In Vitro Pathogenicity of Acanthamoeba Is Associated with the Expression of the Mannose-Binding Protein

Abstract

purpose. To determine whether the expression of Acanthamoeba mannose-binding protein (MBP) is associated with the pathogenicity of the parasite in vitro.

methods. Both active trophozoites and dormant cysts of a pathogenic strain of A. castellanii were analyzed for their ability to bind to corneal epithelium, express MBP, and produce a cytopathic effect (CPE) on host cells. In addition, host cell binding, CPE-inducing ability, and MBP expression pattern of trophozoites of four different isolates of Acanthamoeba with various degrees of in vitro pathogenicity were analyzed. Binding assays were performed with radiolabeled parasites; CPE assays were performed with rabbit corneal epithelial cells as host cells; and the expression of MBP was detected by affinity chromatography of parasite extracts on mannose affinity columns and by immunohistochemical and Western blot analyses.

results. Trophozoites of A. castellanii bound avidly to corneal epithelial cells in a mannose-inhibitable manner, whereas cysts exhibited little binding. The lack of binding of the cysts to host cells was associated with the downregulation of MBP, along with the concomitant loss of CPE. Analysis of trophozoites of five different species of Acanthamoeba exhibiting various degrees of pathogenic potential revealed that the ability of parasites to bind to host cells and produce CPE is directly correlated with the expression of the MBP. Acanthamoeba strains that bound avidly to host cells and produced potent CPE, robustly expressed MBP. In contrast, parasite strains that produced only weak CPE, expressed markedly reduced levels of MBP.

conclusions. The data demonstrating that the pathogenic potential of Acanthamoeba directly correlates with the expression level of the MBP in conjunction with our published studies showing that Acanthamoeba MBP is a major virulence protein suggest that the amoeba lectin has the potential to serve as a marker of pathogenicity.

Acanthamoeba keratitis is a painful, vision-threatening infection caused by pathogenic strains of the protozoan, Acanthamoeba. ^{1 2 3} In healthy individuals, the only known tissue susceptible to infection by Acanthamoeba is the cornea. In immunocompromised individuals, the parasite causes chronic granulomatous amoebic encephalitis (GAE), dermatitis, and/or pneumonitis. ^{4 5} The free-living and opportunistic acanthamoebae are widely found throughout natural and man-made environments. They have been recovered from soil, air, chlorinated swimming pools, hot tubs, tap water, bottled mineral water, and contact lens solutions. ^{6 7 8}  

The lifecycle of Acanthamoeba comprises two distinct stages: trophozoite and cyst. The trophozoite is flat and irregular in shape and measures from 20 to 40 μm. Under favorable environmental conditions, the trophozoite undergoes mitosis and is capable of phagocytosing bacteria and yeast. A cyst is formed from a trophozoite when adverse conditions such as exposure to biocidal agents, starvation, desiccation, and hyperosmolarity prevail. ^{9 10} The presence of a double wall and an average diameter of 10 to 15 μm make cysts morphologically distinguishable from trophozoites. The cyst wall of Acanthamoeba contains largely protein (33%), cellulose (35%), and lipid (4%–6%) and is composed of two layers, an outer exocyst, and inner endocyst. ^{9 11} The endocyst is mainly constituted of cellulose and lipoproteins, whereas most of the cyst wall proteins are localized in the exocyst. ^{9 11} Cysts can excyst and differentiate into trophozoites when the conditions are favorable. ¹²  

Studies designed to characterize the molecular mechanism by which the parasite invades the corneal tissue have suggested that a carbohydrate-based recognition system plays a key role in the adhesion of the parasites to the host cells ^{13 14 15 16} and in the amoeba-induced cytopathic effect (CPE) that occurs subsequent to the adhesion. ¹⁵ More specifically, recent studies have shown that: (1) acanthamoebae express a ∼400-kDa transmembrane mannose-binding protein (MBP) that is constituted by multiple subunits of 130 kDa ^{16 17} and (2) free mannose ¹⁵ and antibodies to Acanthamoeba MBP inhibit the adhesion of the parasite to host cells as well as the amoeba-induced CPE. ¹⁸ These studies suggest that Acanthamoeba MBP is central to the pathogenic property of the parasite and may serve as a marker of pathogenicity. 

