Effects of Mannose on Acanthamoeba castellanii Proliferation and Cytolytic Ability to Corneal Epithelial Cells

Abstract

purpose. Acanthamoeba trophozoites express a mannose binding receptor that facilitates adhesion of trophozoites to mannosylated proteins on corneal epithelial cells. This study was undertaken to determine the role that mannose stimulation has in the amoeba’s growth, secreted products, and ability to desquamate the corneal epithelium.

methods. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose (PYG) and PYG with 100 mM methyl α-d-mannopyranoside or galactose. The proliferation of trophozoites and cysts was examined by optical density and direct counts. The molecular weight of the mannose-stimulated protein was examined by SDS/PAGE. The cytolytic protein was purified by fast protein liquid chromatography (FPLC) size exclusion and ionic exchange and then tested for cytopathic effect (CPE) and collagenolytic activity in vitro. Collagenolytic activity was examined by zymography. Proteases and protease inhibitors were used to characterize the nature of the cytolytic protein.

results. Methyl α-d-mannopyranoside inhibited the growth of A. castellanii by 50% (P < 0.05) and concomitantly induced a threefold increase in the formation of cysts. SDS-PAGE analysis revealed a mannose-induced protein of ∼133 kDa (MIP-133). The MIP-133 protein was found to be highly cytolytic against corneal epithelial cells, but not human intestinal epithelial cells and also degraded collagen in vitro. Serine protease inhibitors abrogated both CPE and collagenolytic activity of the MIP-133 protein (P < 0.001).

conclusions. The results suggest that binding of trophozoites to mannosylated proteins on the corneal surface induces A. castellanii to secrete a ∼133-kDa serine protease that kills both human and hamster corneal epithelium and degrades collagen.

Acanthamoebakeratitis is a vision-threatening infection caused by a free-living, pathogenic amoeba. ^{1 2} Corneal infection was first recognized in 1973, and the main risk factor was found to be contact lens wear. ^{3 4} Characteristic symptoms include disproportionately severe ocular pain, a paracentral ringlike stromal infiltrate, epithelial ulcers, and resistance to many antimicrobial agents. ^{5 6 7} Typical treatment consists of around-the-clock, hourly, topical applications of chlorhexidine, brolene, or polyhexamethylene biguanide, alone or in combination. This regimented therapy may continue for weeks. Even with such treatments, many patients require corneal transplants, which can be reinfected by dormant cysts. ⁵  

Acanthamoeba spp. are ubiquitously distributed in nature and can be readily isolated from swimming pools, hot tubs, soil, dust, drinking fountains, air-conditioning ducts, nasopharyngeal mucosa, and eyewash stations. ^{8 9 10 11 12 13 14 15 16} Although contact lens wear is the predominant risk factor, it is believed that existing trauma to the eye is also necessary. ^{2 4 8 17 18} Moreover, induction of Acanthamoeba keratitis in experimental animals requires corneal abrasion before exposure to infectious trophozoites. ^{18 19 20}  

The pathogenesis of Acanthamoeba keratitis occurs in a sequential manner. Binding of the trophozoites to the corneal epithelial surface is believed to be an essential first step in the infectious cascade. Studies have shown that A. castellanii bind with high affinity to mannose glycoproteins, but not to other sugars. ^{21 22} Moreover, methyl-α-d-mannopyranoside (up to 200 mM) has been shown to inhibit effectively the binding of Acanthamoeba trophozoites to both rabbit and Chinese hamster corneal epithelial (HCORN) cells. ^{22 24 27} When the cornea is injured, part of the wound-healing response is the upregulation of mannosylated glycoproteins on the cell surface. ^{23 24} These proteins may act as ligands to which the amoebae can bind. 

We have shown that although mannose inhibits the binding of Acanthamoeba trophozoites to corneal epithelial cells, the cytopathic activity of trophozoites against the corneal cells is elevated. ²⁷ In the present study, we sought to determine whether this elevated cytopathic effect (CPE) on corneal epithelial cells by mannose-treated trophozoites was due to an increased number of trophozoites that were induced to proliferate in response to mannose, to a reduction in the spontaneous encystment of trophozoites that spontaneously occurs in vitro, or to mannose-induced production of cytolytic proteins by trophozoites. The results of the preliminary experiments revealed that mannose induced the production of a cytolytic protein. Accordingly, the remainder of the study characterized the mannose-induced protein. 

