Michael Hurt

Pathogenic Acanthamoeba spp. Secrete a Mannose-Induced Cytolytic Protein That Correlates with the Ability To Cause Disease

ABSTRACT 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.

Tear IgA and serum IgG antibodies against Acanthamoeba in patients with Acanthamoeba keratitis.

Purpose. 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. Methods. 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. Results. 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. Conclusions. The results suggest that a low level of anti-Acanthamoeba IgA antibody in the tears appears to be associated with Acanthamoeba keratitis.

Exacerbation of Acanthamoeba Keratitis in Animals Treated with Anti-Macrophage Inflammatory Protein 2 or Antineutrophil Antibodies

ABSTRACT Neutrophils are thought to be involved in many infectious diseases and have been found in high numbers in the corneas of patients withAcanthamoeba keratitis. 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 ofAcanthamoeba keratitis 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 combating Acanthamoeba infections in the cornea.

Role of Contact Lens Wear, Bacterial Flora, and Mannose-Induced Pathogenic Protease in the Pathogenesis of Amoebic Keratitis

ABSTRACT The ocular surface is continuously exposed to potential pathogens, including free-living amoebae. Acanthamoeba species are among the most ubiquitous amoebae, yet Acanthamoeba keratitis is remarkably rare. The pathogenesis of Acanthamoeba keratitis is a complex, sequential process. Here we show that Acanthamoeba keratitis is profoundly affected by mannosylated proteins on the ocular surface, which stimulate the amoebae to elaborate a 133-kDa pathogenic protease. The mannose-induced protease (MIP133) mediates apoptosis of the corneal epithelium, facilitates corneal invasion, and degrades the corneal stroma. We show that contact lens wear upregulates mannosylated proteins on the corneal epithelium, stimulates MIP133 secretion, and exacerbates corneal disease. Corynebacterium xerosis, a constituent of the ocular flora, contains large amounts of mannose and is associated with Acanthamoeba keratitis. The present results show that amoebae exposed to C. xerosis produce increased amounts of MIP133 and more severe corneal disease. Oral immunization with MIP133 mitigates Acanthamoeba keratitis and demonstrates the feasibility of antidisease vaccines for pathogens that resist immune elimination.

THE INTERACTION OF ACANTHAMOEBA CASTELLANII CYSTS WITH MACROPHAGES AND NEUTROPHILS

Acanthamoeba castellanii, a free-living amoeba, causes a sight-threatening form of keratitis. Even after extensive therapies, corneal damage can be severe, often requiring corneal transplantation to restore vision. However, A. castellanii cysts are not eliminated from the conjunctiva and stroma of humans and can excyst, resulting in infection of the corneal transplant. The aim of this study was to determine whether elements of the innate immune apparatus, neutrophils and macrophages, were capable of detecting and eliminating A. castellanii cysts and to examine the mechanism by which they kill the cysts. Results show that neither innate immune cell is attracted chemotactically to intact cysts, yet both were attracted to lysed cysts. Both macrophages and neutrophils were capable of killing significant numbers of cysts, yet neutrophils were 3-fold more efficient than macrophages. Activation of macrophages with lipopolysaccharide and interferon-γ did not increase their cytolytic ability. Conditioned medium isolated from macrophages did not lyse the cysts; however, prevention of phagocytosis by cytochalasin D inhibited 100% of macrophage-mediated killing of the cysts. Conditioned medium from neutrophils did kill significant numbers of the cysts, and this killing was blocked by quercetin, a potent inhibitor of myeloperoxidase (MPO). These results indicate that neither macrophages nor neutrophils are chemoattracted to intact cysts, yet both are capable of killing the cysts. Macrophages killed the cysts by phagocytosis, whereas neutrophils killed cysts through the secretion of MPO.

Role of Tear Anti-Acanthamoeba IgA in Acanthamoeba Keratitis

Acanthamoeba keratitis is a sight-threatening corneal disease caused by pathogenic free-living amoebae.1The organisms have been isolated from a wide variety of environments and from nasopharyngeal washes of asymptomatic individuals. 1Contact lens wear, practiced by over 25 million individuals in the United States, is the leading risk factor. Over 85% of the cases of Acanthamoeba keratitis occurred in contact lens wearers.2 Antibodies against Acanthamoeba spp. were detected in 52-100% of normal subjects tested in two serological surveys.3,4 In spite of the ubiquity of Acanthamoeba spp., the large number of contact lens wearers, and the apparent frequency of exposure to Acanthamoeba antigens, Acanthamoeba keratitis is rare. We hypothesized that frequent environmental exposure to Acanthamoeba antigens induces an immunity that protects against corneal infection in most contact lens wearers.