The immunobiology of Acanthamoeba keratitis

Acanthamoeba spp. are free-living amoebae that cause Acanthamoeba keratitis, a blinding corneal infection. The innate immune apparatus is crucial for the resolution of the disease. With the exception of mucosal antibody, elements of the adaptive immune system fail to prevent infection or contribute to its resolution in experimental animals.     

The pathophysiology of Acanthamoeba keratitis

Acanthamoeba keratitis is a sight-threatening infection of the ocular surface that is produced by several free-living amebae of the genus Acanthamoeba. Infection is usually initiated by Acanthamoeba-contaminated contact lenses and produces exquisite pain and ulceration of the ocular surface. The pathophysiology of this infection involves an intricate series of sequential events that includes the production of several pathogenic proteases that degrade basement membranes and induce cytolysis and apoptosis of the cellular elements of the cornea, culminating in dissolution of the collagenous corneal stroma. Targeting such proteases could lead to the development of vaccines that target the disease process rather than the pathogen itself.     

Role of activated macrophages in Acanthamoeba keratitis

The purpose of this study was to determine whether activating the conjunctival macrophages would affect the course of Acanthamoeba spp. keratitis in a Chinese hamster model of this disease. Chinese hamster spleen cells were stimulated with concanavalin A (Con A), and interferon gamma (IFN-gamma) -containing supernatants were collected 24 hr later. The IFN-gamma-containing supernatants were loaded into liposomes, which were fed to peritoneal macrophages in vitro. Macrophage activation was assessed by testing for production of nitric oxide (NO) with the use of Griess reagent. Conjunctival macrophages were activated in situ by subconjunctival injection of liposomes containing Con A-activated spleen cell culture supernatants. Control liposomes were loaded with phosphate-buffered saline (PBS). Macrophages exposed to supernatants from Con A-stimulated spleen cells produced 4-fold-higher amounts of NO than unstimulated macrophages. Activation of macrophages via subconjunctival injection of liposomes containing supernatants from Con A-stimulated spleen cell cultures resulted in rapid resolution of the corneal infection. Approximately 80% of animals treated with PBS-containing liposomes demonstrated evidence of corneal disease at day 14 compared to 10% incidence of infection in the Con A-treated group. Moreover, at all time points examined, the clinical appearance of the keratitis in animals treated with liposomes containing Con A supernatant was significantly reduced compared to the group treated with liposomes containing PBS (P < 0.05). Macrophages stimulated with IFN-gamma-containing supernatants killed significant numbers of the trophozoites in vitro (P < 0.05). Killing was inhibited by cytochalasin D, but not by L-N6-1-iminoethyl-L-lysine dihydrochloride (L-NIL), which is a selective inhibitor of inducible NO synthase (INOS).     

The role of autophagy in the pathogenesis of exposure keratitis

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.     

Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.     

Molecular characterization of Acanthamoeba isolated from amebic keratitis related to orthokeratology lens overnight wear

In an effort to characterize, on the molecular scale, the Acanthamoeba initially isolated from the cornea of an amoebic keratitis patient associated with overnight-wear orthokeratology lens in Korea, we conducted mitochondrial DNA restriction fragment length polymorphism, 18S rDNA sequencing, and drug sensitivity analyses on the isolate (KA/PE1). The patient was treated with polyhexamethylene biguanide, chlorhexidine and oral itraconazole, which resulted in resolution of the patientos ocular inflammation. The majority of the molecular characteristics of the KA/PE1 were determined to be identical, or quite similar, to those of A. castellanii Ma strain, which had been isolated also from amoebic keratitis. The risk of Acanthamoeba keratitis as a potential complication of overnight orthokeratology is briefly discussed.     

Value of nu/nu mice in the production of experimental amoebic keratitis with Acanthamoeba

The intracorneal inoculation of nude mice with Acanthamoeba hatchetti amoebae induced a keratitis with disruption of corneal fibres, inflammatory response, secondary vascularization and amoebic trophozoites.     

Conserved mechanisms of Mycobacterium marinum pathogenesis within the environmental amoeba Acanthamoeba castellanii

Mycobacterium marinum is a waterborne mycobacterial pathogen. Due to their common niche, protozoa likely represent natural hosts for M. marinum. We demonstrate that the ESX-1 secretion system is required for M. marinum pathogenesis and that M. marinum utilizes actin-based motility in amoebae. Therefore, at least two virulence pathways used by M. marinum in macrophages are conserved during M. marinum infection of amoebae.     

The cytochromes of Acanthamoeba castellanii.

1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).     

The covalent structure of Acanthamoeba actobindin

Actobindin is a protein from Acanthamoeba castellanii with bivalent affinity for monomeric actin. Because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of F-actin elongation. The complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, and CNBr. Actobindin contains 2 trimethyllysine residues and an acetylated NH2 terminus. About 76% of the actobindin molecule consists of two nearly identical repeated segments of approximately 33 residues each. This could explain actobindin's bivalent affinity for actin. The circular dichroism spectrum of actobindin is consistent with 15% alpha-helix and 22% beta-sheet structure. A hexapeptide with sequence LKHAET, which occurs at the beginning of each of the repeated segments of actobindin, is very similar to sequences found in tropomyosin, muscle myosin heavy chain, paramyosin, and Dictyostelium alpha-actinin. A longer stretch in each repeated segment is similar to sequences in mammalian and amoeba profilins. Interestingly, the sequences around the trimethyllysine residues in each of the repeats are similar to the sequences flanking the trimethyllysine residue of rabbit reticulocyte elongation factor 1 alpha, but not to the sequences around the trimethyllysine residues in Acanthamoeba actin and Acanthamoeba profilins I and II.     