Materials and Methods

Amoebae and Cell Lines

A. castellanii (ATCC 30868), originally isolated from a human cornea, was obtained from the American Type Culture Collection (Manassas, VA). Amoebae were grown as axenic cultures in peptone-yeast extract glucose (PYG) at 35°C with constant agitation. ¹⁵  

Chinese corneal (HCORN) cells were immortalized with human papillomavirus E6 and E7 genes, as previously described ²⁵ and cultured in complete minimum essential medium (MEM; JRH Biosciences, Lenexa, KS) containing 1% l-glutamine (BioWhittaker, Walkersville, MD), 1% penicillin, streptomycin, amphotericin B (Fungizone; BioWhittaker), 1% sodium pyruvate (BioWhittaker), and 10% fetal calf serum (FCS; HyClone Laboratories, Logan, UT). 

Human corneal epithelial (HCE) cells were a generous gift from Sherry Ward of the Gillette Company (Gillette Medical Evaluation Laboratories, Gaithersburg, MD). Cells were cultured in KGM medium with a kit (Bullet Kit CC-3111; Clonetics, Walkersville, MD). 

Human small intestinal epithelial cells (ATCC CCL-241; Fhs-74) were obtained from the ATTC. Cells were cultured in Hybricare culture medium (46-X; ATCC) supplemented with 10% fetal calf serum. 

Acanthamoeba Growth and Cyst Analysis

Acanthamoeba trophozoites were grown in 200 mL of PYG, either with or without 100 mM methyl-α-d-mannopyranoside (Sigma-Aldrich, St. Louis, MO) on a shaker incubator set at 125 rpm and 35°C. Initial cultures were seeded with 1 × 10⁷ trophozoites. Control experiments were performed with 100 mM galactose and 100 mM lactose. One-milliliter samples were removed approximately every 12 hours for 200 hours, with additional samples taken at 270, 290, and 320 hours. Samples were either immediately examined for trophozoites and cysts by direct counts or examined spectrophotometrically at an optical density of 480 nm (OD₄₈₀). All direct counts were performed by hemocytometer. Assays were performed in triplicate. 

Excystment assays were performed in 100 mL of PYG, either with or without 100 mM methyl-α-d-mannopyranoside on a shaker incubator set at 125 rpm and at 35°C. Initial cultures were seeded with a total of 1 × 10⁷ cysts (0% trophozoites, verified by calcofluor white staining). One-milliliter samples were removed every 24 hours for 10 days and examined by direct count. The experiment was ended on day 10, to prevent the excysted trophozoites from proliferating to stationary phase and beginning to encyst. Control experiments were duplicated with phosphate-buffered saline (PBS). Assays were performed in triplicate. 

Molecular Weight Separation of Supernatants

Supernatants from mannose-stimulated cultures were removed during midlog phase (OD₄₈₀ = 0.26), sterilized using low protein binding 0.22-μm filter units (Corning, Corning, NY), and fractionated using microcentrifugal concentrators with membranes having a molecular mass cutoff of approximately 50 or 100 kDa (Pall-Filtron, Northborough, MA). Three milliliters of culture supernatant were added to the centrifugal concentrator and centrifuged at 1900g in a floor centrifuge (Sorvall RC5C; DuPont, Newtown, CT) for 1 hour. Supernatants were removed from the top chamber at 10× concentration, and 25 μL was used directly in the CPE assays. 

Assay for CPE

Supernatants were added to 96-well plates, with or without confluent monolayers of HCORN, HCE, or Fhs-74 cells, and incubated for 18 hours at 35°C. Each well contained 200 μL of the respective growth medium. Control wells consisted of untreated confluent cells. After incubation, all wells were washed three times with their respective growth media and stained with Giemsa stain (Shandon, Inc., Pittsburgh, PA). After staining, the wells were washed three times with PBS (pH 7.2) and solubilized in 0.1 mL of 5% sodium dodecyl sulfate (SDS) in PBS. Solubilized cells were transferred to a new 96-well plate, and the OD was read at 590 nm in a microplate reader (Molecular Devices, Menlo Park, CA). Percent CPE was calculated according to the following formula: %CPE = 100 − [(OD of experimental well − OD of supernatant alone/OD of control cells alone) × 100]. Assays were performed in triplicate. 

