Mannose induces the release of cytopathic factors from Acanthamoeba castellanii

Abstract

Acanthamoeba keratitis is a chronic inflammatory disease of the cornea which is highly resistant to many antimicrobial agents. The pathogenic mechanisms of this disease are poorly understood. However, it is believed that the initial phases in the pathogenesis of Acanthamoeba keratitis involve parasite binding and lysis of the corneal epithelium. These processes were examined in vitro, using Acanthamoeba castellanii trophozoites. Parasites readily adhered to Chinese hamster corneal epithelial cells in vitro; however, parasite binding was strongly inhibited by mannose but not by lactose. Although mannose prevented trophozoite binding, it did not affect cytolysis of corneal epithelial cells. Moreover, mannose treatment induced trophozoites to release cytolytic factors that lysed corneal epithelial cells in vitro. These factors were uniquely induced by mannose because supernatants collected from either untreated trophozoites or trophozoites treated with other sugars failed to lyse corneal cells. The soluble factors were size fractionated in centrifugal concentrators and found to be > or = 100 kDa. Treatment of the supernatants with the serine protease inhibitor phenylmethylsulfonyl fluoride inhibited most, but not all, of the cytopathic activity. These data suggest that the binding of Acanthamoeba to mannosylated proteins on the corneal epithelium may exacerbate the pathogenic cascade by initiating the release of cytolytic factors.

FIG. 1

Effect of mannose on Acanthamoeba binding to CHCE cells. Adherence of radiolabeled A. castellanii trophozoites to CHCE cells was examined after 2 h of incubation in the presence of either 100 mM mannose (▨) or 100 mM lactose (▧). Percent adherence is calculated by dividing the counts of radiolabel recovered after washing monolayers with Hanks balanced salt solution by the total counts of the initial inoculum. □, medium control.

FIG. 2

Effect of mannose on CPE mediated by A. castellanii trophozoites. CHCE cell monolayers were incubated for 48 h with trophozoites in MEM with (▨) or without (□) 100 mM mannose (A) or with 100 mM lactose (▧) (B). Monolayers were washed and stained with Giemsa stain, and CPE was assessed spectrophotometrically.

FIG. 3

Mannose reconstitution and Acanthamoeba-mediated CPE. CHCE cells were incubated with A. castellanii trophozoites (trophs) in the presence or absence of 100 mM mannose. After 24 h of incubation, the culture medium in one group was removed and replaced with fresh medium containing 100 mM mannose [trophs + mannose (2X)]. The negative control consisted of monolayers cultured in medium without trophozoites. Monolayers were washed and stained with Giemsa stain, and CPE was assessed spectrophotometrically. The differences between the experimental groups were insignificant (P > 0.05).

FIG. 4

Cytolytic activity of supernatants from A. castellanii trophozoites stimulated with mannose. Trophozoites were incubated with 100 mM mannose for 48 h at 35°C. Supernatants were collected, centrifuged, filter sterilized, dialyzed, and added to CHCE cell monolayers. CPE was assessed spectrophotometrically after 48 h. Positive controls consisted of CHCE cell monolayers incubated with 2.5 × 10⁶ trophozoites/ml (trophs). Negative controls consisted of monolayers incubated in medium alone and, by definition, had no CPE (data not shown). ∗, the difference between the undiluted mannose supernatant group (neat) and the medium control (0% CPE; not shown) was significant (P = 0.0078). Trophs + mannose (50%), mannose supernatant diluted 1:2 in complete MEM. Results are expressed as mean ± standard deviation.

FIG. 5

Effects of supernatants from mannose-treated A. castellanii trophozoites on CHCE cell monolayers. Trophozoites were incubated with 100 mM mannose for 48 h at 35°C. Supernatants were collected, centrifuged, filter sterilized, concentrated fourfold, and dialyzed against a 10-kDa-cutoff membrane. The concentrated supernatants were added to CHCE cell monolayers and incubated at 35°C for 48 h (A). MEM containing 100 mM mannose, but not exposed to trophozoites, was similarly treated and added to monolayers (B). Monolayers were photographed 48 h later. Bar = 100 μm.

FIG. 6

Effects of lactose, galactose, and mannose on the release of cytolytic factors by A. castellanii trophozoites. Trophozoites were incubated with complete MEM or complete MEM containing 100 mM mannose, 100 mM lactose, or 100 mM galactose for 48 h at 35°C. Supernatants were collected, centrifuged, filter sterilized, concentrated fourfold, dialyzed against a 10-kDa-cutoff membrane, and added to CHCE cell monolayers. Negative controls consisted of monolayers incubated in medium alone (0% CPE; not shown). CPE was assessed spectrophotometrically 48 h later. Results are expressed as mean ± standard deviation. ∗, the mannose treatment group was significantly different from each of the other groups (P = 0.0001). The lactose and galactose groups were not significantly different from the complete MEM control supernatant (P > 0.05).

FIG. 7

Effect of a serine protease inhibitor on Acanthamoeba-derived cytolytic factors. Trophozoites were incubated with 100 mM mannose for 48 h at 35°C. Supernatants were collected, centrifuged, filter sterilized, concentrated fourfold, and dialyzed against a 10-kDa-cutoff membrane. Supernatants were mixed with 10 μg of PMSF per ml 30 min prior to addition to CHCE cell monolayers. CPE was assessed spectrophotometrically after 48 h. Negative controls consisted of monolayers incubated in medium alone (0% CPE). Results are expressed as mean ± standard deviation. ∗, the difference between the two groups was significant (P = 0.04). □, mannose supernatant; ▨, mannose supernatant + PMSF.

FIG. 8

Molecular weight estimation of Acanthamoeba-derived cytolytic factors. Trophozoites were incubated with 100 mM mannose for 48 h at 35°C. Supernatants were collected, centrifuged, filter sterilized, and size fractionated with centrifugal concentrators having a molecular mass cutoff of either 30 or 100 kDa. Fractions were added to CHCE cell monolayers, and CPE was assessed spectrophotometrically. Results are expressed as mean ± standard deviation.