Acanthamoeba cysts are the dormant stage of the parasite, but it is unknown whether the expression pattern of major virulence proteins is altered during encystment. Considering that the outer walls of the amoebae cysts contain a significant number of proteins, ^{9 11} it is of importance to determine whether the MBP is expressed in the Acanthamoeba cysts. In the present study, we compared trophozoites and cysts of a pathogenic strain of Acanthamoeba for their ability to bind to corneal epithelium, produce CPE, and express MBP. This study revealed that encystment of Acanthamoeba is associated with the downregulation of MBP along with the concomitant loss of the ability of the encysted parasite to bind to host cells and produce in vitro CPE. Moreover, using four additional isolates of Acanthamoeba exhibiting various degrees of CPE-inducing capacity, we demonstrate that the pathogenetic potential of trophozoites of various Acanthamoeba isolates directly correlates with the expression level of the amoeba MBP. 

Materials and Methods

Saccharides, Neoglycoproteins, and Affinity Chromatography Matrices

Methyl-α-d-mannopyrannoside (α-Man), was purchased from Pfanstiehl Laboratories, Inc. (Waukegan, IL). The neoglycoprotein, mannosylated-bovine serum albumin (man-BSA, 15-25 moles of α-D-mannopyrannoside/mole of BSA) and the affinity matrix aminophenyl-α-mannose gel (α-Man gel) were purchased from EY Laboratories, Inc. (San Mateo, CA). 

Preparation of Acanthamoeba Trophozoites and Cysts

For all studies involving the comparison of trophozoites and cysts, an Acanthamoeba strain, MEEI 0184 (an in-house strain derived from an infected human cornea; A. castellanii based on morphologic characteristics) was used. The parasites were axenically cultured in a proteose peptone-yeast, extract-glucose (PYG) medium at 30°C without agitation. ¹⁹ More than 95% of the parasites were recovered as trophozoites when they were subcultured twice a week in these conditions. Encystment was induced by the procedure of Hirukawa et al. ²⁰ Approximately 1 × 10⁶ trophozoites were washed once in encystment medium (95 mM NaCl, 5 mM KCl, 8 mM MgSO₄, 0.4 mM CaCl₂, 1 mM NaHCO₃, and 20 mM Tris-HCl [pH 9.0]) and were incubated in 10 mL of the encystment medium in 150-mm diameter glass dishes at 30°C. The encystment process was monitored daily by observation under a phase-contrast microscope and by staining (Calcofluor White MR2; Sigma-Aldrich Inc., St. Louis, MO) as described by Connell et al. ²¹ In these conditions, more than 95% of the cells encysted during the 120-hour incubation. 

In the present study, in addition to the Acanthamoeba strain MEEI 0184, four Acanthamoeba isolates of various degrees of pathogenic potential (Table 1)were used. These isolates were obtained either from the Culture Collection of Algae and Protozoa (CCAP, Argyll, UK) or from the Public Health Laboratory Service (PHLS) in the United Kingdom. 

Isolation of Acanthamoeba MBP from Trophozoites and Cysts

To isolate Acanthamoeba MBP, frozen cell pellets (2.5 × 10⁷ cysts or trophozoites) were washed in resuspension buffer (25 mM Tris-HCl [pH 7.2], 100 mM NaCl, 20 mM CaCl₂, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) by centrifugation and were then disrupted in lysis buffer (0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS] with 2 mM β-mercaptoethanol in resuspension buffer) by a sonifier (model 250; Branson Ultrasonics Corp., Danbury, CT), using a tapered microtip at an output setting of 20 W. The amoeba extracts were clarified by centrifugation (100,000g, 1 hour, 4°C) and were chromatographed on an α-Man-affinity column at 4°C. The unbound components were removed by washing the column with the lysis buffer, and the bound components were eluted in 0.5 mL fractions with the lysis buffer containing 150 mM α-Man. 