Protein Purification

All purification procedures were performed at room temperature. Protein concentrations were determined by bicinchoninic acid protein assay (BCA; Rockford, IL) with bovine serum albumin used as a standard. ²⁶ Tenfold concentrated PYG, and 10-fold concentrated Acanthamoeba culture supernatants, with or without 100 mM methyl-α-d-mannopyranoside, were analyzed by 4% to 15% SDS-PAGE (Ready Gels; Bio-Rad, Hercules, CA) under both reducing and nonreducing conditions. Supernatants were taken from trophozoites at mid log phase (OD₄₈₀ = 0.35 or 0.26 respectively; see Fig. 1A ). 

For fast protein liquid chromatography (FPLC), culture supernatants of mannose-stimulated Acanthamoeba trophozoites were concentrated 10-fold using centrifuge concentrators with a molecular cutoff of 5 kDa (Ultrafree-15; Millipore, Bedford, MA). Samples were centrifuged at 3000g for 20 minutes and passed in 0.5-mL volumes over a column (Superdex 200; Amersham Pharmacia Biotech, Piscataway, NJ) using PBS (pH 7.2). Fractions were collected every 0.5 mL and examined by 4% to 15% SDS-PAGE. Fractions containing the mannose-induced cytolytic protein were pooled, concentrated 10-fold, and the buffer exchanged three times into 10 mM Tris buffer (pH 8.0), using the centrifuge concentrators. Two hundred microliters containing 1 mg of protein was applied to a diethylaminoethyl (DEAE) ionic-exchange column, using 10 mM Tris buffer (pH 8.0; buffer A). Adsorbed protein was eluted with a gradient of 10 mM Tris buffer (pH 8.0) with 1 M NaCl (buffer B). Fractions were examined for the ∼133-kDa protein by 4% to 15% SDS-PAGE. An initial 15% buffer B step removed contaminating proteins, and the ∼133-kDa protein was eluted between 15% to 30% of buffer B run at 0.1 mL/min. Fractions containing the ∼133-kDa protein were pooled and concentrated 10-fold and washed three times with PBS (pH 7.2) to exchange buffer. Protein test samples were adjusted to 1.5, 7.8, and 15.6 μg in 25 μL PBS (pH 7.2) and immediately used in CPE assays. 

Protein Inhibition and Degradation

Inhibition of the CPE was performed by incubating the purified protein test sample (15.6 μg in 25 μL PBS) in either 0.1 or 1.0 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), 1 mM 1,10-phenanthroline (Sigma-Aldrich), or 1 or 10 μM cystatin (Sigma-Aldrich) for 30 minutes before use in CPE assays, using HCORN cells as the target cells. Control experiments included 10-fold–concentrated PYG, containing 100 mM methyl-α-d-mannopyranoside or the untreated protein test sample. All assays were performed in triplicate. 

Protein degradation involved incubating the protein test samples with pepsin (5 mg/mL, Sigma-Aldrich) and proteinase K (5 mg/mL, Sigma-Aldrich) for 4 hours at 37°C, or incubating the protein test samples at 100°C for 30 minutes. Control treatments included PBS (pH 7.2) at 37°C for 4 hours and pepsin or proteinase K alone applied directly to target cells. All treated samples were immediately used in CPE assays with HCORN target cells. All assays were performed in triplicate. 

Zymography

Zymography overlays consisted of running the purified protein test sample (15.6 μg) on 4% to 15% SDS-polyacrylamide gels at 4°C and then soaking the gels in 1× zymogram renaturation (Bio-Rad) buffer at room temperature for 30 minutes. Protein test samples were either run untreated or pretreated with 1 mM PMSF or 10 μM cystatin for 30 minutes before loading. After 30 minutes, the gel was overlaid onto a 10% gelatin zymogram gel (Bio-Rad) and incubated for 2 hours at 35°C. After incubation, gels were stained with bio-safe Coomassie G250 stain (Bio-Rad). 

