Resistance of Acanthamoeba castellanii cysts to disinfection agents, antimicrobial agents, heat, freeze–thawing, ultraviolet radiation (UV), γ irradiation, and cellulase were evaluated in vitro. Following exposure to different agents, the cysts were removed and cultured for A. castellanii trophozoites for 3–14 days. Solutions containing 20% isopropyl alcohol or 10% formalin effectively killed A. castellanii cysts. Hydrogen peroxide (3%, AOSept Disinfectant) effectively killed A. castellanii cysts after 4 hr of exposure. Polyhexamethylene biguanide (0.02%), clotrimazole (0.1%), or propamidine isethionate (Brolene) were effective in killing A. castellanii cysts in vitro. Acanthamoeba castellanii cysts were resistant to both 250 K rads of γ irradiation and 800 mJ/cm2 of UV irradiation. Excystment of trophozoites was accelerated after exposure to 10, 100, and, 1,000 units of cellulase. These results suggest that A. castellanii cysts benefit by enhanced survival because of their resistance to very harsh environmental conditions.

Exposure to Acanthamoeba species appears to be ubiquitous, as 50% to 100% of healthy human subjects display anti-Acanthamoeba antibodies. However, the presence of specific anti-Acanthamoeba antibodies in the serum and tears of patients has not been investigated. The prevalence of serum immunoglobulin G (IgG) and tear IgA against three species of Acanthamoeba was assessed in healthy subjects and patients with Acanthamoeba keratitis. The level of specific serum IgG and tear IgA against A. castellanii, A. astronyxis, and A. culbertsoni in the sera of 23 patients and 25 healthy subjects was tested by enzyme-linked immunosorbent assays. Total serum IgM, IgG, and IgA concentrations were measured by nephelometry. Acanthamoeba keratitis was diagnosed clinically and confirmed by in vivo confocal microscopy. In some patients, corneal biopsies were also performed and trophozoites were cultured on lawns of Escherichia coli on non-nutrient agar. All healthy subjects and patients with Acanthamoeba keratitis had detectable serum IgG antibodies against all Acanthamoeba antigens. However, patients with Acanthamoeba keratitis had significantly higher anti-Acanthamoeba IgG antibody titers than healthy subjects. In contrast, Acanthamoeba-specific tear IgA was significantly lower in patients with Acanthamoeba keratitis in comparison with healthy subjects. Total serum immunoglobulins did not differ significantly between healthy subjects and patients with Acanthamoeba keratitis. The results suggest that a low level of anti-Acanthamoeba IgA antibody in the tears appears to be associated with Acanthamoeba keratitis.

The pathogenesis of Acanthamoeba keratitis begins when Acanthamoeba trophozoites bind specifically to mannosylated glycoproteins upregulated on the surfaces of traumatized corneal epithelial cells. When Acanthamoeba castellanii trophozoites are grown in methyl -α- d -mannopyranoside, they are induced to secrete a novel 133-kDa protein that is cytolytic to corneal epithelial cells. Clinical isolates of Acanthamoeba spp., and not the soil isolates, were proficient at producing a mannose-induced protein (MIP-133) and generating disease in Chinese hamsters. The purified protein was efficient at killing corneal epithelial cells, the first mechanistic barrier, by inducing apoptosis in a caspase 3-dependent pathway. Subsequent steps in pathogenesis require the amoebae to penetrate and degrade collagen. Only the clinical isolates tested were efficient at migrating through a collagenous matrix in vitro, presumably by MIP-133 degradation of both human type I and human type IV collagen. A chicken anti-MIP-133 antiserum effectively bound to the protein and blocked collagenolytic activity, migration, and cytopathic effects (CPE) against corneal cells in vitro. Chinese hamsters orally immunized with MIP-133 displayed a >30% reduction in disease. Immunoglobulin A isolated from immunized animals bound MIP-133 and blocked CPE on corneal cells in vitro. Animals induced to generate severe chronic infections displayed significant reductions in disease symptoms upon oral immunization postinfection. These data suggest that MIP-133 production might be necessary to initiate corneal disease and that it may play an important role in the subsequent steps of the pathogenic cascade of Acanthamoeba keratitis. Furthermore, as antibodies produced both prior to and after infection reduced clinical symptoms of disease, the protein may represent an important immunotherapeutic target for Acanthamoeba keratitis.

Neutrophils are thought to be involved in many infectious diseases and have been found in high numbers in the corneas of patients withAcanthamoebakeratitis. Using a Chinese hamster model of keratitis, conjunctival neutrophil migration was manipulated to determine the importance of neutrophils in this disease. Inhibition of neutrophil recruitment was achieved by subconjunctival injection with an antibody against macrophage inflammatory protein 2 (MIP-2), a powerful chemotactic factor for neutrophils which is secreted by the cornea. In other experiments, neutrophils were depleted by intraperitoneal injection of anti-Chinese hamster neutrophil antibody. The inhibition of neutrophils to the cornea resulted in an earlier onset and more severe infection compared to controls. Anti-MIP-2 antibody treatment produced an almost 35% reduction of myeloperoxidase activity in the cornea 6 days postinfection, while levels of endogenous MIP-2 secretion increased significantly. Recruitment of neutrophils into the cornea via intrastromal injections of recombinant MIP-2 generated an initially intense inflammation that resulted in the rapid resolution of the corneal infection. The profound exacerbation ofAcanthamoebakeratitis seen when neutrophil migration was inhibited, combined with the rapid clearing of the disease in the presence of increased neutrophils, strongly suggests that neutrophils play an important role in combatingAcanthamoebainfections in the cornea.

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.