Western Blot Analysis

Polyclonal anti-MBP IgY antibody was prepared in chicken as described in our previous work using affinity-purified MBP. ¹⁷ Briefly, anti-MBP IgY was purified from the egg yolk using a proprietary method (Aves Laboratories Inc., Tigard, OR). The purified preparation was >95% IgY, as determined by Coomassie staining of SDS-PAGE gels. ²² For comparison purposes, preimmune IgY was isolated from the egg yolk of the same hen collected before immunization. For Western blot analysis, affinity-purified MBP was electrophoresed in 8% reducing SDS-polyacrylamide gels. Proteins from the gels were transferred onto nitrocellulose filters ²³ and stained with ponceau S to visualize protein bands. ²⁴ The protein blots were treated overnight at room temperature with 5% dry milk in PBS to block nonspecific binding sites and were then sequentially incubated with anti-MBP IgY (20 μg/mL, 37°C, 1 hour), horseradish peroxidase-linked goat anti-chicken IgY (0.2 μg/mL, 37°C, 1 hour; Aves Laboratories Inc.), and a freshly prepared solution of diaminobenzidine-H₂O₂ reagent (0.05% diaminobenzidine-0.01% H₂O₂-0.04% nickel chloride in Tris-HCl buffer [pH 7.2]). The membranes were washed extensively with PBS containing 0.1% Tween-20 after each incubation step. Control reactions were performed as described above, except that the blots were incubated with preimmune IgY instead of the anti-MBP IgY. 

Immunostaining of Acanthamoeba Cysts and Trophozoites by Anti-MBP

Trophozoites and cysts were immunostained in cell suspension using anti-MBP IgY (1 × 10⁶ cells in 1 mL of PBS + 80 μg of anti-MBP IgY or preimmune IgY/mL 0.5% BSA in PBS, 30 minutes) and FITC-labeled goat anti-chicken IgY (10 μg/mL 0.5% BSA in PBS, 30 minutes; Aves Laboratories, Inc). After immunostaining, the cells were smeared onto glass slides, fixed with 2.5% paraformaldehyde, and visualized by microscope (Eclipse TE200; Nikon, Tokyo, Japan). 

Analysis of the Effect of Anti-MBP on the Adhesion of Acanthamoeba Trophozoites and Cysts to Man-BSA and to Corneal Epithelial Cells

Acanthamoeba parasites (>95% trophozoites) were radiolabeled with ³⁵S-methionine (2 cpm/trophozoite) and were used for adhesion assays either directly or after encystment (>95% cysts, 1.5 cpm/cyst as determined after 120 hours of encystment). Radiolabeled trophozoites and cysts (1 × 10⁶ cells/mL in PBS) were incubated with anti-MBP IgY or preimmune IgY (200 μg/mL 0.5% BSA in PBS, 30 minutes, 37°C), and binding assays were performed as described in published protocols. ^{15 16} Briefly, to assess binding to man-BSA, wells of microtiter plates were coated with man-BSA, nonspecific binding sites were blocked with 5% BSA in PBS (1 hour, room temperature), and a 50-μL aliquot of ³⁵S-labeled Acanthamoeba trophozoites or cysts (5 × 10⁵ cells/mL in PBS) pretreated with either anti-MBP IgY or preimmune IgY was added to each well (four wells/group), and the plates were incubated for 1 hour at room temperature. At the end of the incubation period, the plates were rinsed with PBS to remove unbound acanthamoebae, 0.1 mL of 1% SDS was added to each well, and the radioactivity in the solubilized material was determined in a scintillation counter. 

For assessing Acanthamoeba binding to epithelial cells, we prepared immortalized cultures of rabbit corneal epithelial cells, as described elsewhere. ¹⁵ The binding assays were performed as described except that instead of microtiter wells coated with man-BSA, confluent cultures of rabbit corneal epithelial cells in 24-well plates were used, and the blocking step was omitted. 

Cytopathic Effect Assay

CPE assays were performed as described in our published study, ¹⁵ with minor modifications. Briefly, the parasites (>95% trophozoites or >95% cysts) were rinsed three times in a serum-free medium supplemented with 0.4% BSA and aliquots of the parasite suspension (1 × 10⁶ parasites/mL; 50 μL/well of 48-well plates) were added to duplicate wells of confluent cultures of rabbit corneal epithelium that had been rinsed and preincubated in the serum-free medium for 2 hours. The plates were then incubated at 37°C for 1, 3, and 6 hours in a CO₂ incubator, examined under a phase-contrast microscope for the presence of cell-free plaques in the monolayer, and were stained with Giemsa (Diff-Quik; Dade Diagnostic Inc., Aguada, PR). Approximate cell density in each well was estimated (Plot Density Tool of Quantity One software (Bio-Rad, Hercules, CA), and results were expressed as %CPE (total loss of cells, 100% CPE). To test the effect of anti-MBP, parasites pretreated with anti-MBP IgY or preimmune IgY (0.2 mg/mL, 37°C, 30 minutes) were used. Statistical analysis was performed by one-way analysis of variance (ANOVA) of the mean ± SD results of triplicate experiments. 

