-independent mechanisms, ACM prepared in the presence and absence of the saccharides (50 mM) were also analyzed by zymography.
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RESULTS |
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The Amoeba Lectin Has the Highest Affinity for -Man and
Man(
1-3)Man Units
In solid-phase assays, binding of amoebae to Man-BSA was
dose-dependent (Fig. 1).
Amoebae did not bind to a number of other neoglycoproteins including
galactose-BSA, fucose-BSA,
N-acetyl-D-galactosamine-BSA, and GlcNAc-BSA
(Fig. 1). To characterize the carbohydrate binding properties of the
amoeba lectin, sugar inhibition studies were performed. Concentrations
of sugars required for 50% inhibition were calculated from inhibition
curves and are listed in Table I. Among
various monosaccharides tested, -Man was the most potent inhibitor
of amoeba binding to Man-BSA. Compared with
-Man the inhibitory
potency of methyl-
-mannopyranoside and D-mannose was 8- and 10-fold less, respectively. Epimers of D-mannose,
i.e. altrose (C-3) and talose (C-4), were not inhibitory but
glucose (C-2) was a weak inhibitor. Compared with
D-mannose, the inhibitory potency of D-glucose
was 3.5-fold lower. D-Lyxose, which is equivalent to
D-mannose with the C-5 substituent missing, was not
inhibitory. The reduction of D-mannose to
D-mannitol by sodium borohydride completely destroyed its
ability to inhibit the amoeba binding to Man-BSA. Neither
D-mannosamine nor
N-acetyl-D-mannosamine were inhibitory.
D-Glucosamine was also not inhibitory. However, GlcNAc and
-L-fucose were weak inhibitors. Their inhibitory
potencies were 26- and 14-fold lower, respectively, compared with that
of
-Man. Both mannobioses, the mannotriose, and the mannopentose were also potent inhibitors; the inhibitory potency of
1-3-D-mannobiose was approximately three times
greater than that of
1-6-D-mannobiose and
-Man
alone.
1-3,
1-6-D-mannotriose and
1-3,
1-6,
1-3,
1-6-D-mannopentose were
slightly better inhibitors than
1-3-D-mannobiose
(Table I).
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Mannose-mediated Host-Parasite Interactions Play a Role in Acanthamoeba-induced Cytopathogenic Mechanisms
Acanthamoeba trophozoites have been shown to produce a CPE on a variety of cultured mammalian cells (5, 9-13). To determine whether the carbohydrate-mediated adhesion of Acanthamoeba to corneal epithelium is a prerequisite for the parasite-induced target cell damage, CPE assays were performed in the presence and absence of various saccharides. The pathogenic ocular isolate of Acanthamoeba used in the present study produced extensive CPE on both immortalized (Fig. 2) as well as primary (Fig. 3) rabbit corneal epithelial cells in culture. Within 8-10 h incubation with the parasites, small cell-free plaques were seen in the monolayers (Fig. 2, 8 h). With the continued incubation with the amoebae, the size of the cell-free areas increased (Fig. 2, 16 h), and eventually the monolayer surrounding the large plaques lifted entirely from the culture dish resulting in almost complete loss of the cell layer (Fig. 2, 24 h). The extent of the monolayer destruction depended on the concentration of amoebae, the nature of cell cultures (primary versus immortalized), the length of the incubation period, and the composition of the media used for the assay. When immortalized cultures were used, an amoeba concentration of 2 × 105 cells/ml was required to completely destroy the monolayer within 20 h. All CPE assays were performed in a serum-free medium supplemented with 0.4% BSA. If BSA was omitted from the media, the destruction of the monolayer occurred approximately twice as fast (data not shown).
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Upon phase-contrast microscopy, it was evident that when amoebae were
incubated with monolayers, they adhered to the cells within minutes.
All saccharides which inhibited amoeba binding to Man-BSA also
inhibited the amoeba binding to corneal epithelial cells (not shown).
In vitro CPE assays, for the most part, revealed a direct
correlation between the ability of the sugar to inhibit the
Acanthamoeba binding to Man-BSA and to inhibit the
amoeba-induced CPE (Table I). -Man, both mannobioses, the
mannotriose, and the mannopentose which were potent inhibitors of
amoeba binding to Man-BSA were also potent inhibitors of amoeba-induced
CPE; saccharides such as mannitol, mannosamine,
N-acetyl-D-mannosamine, methyl-
- and
methyl-
-galactopyranoside which did not inhibit amoeba binding to
Man-BSA also did not inhibit amoeba-induced CPE. One striking exception
was GlcNAc which was a weak inhibitor of the amoeba adhesion to Man-BSA
but a potent inhibitor of CPE (see below).