Dot zymography was performed by cutting holes in 10% gelatin zymogram gels with a 3-mm trephine. Test samples included 1.5 and 7.8 μg (in 10 μL) of the purified protein. Protease inhibition included pretreatment of 7.8 μg of the purified protein with 1 mM PMSF or 10 μM cystatin for 30 minutes before addition to the zymogel. Proteolytic activity was determined by comparison with a standard curve generated by using serial dilutions of collagenase (Sigma-Aldrich). Additional controls included undiluted PYG and PBS. All samples were in 10-μL volumes. Samples were applied in the 3-mm wells and incubated for 18 hours at 35°C. After incubation, gels were stained with bio-safe Coomassie G250 stain. 

Statistics

All statistical analyses were performed by using an unpaired Student’s t-test. 

Results

Effects of Mannose on Trophozoite Proliferation, Cyst Development, and Excystment

Before examining the effects that mannose may have on the pathogenic cascade of A. castellanii, it was important to determine how it might affect the growth of the organism. 

A. castellanii cultures grown in medium that contained mannose consistently produced approximately 50% fewer trophozoites than cultures that did not contain mannose (Figs. 1A 1B) . Trophozoites grown in 100 mM galactose or 100 mM lactose were not significantly different from amoebae grown in PYG alone (Fig. 1A) . Stationary phases were reached at the same time in both groups (∼125 hours). 

After reaching the stationary phase, cultures stimulated with mannose produced nearly three times more cysts (Fig. 1B) . This increase was sustained during the remaining times that were monitored. Moreover, the mannose cultures produced cysts approximately 24 hours earlier then cultures grown without mannose. The results indicate that mannose induced encystment. 

To assess whether the presence of mannose affects excystment, cultures containing 100% cysts were placed in PYG, PYG with 100 mM methyl-α-d-mannopyranoside, or PBS (Fig. 1C) . Acanthamoeba cysts in PYG displayed a consistent reduction in the number of cysts until approximately day 7, when they became too few to count, whereas the number of trophozoites increased proportionately. By contrast, cysts incubated in PYG with mannose exhibited a slower rate of excystment, and by day 10, approximately 50% of the cysts had excysted. Cyst populations in PBS remained constant throughout the times monitored. 

Molecular Weight Characterization of Mannose-Induced Cytolytic Factors

Recent studies have shown that soluble mannose is effective at blocking the binding of Acanthamoeba trophozoites to rabbit and HCORN cells, yet CPE was not reduced. ^{22 27} Supernatants from mannose-treated trophozoites produced 95% cytolysis of both HCE and HCORN cells, as measured by ⁵¹Cr release (data not shown). This was a 40% increase in cytolytic activity when compared with supernatants from trophozoite cultures not stimulated with mannose. Moreover, in agreement with a previous report, ²⁷ trophozoites treated with an unrelated sugar, lactose, did not demonstrate increased cytopathic activity against either human or Chinese HCORN cells (data not shown). It is noteworthy that Fhs-74 cells were not susceptible to any of the supernatants tested (data not shown). 

Size-fractionated supernatants displayed killing of the HCE and HCORN cells in both upper fractions from the 50- and 100-kDa membrane concentrators (Figs. 2A 2B) . Unfractionated 10× concentrated supernatants from mannose-stimulated trophozoites displayed approximately 90% CPE, whereas 10× concentrated PYG supplemented with mannose did not produce cell death. In contrast, Fhs-74 cells were not killed by either the upper or lower fractions tested (Fig. 2C) . 

Isolation of Mannose-Induced Cytolytic Factor

Initial examination involved SDS-PAGE analysis of concentrated supernatants. Mannose-stimulated supernatants contained two new bands at approximately 133 kDa and 70 kDa (Fig. 3) . Both the 133-kDa and 70-kDa bands appeared under nonreducing and reducing conditions. Supernatants from A. castellanii grown without mannose did not produce the two bands (Fig. 3) . 

Initial purification of the 133-kDa protein by size-exclusion FPLC (Superdex 200; Amersham Pharmacia Biotech) generated high yields of the protein as seen by SDS-PAGE (Fig. 4A) . Further separation by DEAE ionic exchange produced a single band between a 15% and 30% elution gradient (Fig. 4B) . Fractions collected between 0% and 14% and 31% and 100% did not contain the 133-kDa protein. 