Results

Binding of Acanthamoeba Trophozoites and Cysts to Man-BSA and Epithelial Cells

Acanthamoeba trophozoites have been shown to bind and produce a potent CPE on host cells. ^{15 25 26 27} To determine whether Acanthamoeba cysts lack the capacity to bind and cause destruction of the host cells, comparative binding and CPE assays were performed with Acanthamoeba cysts and trophozoites. In solid-phase assays, the cysts derived from the pathogenic ocular isolate of A. castellanii showed little binding to man-BSA (Fig. 1A , left panel) or to epithelial cells (Fig. 1A , right panel) when compared with trophozoites cultured from the same isolate. Compared to trophozoites, binding of cysts to man-BSA and to epithelial cells was 8.9% and 2.3%, respectively. As previously reported, ¹⁵ trophozoites did not bind to control neoglycoproteins (galactose-BSA and fucose-BSA) and their binding to man-BSA and corneal epithelial cells was inhibited by α-Man but not by other control saccharides, including α-galactose and mannitol (data not shown). Also, unlike trophozoites which produced a potent CPE on epithelial cells, Acanthamoeba cysts produced little, if any, detectable CPE on host cells (Fig. 1B) . 

Reduced MBP Expression in Acanthamoeba Cysts Compared with Trophozoites

In previous studies, we have shown that Acanthamoeba trophozoites express an MBP (130-kDa subunit molecular mass) that mediates the adhesion of parasites to host cells and subsequent CPE. ^{15 16 17} Having established that the Acanthamoeba cysts lack the capacity to bind to corneal epithelial cells, it was of interest to determine whether the expression of Acanthamoeba MBP is attenuated during encystment. Toward this end, we stained cell membranes of both cysts and trophozoites with anti-MBP IgY. Polyclonal anti-MBP IgY antibody was produced in chicken, as described in our previous work with affinity-purified MBP. ¹⁷ This antibody is highly specific for the 130-kDa component present in the reducing SDS-PAGE gels of the affinity-purified MBP fraction and it does not react with numerous components present in the total extract of the parasites. ¹⁷ Cell membranes of Acanthamoeba trophozoites stained intensely with anti-MBP IgY (Fig. 2A , middle). In contrast, cell membranes of Acanthamoeba cysts either did not stain or stained weakly with anti-MBP IgY (Fig. 2B , middle). Preimmune IgY did not react with either trophozoites or cysts (Fig. 2A 2B , right). To further confirm that the expression of MBP is reduced in cysts, membrane extracts were prepared from both cysts and trophozoites (2.5 × 10⁷ cells each), and MBP was isolated by affinity chromatography on α-Man gel, as described in our previous studies. ¹⁷ Proteins bound to the affinity column were eluted by α-Man, electrophoresed on SDS-polyacrylamide gels, and visualized by silver staining. When membrane extracts of trophozoites were chromatographed on α-Man gel, a 130-kDa component was detected in the bound fraction eluted with α-Man (Fig. 2C , MBP, right lane). In contrast, when membrane extracts of cysts were chromatographed on the α-Man gel, the 130-kDa component was not detected in the fraction eluted with α-Man (Fig. 2C , MBP, left lane). Thus, the expression of MBP is associated specifically with the active stage of trophozoite in A. castellanii. 