Primary cultures were relatively less susceptible to amoeba-induced CPE
in that 1 × 106 parasites/ml were required to
completely destroy the monolayer within 24 h. This parasite
concentration is five times higher than that required to produce the
same degree of CPE when immortalized cultures were used as target
cells. Only a limited number of saccharides were tested for their
ability to inhibit the amoeba-induced CPE on primary cultures. At a
sugar concentration of 5 mM or less, -Man, both
mannobioses, the mannotriose, the mannopentose, and GlcNAc inhibited
CPE. Methyl-
-D-galactopyranoside,
methyl-
-D-galactopyranoside, and
-L-fucose were not inhibitory up to 100 mM
concentration (Fig. 3).
-Man and GlcNAc Modulate Acanthamoeba-induced Cytopathic Effect
by Distinct Mechanisms
As described above, GlcNAc, a weak inhibitor of amoebae adhesion
to Man-BSA, was a potent inhibitor of amoeba-induced CPE. The
inhibitory potency of GlcNAc in the amoeba adhesion assay was 26 times
less than that of -Man (Fig. 4,
top panel; Table I). In contrast, in the CPE assays, the
inhibitory potency of both
-Man and GlcNAc was nearly identical
(Fig. 4, bottom panel; Table I). Amoebae did not adhere to
the cell monolayer when
-Man was present. In addition,
-Man
almost completely inhibited amoeba-induced CPE. Cell-free plaques were
not detected in the presence of
-Man (Figs. 3 and Fig.
5, top panel, group M).
-Man, however, did not inhibit the lifting of the intact monolayer
from the cell substratum upon "prolonged" (24-28 h) incubation of
the cultures with amoebae (Fig. 5, middle and bottom
panels, groups M). In the absence of
-Man, nearly complete loss
of cell layer occurred around 20 h; in the presence of
-Man (50 mM), after an approximate 24-h incubation with the amoebae,
the cell layer began to lift from the periphery (Fig. 5, middle
panel, group M) and continued to roll inwards until the entire
layer was lifted from the dish (Fig. 5, bottom panel, group
M). This occurred more than 4 h after the complete loss of
the cell layer had occurred in the cultures incubated without the
sugar. When GlcNAc (50 mM) was present in the media,
amoebae adhered tightly to the monolayer. Despite this, the cell layer
remained attached to the culture dish during the entire assay period of
28 h (Fig. 5, middle and bottom panels, groups
G). GlcNAc markedly delayed, but did not completely inhibit plaque
formation. Upon careful examination of the cultures incubated with
amoebae in the presence of GlcNAc for 28 h, small plaques could be
seen in the monolayer (Fig. 5, bottom panel, group G). Since
CPE assays were performed in the serum-free media, incubations were not
continued beyond 28 h.
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-Man but Not GlcNAc Is a Potent Inhibitor of Amoeba-induced
Cytolysis--
Cell lysis as measured by 51Cr release
revealed that Acanthamoeba possesses cytolytic activity
(Fig. 6, E+A). The extent of cytolysis, however, did not correlate with the extent of CPE; when
approximately 50% cell loss had occurred in the CPE assays, percent
specific 51Cr release was about 12.5%. Greater than 70%
of amoeba-induced cytolysis was inhibited by
-Man (50 mM) (Fig. 6, E+A+M). In contrast, GlcNAc was
only a weak inhibitor of amoeba-induced cytolysis with inhibition
values ranging between 10 and 20% (Fig. 6, E+A+G). Similar
observations were made using LDH release assays (data not shown).
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GlcNAc but Not -Man Inhibits ACM-induced CPE--
Early studies
have shown that cell-free ACM also has the ability to produce CPE. The
CPE-inducing ability of ACM is likely to be independent of
host-parasite interactions, and related to the cytotoxic secretory
factors of Acanthamoeba. To determine the role of secretory
factors of the parasites in the amoeba cytopathogenic mechanisms,
51Cr release and cytopathic assays were also performed
using cell-free ACM. 51Cr release assays revealed that ACM
lacks cytolytic activity (Fig. 6, E+ACM). Also, ACM failed
to produce cell-free plaques in the monolayer (Fig.