In Vitro CPE Analysis of the Purified 133-kDa Protein

To test the cytopathic activity of the purified protein, samples were adjusted to 1.5, 7.8, and 15.6 μg of protein in 25 μL of PBS (pH 7.2). 

The HCE cells were killed by the 133-kDa protein in a dose-dependent manner (Fig. 5A) . The lowest concentration tested (1.5 μg) killed approximately 20% of the corneal cells, whereas the maximum dose (15.6 μg) killed approximately 90% of the target cells. Microscopic examination revealed piecemeal death of individual corneal cells, as opposed to entire layers of cells lifting up from the bottom of the wells (Fig. 6A) . In contrast to the HCE cells, HCORN cells were effectively killed by all three concentrations of the protein tested (Figs. 5B 6B) . As before, the human intestinal epithelial cells (Fhs-74) were not susceptible to any of the protein samples tested (Figs. 5C , Fig. 6C ). Fractions collected by DEAE ionic exchange eluted between 0% and 14% and 31% and 100% did not induce significant CPE (data not shown). In all three treatment groups, the PBS control did not significantly kill any of the cell lines tested. 

Protein Inhibition and Degradation

Previous studies in our laboratory have shown that the CPE produced by crude mannose-stimulated supernatants of A. castellanii could be inhibited by the serine protease inhibitor PMSF. ²⁷ Figure 7A shows that the 0.1-mM PMSF treatment inhibited the cytolytic function of the 133-kDa protein by 20%, whereas the higher dose (1 mM) inhibited CPE by approximately 60%. Treatments with 1 mM 1,10-phenanthroline inhibited CPE 70%, and in combination with 1 mM PMSF, completely inhibited CPE. PMSF and 1,10-phenanthroline alone did not affect cell viability (data not shown). In contrast, cystatin did not inhibit the cytolytic activity of the sample protein (Fig. 7B) . 

To determine whether proteases could reduce the CPE of the 133-kDa protein, the protein test samples were digested with pepsin and proteinase K. Figure 7C shows that both pepsin and proteinase K significantly (P < 0.001) reduced the CPE of HCORN cells by 80% and 70%, respectively. Proteinase K and pepsin alone did not affect cell viability (data not shown). The cytolytic activity of the samples incubated at 100°C for 30 minutes was reduced by approximately 90%. PBS treatment did not significantly reduce the cytolytic activity of the protein compared with the untreated control. 

Zymography

To determine whether the purified protein had protease activity, the protein test samples were incubated with gelatin-impregnated zymogels. 

The zymography overlay using 15.6 μg of the purified protein displayed clear lytic ability against the gelatin in the zymogels (Fig. 8A) . Within 2 hours, lytic zones were readily visualized. Dot zymography of the 1.5- and 7.8-μg purified protein samples displayed 7- and 10-mm lysis diameters, respectively (Fig. 8B) . Proteolytic activity was comparable to 0.1 mg collagenase (approximately 41 units) under the conditions tested. All samples pretreated with PMSF displayed 100% inhibition of lysis. Samples pretreated with cystatin produced the same lysis as the untreated protein samples on the overlay and the dot zymography (10 mm). Neither the PYG nor the PBS control was found to be lytic against gelatin. 

Discussion

The purpose of this study was to examine the effect of mannose on the proliferation, encystment, and secretion of cytopathic factors by A. castellanii. Moreover, the mannose-induced 133-kDa protein was purified and examined for cytopathic ability against corneal epithelial cells. 

A prerequisite to Acanthamoeba keratitis is the amoeba’s ability to bind to the corneal surface. This binding is affected by the upregulation of mannosylated glycoproteins that occurs after corneal trauma. ^{23 24} After binding, trophozoites penetrate the corneal epithelial layers and gain entry into the stroma. ^{24 27 28 29} Before this study, it was not known whether the binding to the mannosylated glycoproteins affects trophozoite proliferation. Induction of trophozoite proliferation could exacerbate the disease. However, the results of the proliferation assays show that mannose reduced, rather than stimulated, trophozoite proliferation within 24 hours. Moreover, trophozoite proliferation remained depressed throughout all times monitored. After 6 days, cyst production peaked, displaying three times more cysts in cultures that contained mannose. Because trophozoite proliferation was reduced by approximately 50% and cyst production was increased in the mannose-stimulated cultures, the destruction of the corneal cells in vitro was most likely due to a greater cytopathic potential on a per trophozoite basis rather than an increase in their numbers. In addition, the presence of mannose delayed the conversion of cysts to trophozoites by twofold. 