Correlation of Pathogenic Potential of Various Isolates of Acanthamoeba with the Expression of Acanthamoeba MBP

To elucidate further our hypothesis that Acanthamoeba MBP is a major virulence protein, we analyzed trophozoites of four additional isolates of Acanthamoeba (Table 1)with varying degrees of pathogenic potential for their MBP expression levels. First, these isolates were tested for their ability to bind to man-BSA and produce a CPE on host epithelial cells. Of the four isolates tested (Table 1) , A. palestinensis showed markedly low binding to man-BSA and exhibited low capacity to produce CPE (9.8% binding and 15% CPE compared to A. castellanii trophozoites, Fig. 3 , Media); A. royreba exhibited a moderate binding to man-BSA and moderate capacity to produce CPE (44.3% binding and 32% CPE compared to A. castellanii, Fig. 3 , Media) and two amoeba isolates, Acanthamoeba ssp. (strains Esbc4 and Shi) exhibited a high level of binding to man-BSA and a high capacity to produce CPE (A. spp. [strain Esbc4]: 76.3% binding and 92% CPE; A. spp. [strain Shi]: 90.7% binding and 94% CPE compared with A. castellanii; Fig. 3 , Media). 

To determine whether MBP mediates binding of these different strains of Acanthamoeba to man-BSA, Acanthamoeba trophozoites pretreated with anti-MBP IgY or preimmune IgY were allowed to bind to man-BSA-coated wells. Compared with control wells incubated with media alone, inhibition of binding to man-BSA by anti-MBP was 82% for A. castellanii, 65% for A. royreba and Acanthamoeba spp. (strain Shi), and 57% for Acanthamoeba spp. (strain Esbc4; Fig. 3A , Anti MBP IgY). In contrast, preimmune IgY showed only weak inhibition of the amoebae binding to man-BSA (Fig. 3A , Preimmune IgY). Likewise, anti-MBP IgY (Fig. 3B , Anti-MBP IgY) but not preimmune IgY (Fig. 3B , Preimmune IgY) significantly inhibited the CPE induced by various Acanthamoeba strains. Also, the addition of free α-Man inhibited the CPE induced by each strain of amoeba (Fig. 3B , Man) further confirming the role of MBP-mediated host parasite interactions in the induction of amoeba-induced CPE. 

To elucidate further whether the pathogenicity of Acanthamoeba is associated with the expression of MBP, the expression levels of MBP were compared in various strains. For this, membrane extracts of different strains of Acanthamoeba derived from 2.5 × 10⁷ parasites each were chromatographed on α-Man gel, and the material bound to the column was eluted by free α-Man and was analyzed by gel electrophoresis and silver staining. In the bound fraction eluted with α-Man, a 130-kDa component was seen as an intense band in preparations of strains exhibiting high in vitro pathogenicity (A. spp. [strain Esbc4], A. spp. [strain Shi], and A. castellanii, Fig. 4A, right); whereas only a weak band was seen in preparations from isolates exhibiting weak–moderate CPE producing capacity (A. royreba or A. palestinensis; Fig. 4A , panel]. A Western blot analysis was performed, to ensure that the 130-kDa component (Fig. 4A)is indeed authentic amoeba MBP. For this, electrophoresis blots of the affinity-purified 130-kDa component derived from 10⁹ parasites of each strain were first stained with ponceau S to visualize protein bands and were probed with anti-MBP. In the ponceau S-stained blots, the 130-kDa component was detected in preparations of strains exhibiting high in vitro pathogenicity (A. ssp. [strain Esbc4], A. ssp. [strain Shi], and A. castellanii, Fig. 4B , left panel). Again, the 130-kDa component reacting strongly with anti-MBP (Fig. 4B , right) but not with preimmune IgY (not shown) was present in preparations of strains exhibiting high in vitro pathogenicity (A. ssp. [strain Esbc4], A. ssp. [strain Shi], and A. castellanii, Fig. 4B , right). In ponceau S as well as Western blot analysis, the 130-kDa component was either not detected or was detected as only a weak, equivocal band in preparations of strains exhibiting low to moderate in vitro pathogenicity (A. royreba or A. palestinensis). This finding is most likely due to low sensitivity of ponceau S and Western blot staining compared with silver staining which indicated the presence of a weak 130-kDa band in MBP preparations of both A. royreba or A. palestinensis. The yield of MBP, as estimated by protein determination of the purified preparation of MBP isolated by affinity chromatography on α-Man gel and corroborated by the density of the bands in the stained gel was four- and fivefold higher in the strains exhibiting high CPE (Acanthamoeba spp. [strain Esbc4], A. spp. [strain Shi], and Acanthamoeba castellanii) compared with A. royreba or A. palestinensis, respectively. Comparative analysis of the CPE effect of different strains revealed that the CPE-inducing capacity of highly pathogenic strains (Acanthamoeba spp. [strain Esbc4], Acanthamoeba spp. [strain Shi], and A. castellanii) was nearly three and six times higher than that of A. royreba and A. palestinensis, respectively (Figs. 3A 3B) . 