7, top panel). However,
approximately 6-8 h following the incubation of the monolayer with the
ACM, the cell sheet at the edges of the monolayer began to lift and
roll inwards (Fig. 7, top panel, 8 h) and eventually the
entire monolayer lifted en bloc from the dish (Fig. 7,
top panel, 16 h). These observations were similar to those
seen when the monolayers were incubated with trophozoites in the
presence of
-Man for prolonged periods (Fig. 5). When saccharides
(50 mM) were added to ACM after it was produced, neither
-Man nor GlcNAc had an inhibitory effect on the ACM-induced CPE
(Fig. 7, middle panel, groups ACM+M and ACM+G).
Also, ACM produced in the presence of
-Man exhibited potent CPE
(Fig. 7, middle panel, group ACM+M'). In contrast, the ACM
produced in the media containing GlcNAc did not produce CPE (Fig. 7,
middle panel, group ACM+G'). To further determine whether
GlcNAc inhibits the ACM-induced CPE directly by itself or indirectly by
influencing the expression of CPE producing factors of amoeba, filtrate
and retentate obtained after filtering the ACM in Centricon 3 microconcentrators were tested for their ability to produce CPE. From
ACM produced in the absence of sugar, almost all of the CPE inducing
ability was recovered in the retentate obtained after Centricon
filtration (Fig. 7, bottom panel). The ACM produced in the
presence of GlcNAc could not be caused to acquire the CPE inducing
ability after removing GlcNAc by Centricon filtration (Fig. 7,
bottom panel).
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Mannose-mediated Adhesion of Acanthamoebae to Host Cells Induces
Expression and/or Secretion of a Metalloproteinase--
To
characterize the mechanism by which the mannose-mediated host-parasite
interactions influence the amoeba-induced CPE, conditioned media
obtained after incubating amoebae with the host cells for 6-8 h in the
presence and absence of -Man were analyzed for proteinases by
zymography on gelatin gels. Four components (P1, 230-kDa; P2, 97-kDa;
P3, 80-kDa; and P4, 55-kDa; average values from four gels) were
detected in the samples obtained after incubating amoebae with primary
cultures of corneal epithelium in media alone for 6 h (Fig.
8, panel A, lane E+A). Of
these four components, the expression and/or secretion of the component
P3 was dependent on the mannose-mediated adhesion of amoeba to host
cells; if the adhesion of amoebae to the host cells was prevented by
adding
-Man in the media, component P3 was not detected (Fig. 8,
panel A, lane E+A+M). Nor was proteinase P3 detected in
media conditioned by Acanthamoeba or epithelial cells alone
(Fig. 8, panel A, lanes ACM and EM). Presence of
GlcNAc in the co-culture either did not alter or reduced only slightly
the levels of P3 (not shown), but had a dramatic effect on the levels
of P1, P2, and P4. In the ACM prepared in media alone, mainly
components P1 and P4 with trace amounts of component P2 were seen (Fig.
8, panels A and B, lanes ACM). The presence of
-Man in the media used to prepare ACM did not influence the levels
of P1, P2, or P4 (not shown, pattern indistinguishable from ACM lanes
shown in panels A and B). In contrast, in the ACM
prepared in the presence of GlcNAc, P2 was not detected and P1 and P4
were seen in markedly reduced amounts (Fig. 8, panel B, lane
ACM+G). Components P1, P2, and P4 were susceptible to PMSF (Fig.
8, panel C, lanes ACM and E+A), but were
resistant to 1,10-phenanthroline (Fig. 8, panel D, lanes ACM
and E+A). In contrast, component P3 was susceptible to
1,10-phenanthroline and resistant to PMSF (Fig. 8, panels C
and D, lanes E+A). In the media conditioned by epithelial
cells alone, two components were seen (E1, 93-kDa;
E2, 63-kDa; average values of two gels) (Fig. 8, panel
A, lane EM), both of which were resistant to PMSF (Fig. 8,
panel C, lane EM) and susceptible to 1,10-phenanthroline (Fig. 8, panel D, lane EM). Proteinases P1, P2, and P4 were
susceptible whereas P3, E1, and E2 were resistant to aprotinin, another
serine proteinase inhibitor (not shown, data identical to those shown for PMSF).
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Proteinase Inhibitors Inhibit both Amoebaand ACM-induced CPE
To further determine the role of proteinases in the cytopathogenic mechanisms of Acanthamoeba, the effect of proteinase inhibitors on amoeba- and ACM-induced CPE on corneal epithelial cells was analyzed. Serine proteinase inhibitors, PMSF (0.5 mM) as well as aprotinin, (5 units/ml), almost completely inhibited both amoeba- and ACM-induced CPE (not shown, results identical to those of CPE assays performed in the presence of GlcNAc, Fig. 5). The metalloproteinase inhibitor 1,10-phenanthroline was toxic to corneal epithelial cells, and therefore it was not possible to evaluate its CPE inhibitory potential.