The increasing importance of Acanthamoeba infections

Free-living amebae belonging to the genus Acanthamoeba are the causative agents of granulomatous amebic encephalitis, a chronic progressive disease of the central nervous system, and of amebic keratitis, a chronic eye infection. Granulomatous amebic encephalitis occurs more frequently in immunocompromised patients while keratitis occurs in healthy individuals. The recent increased incidence in Acanthamoeba infections is due in part to infection in patients with acquired immune deficiency syndrome, while that for keratitis is due to the increased use of contact lenses. Understanding the mechanism of host resistance to Acanthamoeba is essential since the amebae are resistant to many therapeutic agents. Studies in our laboratory as well as from others have demonstrated that macrophages from immunocompetent animals are important effector cells against Acanthamoeba. We have demonstrated also that microglial cells, resident macrophages of the brain, elicit cytokines in response to A. castellanii. Neonatal rat cortical microglia from Sprague-Dawley rats co-cultured with A. castellanii produced mRNA for the inflammatory cytokines, interleukin 1alpha, interleukin 1beta, and tumor necrosis factor alpha. In addition, scanning and transmission electron microscopy revealed that microglia ingested and destroyed A. castellanii in vitro. These results implicate macrophages as playing an effector role against Acanthamoeba and suggest immune modulation as a potential alternative therapeutic mode of treatment for these infections.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Herpes simplex virus-induced keratitis: evaluation of the role of molecular mimicry in lesion pathogenesis

Viruses are suspected but usually unproven triggering factors in autoimmunity. One favored mechanism to explain the role of viruses in the genesis of autoimmunity is molecular mimicry. An immunoinflammatory blinding lesion called herpetic stromal keratitis (HSK) that follows ocular infection with herpes simplex virus (HSV) is suggested to result from a CD4(+) T-cell response to a UL6 peptide of HSV that cross-reacts with a corneal autopeptide shared with the immunoglobulin G2a(b) (IgG2a(b)) isotype. The present report reevaluates the molecular mimicry hypothesis to explain HSK pathogenesis. Our results failed to reveal cross-reactivity between the UL6 and IgG2a(b) peptides or between peptide reactive T cells and HSV antigens. More importantly, animals infected with HSV failed to develop responses that reacted with either peptide, and infection with a recombinant vaccinia UL6 vector failed to cause HSK, in spite of generating UL6 reactivity. Other lines of evidence also failed to support the molecular mimicry hypothesis, such as the failure to affect HSK severity upon tolerization of susceptible BALB/c and B-cell-deficient mice with IgG2a(b) or UL6 peptides. An additional study system revealed that HSK could be induced in mouse strains, such as the OT2 x RAG1(-/-) mice (T cell receptor transgenic recognizing OVA(323-339)) that were unable to produce CD4(+) T-cell responses to any detectable HSV antigens. Our results cast doubt on the molecular mimicry hypothesis as an explanation for the pathogenesis of HSK and indicate that if autoimmunity is involved its likely proceeds via a bystander activation mechanism.     

The amino acid sequence of Acanthamoeba profilin

The complete amino acid sequence of Acanthamoeba profilin was determined by aligning tryptic, chymotryptic, thermolysin, and Staphylococcus aureus V8 protease peptides together with the partial NH2-terminal sequences of the tryptophan-cleavage products. Acanthamoeba profilin contains 125 amino acid residues, is NH2-terminally blocked, and has trimethyllysine at position 103. At five positions in the sequence two amino acids were identified indicating that the amoebae express at least two slightly different profilins. Charged residues are unevenly distributed, the NH2-terminal half being very hydrophobic and the COOH-terminal half being especially rich in basic residues. Comparison of the Acanthamoeba profilin sequence with that of calf spleen profilin (Nystrom, L. E., Lindberg, U., Kendrick-Jones, J., and Jakes, R. (1979) FEBS Lett. 101, 161-165) reveals homology in the NH2-terminal region. We suggest, therefore, that this region participates in the actin-binding activity.     

The growth-promoting action of Iron upon Acanthamoeba

Summary The growth-promoting effect of iron salts upon Acanthamoeba sp. Neff. was analyzed and it was found that this action persists after ultrafiltration and thus does not depend upon phagocytosis of particulate material, as has been suggested. Neither is the ultrafiltrable growth-promoting factor in proteose peptone of particulate nature but should have a gel structure. The mitogenetic action of iron was different for amoebae cultivated in neopeptone and proteose peptone, respectively. In neopeptone mainly the duration of growth is prolonged, while in proteose peptone only the rate of growth is increased. The growth-promoting actions of iron and phytohemoagglutinin are compared and found to he different. The iron may interfere with the ultrastructure of the nuclear membrane.Valuable technical assistance was given by Miss Brita Nilsson.     

The Glyoxylate Pathway in Acanthamoeba Sp.*

SYNOPSIS. Cell-free extracts of encysting Acanthamoeba were assayed for the key enzymes of the glyoxylate pathway, viz., isocitrate lyase and malate synthase. Both enzymes were present at the onset of encystment but their activities changed as cyst-wall formation proceeded to completion. Isocitrate lyase activity decreased during the first 4 hr of encystment to a minimum at 4 hr which was 70% of its initial activity. Activity then increased reaching a maximum at 9 hr which was 144% of its initial activity. After 9 hr a decrease in isocitrate lyase activity began which reached 70% of its initial activity at 35 hr. Malate synthase activity slowly decreased throughout encystment to 50% of its initial activity after 35 hr. From these data and others cited, it is concluded that this small soil amoeba has a functional glyoxylate pathway.