Discussion

In the present study, we demonstrate that the pathogenic potential of Acanthamoeba correlates directly with the expression level of the parasite MBP. The first step in the pathogenesis of infection is the adhesion of parasites to the host cells. We studied both the active pathogenic trophozoites and the dormant cysts of A. castellanii for their ability to bind to man-BSA and to epithelial cells. Whereas trophozoites bound avidly to man-BSA and to epithelial cells in a mannose-inhibitable manner, cysts exhibited little binding. As previously described, induction of CPE is a multistep process that involves binding to host cells, secretion of proteases and target cell apoptosis. ^{15 16 25 26 27 28 29} Binding is the first key step in the target cell damage; accordingly, we demonstrated that the lack of binding of the cysts to host cells was associated with their inability to induce target cell damage. It is known that the encystment process alters the shape, metabolism and the protein pattern of the amoeba; nevertheless, a significant number of proteins are present on the exocyst, the outer cyst wall. ^{9 11} Therefore, it was of interest to determine whether the lack of binding of Acanthamoeba cysts to host cells is associated with reduced expression of MBP. Our results showed that Acanthamoeba cysts express markedly reduced levels of MBP compared with the trophozoites. 

Acanthamoeba cysts are frequently seen in the corneal stroma of patients with established Acanthamoeba keratitis, largely because of the unfavorable conditions that prevail for parasites in response to treatment with antimicrobial drugs. ^{2 30} Because cysts are highly resistant to treatment with biocidal agents and are able to survive harsh conditions, they persist into the stroma through the course of treatment and excyst when the conditions become favorable with the concomitant return of robust infection. Although the mechanisms leading to encystment of Acanthamoeba remain largely unknown, it is worth noting that the galactose-specific lectin of Entamoeba, a different parasite that produces infection of the gut, plays a role in encystment of the parasite in addition to its well-known role in mediating the adhesion of the parasite to host cells and the pathogenicity of the parasite. ^{31 32 33} Thus, it is tempting to speculate that in addition to its well-defined role in pathogenicity, Acanthamoeba MBP may also play a role in encystment of the parasite. Clearly, an understanding of the stimuli that triggers encystment of Acanthamoeba will aid in the eventual design of strategies to interrupt the parasite’s lifecycle and thereby prevent recurrent infection. 

The genus Acanthamoeba consists of both pathogenic and nonpathogenic isolates. In the present study, analysis of five different species of Acanthamoeba with various degrees of pathogenic potential revealed that the ability of parasites to bind to host cells and produce a CPE is directly correlated with expression levels of MBP. Acanthamoeba strains that bound avidly to host cells and produced potent CPE (Acanthamoeba ssp. [strain Esbc4], Acanthamoeba ssp. [strain Shi], and A. castellanii), robustly expressed MBP. In contrast, parasite strains that produced only weak to moderate CPE (A. royreba and A. palestinensis), expressed markedly reduced levels of MBP. These data are consistent with our earlier studies, performed with a single clinical isolate of A. castellanii, that suggested that the amoeba MBP is a major virulence protein. ^{15 16} It is noteworthy, however, that of the two isolates exhibiting reduced capacity to produce CPE in vitro, A. royreba induced twice the CPE on host cells in vitro compared with A. palestinensis, but the expression level of the MBP was similar in both strains (Fig. 4A , right). A. palestinensis MBP was clearly not defective in its carbohydrate-binding activity, because it was isolated by affinity chromatography on α-Man affinity columns. These data lead us to speculate that the nonpathogenic strains of Acanthamoeba may not be deficient in the MBP gene and that the pathogenicity of the parasite is regulated by factors that either control the MBP expression and/or provide stability to the expressed protein. It remains to be determined whether A. palestinensis expresses an allele of MBP gene that is more readily degraded by the host cell factors compared with the MBP of highly pathogenic strains. Also, at present, nothing is known about the factors regulating the MBP gene expression. 