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DISCUSSION |
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The goals of the present study were to define more precisely the
carbohydrate binding properties of the Acanthamoebae
mannose-specific lectin and to determine whether carbohydrate-mediated
adhesion of amoebae to host cells is a necessary prerequisite for the
parasite-induced CPE. The results of the saccharide inhibition assays
revealed that among monosaccharides, the amoebic lectin has the highest affinity for -Man. The inhibitory potency of D-mannose
was 10-fold less than that of
-Man. Our results, that the epimers of
D-mannose, altrose (C-3), and talose (C-4) were not
inhibitory and glucose (C-2) was only a weak inhibitor, suggest that
the configuration of free hydroxyl groups at C-2, C-3, and C-4
positions of D-mannose is necessary for optimal binding
interactions. The fact that D-lyxose was not inhibitory
supports the notion that the CH2OH substituent is also
essential for binding. The results of the inhibitory potencies of
both mannobioses, the mannotriose, and the mannopentose suggest that
Man(
1-3)Man disaccharide is the most complimentary to the carbohydrate-binding site of the amebic lectin being three times more
potent than the corresponding
1-6-disaccharide. Elongation of the
Man(
1-3) chains by additional mannose residues increased the
affinity only slightly.
In vivo, pathology of Acanthamoeba infection is characterized by the infiltration and necrosis of the infected tissue (1, 2) and, as described earlier, the parasite has also been shown to cause CPE on a variety of cell types in vitro (5, 9-13). Although it has been presumed that the adhesion of amoeba to host cells is a critical first step in the pathogenesis of infection, several studies have reported that cell-free ACM contain destructive proteinases (23-25) and phospholipases (26). Thus, one could argue that if the amoebae elaborate digestive enzymes, target cell loss may well be independent of host-cell contact. The present study revealed that the trophozoite-induced target cell loss resulted from at least four sequential steps: (i) adhesion of amoeba to target cells; (ii) cytolysis of target cells; (iii) development of cell-free plaques in the monolayer which increase in size with time; and (iv) detachment of the monolayer surrounding the plaques from the culture dish.
What is the likely mechanism by which amoeba produce cell-free plaques
in the monolayer? It appears that the first step in the plaque
formation is carbohydrate-mediated contact-dependent cytolysis. The cytolysis, in turn, makes the cell substratum in the
localized areas accessible to one or more proteinases responsible for
the detachment of the cells from the culture dish. This mechanism of
plaque formation is supported by our findings that: (i) inhibition of
the parasite-induced cytolysis by -Man also inhibited the plaque
formation and (ii) ACM which lacks cytolytic activity did not produce
cell-free plaques. The fact that despite the lack of the ability to
induce plaque formation, the ACM results in the detachment of the
monolayer suggests that the factors responsible for the detachment of
the monolayer are secreted by amoebae and are independent of the
mannose-mediated adhesion of amoeba to the host cells. This would
explain why prolonged incubation of target cultures with amoebae
resulted in the detachment of the monolayer from the substratum despite
the presence of
-Man in culture media which inhibited: (i) adhesion
of amoebae to host cells; (ii) cytolysis of target cells; and (iii)
plaque formation.
The actual mechanisms of Acanthamoeba-induced cytolysis and CPE are not known. It has been reported that pathogenic strains of Acanthamoebae produce significantly higher quantities of phospholipases (26), fibrinolytic enzymes (23), and cysteine proteinases (25) compared with nonpathogenic strains. Moreover, a correlation between pathogenicity and proteinase activity has been reported for a number of protozoans (27) including Entamoeba histolytica (28, 29), Giardia lamblia (30), Leishmania amazomen (31), and Trypanosoma cruzi (32). In the present study, we found, not only that Acanthamoebae secrete proteinases but also that carbohydrate-mediated adhesion of amoebae to host cells has a profound effect on the nature of the proteinases secreted by the parasite and/or host cells. We found that a specific metalloproteinase (P3) was secreted into the culture media only upon mannose-mediated direct contact of the parasite to target cells. Although it remains to be determined whether P3 is produced by the host cells or the parasite and whether it is the expression, the activity, or merely the secretion of P3 which is elevated upon the adhesion of amoeba to the host cells, it is clear that contact-mediated cross-talk between the parasite and the host cells is a key component of Acanthamoeba infection.