Entamoeba histolytica trophozoites bind to intestinal epithelial cells through a galactose-binding lectin, and on binding, elaborate a host of cytolytic factors, such as the pore-forming amoebapores. ^{30 31 32 33} The secretion of these cytolytic factors is key to E. histolytica–induced disease. However, soluble galactose inhibits the secretion of cytolytic factors responsible for Entamoeba-mediated cytolysis of the intestinal epithelial cells. ³⁴ By contrast, our studies indicated that blocking the binding of A. castellanii to corneal cells with mannose did not impair the killing in vitro. ²⁷ Because Acanthamoebae adhere to corneal epithelial cells through mannosylated glycoproteins, we hypothesized that binding induces the secretion of cytopathic factors. Examination of supernatants from mannose-stimulated cultures by SDS-PAGE revealed the appearance of two new protein bands at approximately 70 and 133 kDa. Initial molecular weight separation showed that CPE to both human and HCORN cells in vitro was almost entirely in the more than 100-kDa fraction. We therefore used FPLC size exclusion and DEAE ionic exchange to isolate the 133-kDa protein. The purified protein was found to be highly cytopathic to the human and HCORN cells, yet had no effect on small intestine epithelial cells. This absence of cytopathic activity may indicate a specificity for corneal cells that goes beyond specificity in binding ligands, as the purified secreted protein did not kill the small intestinal epithelial cells. 

Initial studies with crude supernatants from mannose-stimulated cultures revealed that PMSF inhibits the cytolytic activity. ²⁷ Pretreatment of the purified protein with PMSF and 1,10-phenanthroline (serine protease inhibitors) blocked killing of HCORN cells by 60% and 70%, respectively. However, pretreatment with the cysteine protease inhibitor cystatin did not inhibit cytopathic activity. The results suggest that the MIP-133 protein is a serine protease and not a cysteine protease. In addition, treatment of the purified protein with the proteases pepsin and proteinase K nearly abolished the cytopathic ability. Incubation of the protein at 37°C did not reduce killing, indicating that it is heat-stable at that temperature, yet treatment at 100°C for 30 minutes abolished the CPE. In addition, the purified protein was found to be proteolytic to gelatin, as lysis was observed by both dot and overlay zymography. As we expected from the CPE data, pretreatment with PMSF inhibited 100% of the lytic ability, whereas pretreatment with cystatin did not inhibit lysis. 

Binding is an essential step in the pathogenic cascade that leads to Acanthamoeba keratitis, and the present results imply that this binding is not responsible for generating an increase in proliferation, yet it is responsible for initiating the secretion of cytopathic factors. The secretion of a 133-kDa protein was significantly upregulated by mannose, and the induced factor was highly cytolytic to both human and corneal epithelial cells, but not small intestinal epithelial cells. The collagenolytic activity of the mannose-induced protease may also contribute to the pathogenesis of Acanthamoeba keratitis by facilitating trophozoite penetration of the Bowman’s membrane. 

We are currently attempting to sequence the protein. Mass spectrometer analysis and preliminary sequence data from digested peptides confirm that the mannose-induced protein has not yet been reported to the databases. With this information in hand, it may be possible to neutralize the mannose-induced protein, either pharmacologically or immunologically, as an effective strategy for the management of Acanthamoeba keratitis. 

 Supported by National Eye Institute Grant EY09756, National Institute of Allergy and Infectious Diseases Grant AI07520, and an unrestricted grant from Research to Prevent Blindness.

 Submitted for publication January 9, 2003; revised March 5, 2003; accepted March 11, 2003.

 Disclosure: M. Hurt (P); J. Niederkorn (P); H. Alizadeh (P)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Effects of mannose on A. castellanii proliferation, encystment, and excystment. (A) Trophozoites were grown in PYG, PYG with 100 mM methyl-α-d-mannopyranoside (Man), lactose (Lac), or galactose (Gal). Samples were examined spectrophotometrically at OD₄₈₀. (B) Trophozoites were grown in PYG or PYG with 100 mM methyl-α-d-mannopyranoside. Samples were examined for trophozoites (Trophs) and cysts by direct counts. (C) Cysts were incubated in PYG, PYG with 100 mM methyl-α-d-mannopyranoside, and PBS. Samples were examined by direct counts. The results are representative of three separate experiments.