Although the factors regulating pathogenesis of Acanthamoeba parasites in vitro may not parallel those in vivo, it is most likely that the MBP plays a major role in the pathogenesis of Acanthamoeba keratitis in vivo. In this respect, in a recent study, we found that oral immunization with rMBP protects against Acanthamoeba keratitis in a hamster animal model and that this protection correlates with the appearance of MBP-specific IgA in tears of immunized animals (Garate M et al., manuscript in preparation). The MBP-specific IgA in tears is likely to provide protection by preventing the parasite binding to the corneal epithelium. In addition, we have also shown that tears of normal, healthy individuals contain the MBP-specific IgA (Garate M et al., manuscript in preparation). Moreover, the three Acanthamoeba strains that bound avidly to host cells and produced potent CPE (Acanthamoeba ssp. [strain Esbc4], Acanthamoeba ssp. [strain Shi], and A. castellanii), were all derived from patients with Acanthamoeba keratitis (Table 1)and are therefore likely to be relevant to pathogenicity in vivo. 

Thus far, no reliable markers of Acanthamoeba pathogenicity have been described. Several techniques have been used in an effort to classify the different isolates of Acanthamoeba, and attempts have been made to correlate these classifications with the pathogenicity. ^{34 35 36 37} Thus far, nearly two dozen named species have been identified based on morphology. These have been reclassified into 15 different genotypes (T1–T15) based on rDNA sequence analysis. ^{38 39 40 41} Of note, all keratitis isolates belong to only five of these 15 genotypes: T1, T3, T4, T6, and T11. This suggests that the pathogenicity of Acanthamoeba is confined to a subset of genotypes. Our data demonstrating that the pathogenic potential of Acanthamoeba directly correlates with the expression level of the MBP, in conjunction with our published studies showing that Acanthamoeba MBP is a major virulence protein, ^{15 16} suggest that the amoeba lectin has the potential to serve as a marker of pathogenicity. We propose that classification of pathogenic and nonpathogenic strains of the Acanthamoeba genus based on the MBP expression should be given a serious consideration. 

 Supported by National Eye Institute Grants EY09349 (NP) and a core grant EYP30-13078 for Vision Research, the Massachusetts Lions Eye Research Fund, and the New England Corneal Transplant Research Fund.

 Submitted for publication April 17, 2005; revised September 19 and October 25, 2005; accepted January 19, 2006.

 Disclosure: M. Garate, None; J. Marchant, None; I. Cubillos, None; Z. Cao, None; N. A. Khan, None; N. Panjwani, None

 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.

 

Table 1.

 

Acanthamoeba Isolates

Species	Strain	Genotype 8 31 32	Source
A. palestinensis	CCAP 1501/3C	T2	Freshwater, old distilled water carboy, USA
A. royreba	CCAP 1501/7	T4	BeWo tissue culture, USA
Acanthamoeba spp.	Esbc4, PHLS	T4	UK keratitis
Acanthamoeba spp.	Shi, PHLS	T4	UK keratitis
A. castellanii	MEEI0184	ND	USA keratitis

 CCAP, Culture Collection of Algae and Protozoa (UK); PHLS, Public Health Laboratory Service (UK); ND, not determined.

Figure 1.

 

Acanthamoeba trophozoites but not cysts bound to man-BSA and to epithelial cells and produce CPE. (A) Binding of Acanthamoeba castellanii to man-BSA and to epithelial cells. To assess amoeba binding to man-BSA, wells of microtiter plates were coated overnight with the neoglycoprotein (0.1 μg/well), blocked with 5% BSA in PBS and incubated with ³⁵S-labeled trophozoites or cysts (1 × 10⁵ parasites/well) for 30 minutes. Bound parasites were estimated as the radioactivity associated to the wells after five washes with PBS. For assessment of binding to host cells, confluent cultures of rabbit corneal epithelial cells in 96-well plates were used instead of man-BSA coated wells. Results are presented as the mean ± SD of three experiments (n = 12 for each group). Note that trophozoites but not cysts bound to man-BSA and epithelial cells. (B) A CPE assay was used as an index of pathogenicity. Aliquots of cysts or trophozoites (1 × 10⁶ cells/mL) were added to wells of confluent cultures of epithelium. The plates were incubated at 37°C for 1, 3, and 6 hours in a CO₂ incubator, examined under a phase-contrast microscope for the presence of cell-free plaques in the monolayer, stained with Giemsa, and photographed. Left: clear, unstained regions in the photographs indicate loss of cells; dark-stained areas indicate the presence of cells. Right: the approximate cell density in each well, presented as the mean percentage of CPE ± SD of three experiments (n = 6). *P < 0.0001 compared to trophozoite groups.