Studies by Alizadeh and co-workers (33) have shown that the Acanthamoeba-induced cell death is due, at least in part, to apoptosis. A recent study has shown that target cells killed by E. histolytica also undergo DNA fragmentation characteristic of apoptotic death (34). Killing of the host cells by E. histolytica in vitro occurs only upon direct contact which is mediated by a Gal/GalNAc-specific amoebic surface lectin (35). The contact-dependent cytolysis of E. histolytica has been attributed at least in part to amoebic pore-forming proteins and phospholipase A activity, both of which are thought to influence cytopathogenicity by disrupting target cell membranes thereby rendering the cell permeable to attack by other amoebic enzymes or toxins (36, 37). The mechanism by which Acanthamoebae induce apoptotic death of the target cells has not been elucidated. Our studies suggest that the mannose-mediated adhesion of amoebae to host cells followed by secretion of proteinases, especially P3, are likely to be key events.
An unexpected finding of the present study was the observation that
GlcNAc which is neither an inhibitor of the adhesion of amoeba to
target cells nor an inhibitor of amoeba-induced cytolysis is a potent
inhibitor of amoeba-induced CPE. It appears that GlcNAc inhibits CPE
indirectly by influencing the expression and/or secretion of the
molecules involved in cytopathogenic mechanisms of
Acanthamoeba because: (i) the GlcNAc only inhibited the CPE
when it was added to the culture medium used to produce ACM and not
when it was added to the ACM after it was produced in the media alone;
and (ii) removal of GlcNAc from the ACM by Centricon filtration did not
result in the loss of CPE inhibitory activity of the ACM. Indeed we
found that the ACM prepared in the presence of GlcNAc contain markedly
diminished levels of serine proteinases compared with that prepared in
the media alone or in the media containing -Man. Although it remains
to be determined whether GlcNAc inhibits the expression and/or
secretion or merely the activity of Acanthamoeba proteinases, it is tempting to speculate that
O-GlcNAc-bearing nuclear or cytoskeletal proteins may be
involved in the regulation of amoeba serine proteinases.
O-GlcNAc is a major protein modification in almost all
eukaryotes (38, 39), including parasites (40). It is known that many
transcription factors are post-translationally modified with
O-linked N-acetylglucosamine monosaccharide on
serine and/or threonine residues (38, 39). Most
O-GlcNAc-bearing proteins are phosphoproteins, and it has
been shown that phosphorylation could occur at the same
serine/threonine residues that are targeted for glycosylation. It is
therefore thought that O-GlcNAc modification has a
regulatory role in gene expression, possibly by controlling phosphorylation at identical or nearby sites. Indeed, exogenous GlcNAc
can penetrate the interior of the cell (41) leading to an increase in
the intracellular concentration of UDP-GlcNAc, a substrate for protein
glycosylation. This in turn, may hyperglycosylate one or more
transcription factor(s) which modulate the expression of the molecules
involved in the pathogenic mechanisms of Acanthamoeba. It is
conceivable that the glycosylation of the sites which otherwise would
have been phosphorylated could result in the loss of the function of
the transcription factor. Regardless of the mechanism, the use of
GlcNAc in conjunction with
-Man-based saccharides may have
therapeutic potential specially because the saccharides are nontoxic
and can be delivered topically to the eye either in the form of eye
drops or from contact lenses designed to deliver small continuous doses
of drugs into the eye.
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ACKNOWLEDGEMENTS |
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We thank Drs. Pamela Stanley, William Petri Jr., James F. Dice Jr., and Elizabeth Fini for helpful discussions, Dr. Tung-Tien Sun for providing mAb AE5, and Irfan Khan for help with the sugar inhibition assays.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grants EY07088 and EY09349.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom all correspondence should be addressed: Dept. of Ophthalmology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6776; Fax: 617-636-0348; E-mail: NPanjwani{at}infonet.tufts.edu.
1
The abbreviations used are: CPE, cytopathic
effect; -Man, methyl-
-D-mannopyranoside; Man-BSA,
-D-mannopyranosylphenylisothiocyanate/bovine serum
albumin; GlcNAc-BSA,
N-acetyl-D-glucosamine-bovine serum albumin;
ACM, Acanthamoeba-conditioned medium; PMSF,
phenylmethylsulfonyl fluoride.
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REFERENCES |
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