Figure 2.

 

Molecular weight estimation of cytopathic factors in supernatants derived from A. castellanii trophozoites stimulated with 100 mM methyl-α-d-mannopyranoside. Supernatants were collected at mid log phase and size fractionated, with centrifuge concentrators having a molecular mass cutoff of either 50 or 100 kDa. Fractions were added to HCE cells (A), HCORN cells (B), or human small intestine epithelial (Fhs-74) cells (C) for 18 hours, and CPE was assessed spectrophotometrically. Data are the mean ± SE of triplicate counts.

Figure 3.

 

Protein analysis of Acanthamoeba supernatants. Lane L: MW ladder; lane A: PYG concentrated 10-fold; lane B: supernatants from trophozoites grown in PYG alone and concentrated 10-fold; lane C: supernatants from trophozoites grown in PYG supplemented with 100 mM methyl-α-d-mannopyranoside and concentrated 10-fold. Supernatants were collected at mid log phase.

Figure 4.

 

Purification of the 133-kDa protein. Supernatants from trophozoite cultures grown in 100 mM methyl-α-d-mannopyranoside and size excluded by FPLC, pooled, and separated by ionic DEAE. (A) MW ladder (lane L), 10-fold concentrated supernatant (lane 1), unconcentrated supernatant (lane 2), single fraction of 133-kDa protein from a fractionation column (lane 3), and pooled fractions of 133-kDa protein from the column (lane 4). (B) MW ladder (lane L), individual fractions eluted from DEAE resin representing the 15% clearance step (lanes 1, 2), and fractions eluted by 15% to 30% gradient at 0.1 mL/min (lanes 3– 8).

Figure 5.

 

CPE assays, using the purified 133-kDa mannose-induced protein. Test samples were adjusted to 1.5, 7.8, and 15.6 μg of protein in 25 μL of PBS before addition to HCE (A), HCORN (B), or Fhs-74 (C) cells in 96-well microtiter plates for 18 hours. CPE was assessed spectrophotometrically. Data are the mean ± SE of triplicate counts. **P < 0.01 and ***P < 0.001, significantly different from the untreated control.

Figure 6.

 

Light microscopy photographs of target cells incubated with the purified 133-kDa mannose-induced protein. Target cells examined were HCE (A), HCORN (B), and Fhs-74 (C) cells. Cells were then treated with PBS (Con) or 1.5 (row 1), 7.8 (row 2), or 15.6 (row 3) μg of the purified mannose-induced protein for 18 hours. Magnification, ×10.

Figure 7.

 

Effects of protease inhibitors and protease degradation on CPE. The purified 133-kDa protein was pretreated with 0.1 or 1 mM PMSF, 1 mM 1,10-phenanthroline (1,10-P), or 1 mM PMSF and 1 mM 1,10-phenanthroline combined (A) and 1 or 10 μM cystatin (B) for 30 minutes before addition to HCORN target cells. Protein degradation was assessed by incubating the protein test samples with either 10 μg pepsin or 10 μg proteinase K before addition to HCORN target cells for 18 hours (C). CPE was assessed spectrophotometrically. Data are the mean ± SE of triplicate counts. *P < 0.05 and ***P < 0.001, significantly different from treatments with protein test samples.

Figure 8.

 

Proteolytic activity of the purified mannose-induced protein. (A) Purified protein (15.6 μg) was electrophoresed in 4% to 15% SDS-polyacrylamide gels and then overlaid onto 10% zymogels for 2 hours. Lane 1: untreated protein sample: lane 2: pretreatment with 10 μM cystatin: lane 3: pretreatment with 1 mM PMSF. (B) Zymogels (10%) with 3-mm cutouts were incubated with 1.5 and 7.8 μg of the purified protein. Some samples were pretreated with 1 mM PMSF or 1 μM cystatin for 30 minutes before addition. Additional controls included PYG and PBS alone.

The authors thank Robert Ritter for assistance with the FPLC and scientific discussions and Vincent Proy for technical assistance.