Figure 2.

 

Acanthamoeba cysts exhibited markedly reduced MBP expression compared with trophozoites. Acanthamoeba (A) trophozoites and (B) cysts (1 × 10⁶ cells) were incubated in suspension with purified anti-MBP IgY or preimmune IgY (room temperature, 30 minutes), and subsequently with fluorescein-labeled goat anti-chicken IgY. After they were rinsed thoroughly with PBS, the cells were smeared onto coverslips, fixed in 2.5% paraformaldehyde, rinsed three times, and mounted. Left: phase-contrast micrographs; middle and right: photographs of cells treated with anti-MBP IgY and pre-immune IgY, respectively. Note that trophozoites but not cysts stained robustly with anti-MBP IgY. (C) Left: proteins extracted from cell membranes of Acanthamoeba cysts and trophozoites (5 μg each) were electrophoresed in 8% SDS-polyacrylamide gels and silver stained. Right: MBP was isolated by affinity chromatography of the membrane extracts of cysts and trophozoites (2.5 × 10⁷ cells each) on α-Man gel, the bound material was eluted from the column by α-Man, electrophoresed in 8% SDS-polyacrylamide gels and silver stained. Note that MBP is robustly expressed in trophozoites but is hardly detectable in cysts. Magnification bars, 50 μm.

Figure 3.

 

Pathogenic potential of Acanthamoeba is associated with the MBP expression. Comparison of five different isolates of Acanthamoeba. (A) Analysis of binding of five different Acanthamoeba isolates to man-BSA. Wells of microtiter plates coated with man-BSA (0.1 μg/well) were incubated with ³⁵S-labeled trophozoites (1 × 10⁵ parasites/well, 30 minutes) which had been pretreated with medium, purified anti-MBP IgY or preimmune IgY. Bound parasites were estimated as the radioactivity associated to the wells after five washes with PBS. Results are presented as the mean ± SD of three experiments (n = 12 for each group). (B) Analysis of CPE-producing ability of various Acanthamoeba isolates. Left: Rabbit corneal epithelial cells grown to a monolayer in 48-well plates were incubated in duplicates with trophozoites (1 × 10⁶ cells/mL; 50 μL/well, 6 hours) that had been preincubated with: medium, anti-MBP IgY, or preimmune IgY. At the end of the incubation period, cultures were examined under a phase-contrast micro-scope for the presence of cell-free plaques in the monolayer, stained with Giemsa, and photographed. Right: approximate cell density in each well, presented as the mean percentage of CPE ± SD of three experiments (n = 6 for each group). *P < 0.0001 compared to media groups.

Figure 4.

 

Comparison of MBP expression levels in various species of Acanthamoeba trophozoites. (A) Left: proteins extracted from cell membranes of various Acanthamoeba isolates (5 μg each) were electrophoresed on 8% SDS-polyacrylamide gels and silver stained. Right: MBP was isolated from the membrane extracts of Acanthamoeba isolates (2.5 × 10⁷ parasites each) by affinity chromatography on α-Man-gel, material bound to the affinity column was eluted by α-Man, electrophoresed in 8% SDS-polyacrylamide gels and silver stained. Note that a 130-kDa component is robustly expressed in the three Acanthamoeba isolates that exhibited a potent CPE (see Figs. 1 3 ). In contrast, this component was detected in markedly reduced amounts in Acanthamoeba strains exhibiting mild to moderate CPE-inducing ability (see Figs. 1 3 ). (B) Western blot analysis of the affinity-purified MBP. To confirm further that the 130-kDa component detected by silver staining (A, right) is MBP, protein blots of MBP preparations derived from various Acanthamoeba strains (10⁹ parasites each) were stained with ponceau S and then processed for immunostaining with anti-MBP IgY. The IgY antibody but not preimmune IgY (not shown) reacted with the 130-kDa component isolated from all three pathogenic strains of Acanthamoeba.

The authors thank Honorine Ward and Gregory Booton for helpful discussions.