Acanthamoeba keratitis is a progressive sight-threatening corneal disease caused by pathogenic, free-living amoebae Acanthamoeba species. The infection is highly resistant to topical and systemic therapy, leading to serious visual impairment or even loss of vision. The pathogenesis of Acanthamoeba occurs in a multistep process that begins with the binding of the trophozoites to the corneal epithelium (Niederkorn et al., 1992; Yang et al., 1997). Following binding on the corneal epithelial surface, trophozoites invade deeper regions of the corneal epithelium and penetrate the stroma, resulting in necrosis, edema, disruption of the stromal lamellae, and an intense polymorphonuclear inflammatory response (Mathers et al., 1987). During this process, Acanthamoeba trophozoites elicit several cytopathic factors that induce considerable corneal damage and allow the parasite to invade the stroma (He et al., 1990; Mitro et al., 1994; Leher et al., 1998a; Na et al., 2001).

Proteinases of parasites have been known to be important virulence factors in the pathogenicity of parasitic infections either by inducing tissue damage and facilitating invasion or by empowering the parasites to salvage metabolisms from host proteins (McKerrow, 1989). As other parasitic protozoa, pathogenic Acanthamoeba trophozoites produce a variety of proteinases (He et al., 1990; Mitro et al., 1994; Mitra et al., 1995; Park and Song, 1996; Cho et al., 2000; Kong et al., 2000; Na et al., 2001). Although the precise role of these proteinases in the pathogenesis of corneal infection of Acanthamoeba is not clear at present, some evidence suggests that the pathogenic roles of the proteinases as important virulence factors in Acanthamoeba keratitis. The secretory products from trophozoites of Acanthamoeba have been shown to have collagenolytic activity, which damages collagen shield in an in vitro assay and rat cornea in vivo (He et al., 1990; Mitro et al., 1994). Furthermore, Acanthamoeba proteinases can break down a number of host proteins, including major structural proteins such as collagen, fibronectln and laminin (Park and Song, 1996; Cho et al., 2000; Kong et al., 2000; Na et al., 2001). Importantly, intrastromal injection of culture medium and purified proteinase of A. castellanii into the corneas of rabbits produced characteristic ring infiltrates and corneal lesions that clinically and histopathologically resembled those found in patients with Acanthamoeba keratitis (He et al., 1990; Na et al., 2001). These results partially suggest that Acanthamoeba proteinases are closely associated with the pathogenesis of Acanthamoeba by facilitating penetration of the parasites into the corneal stroma.

However, to elucidate the pathogenic role of the Acanthamoeba proteinase in Acanthamoeba infection more definitely, more detailed studies on the pathogenic mechanism of the proteinase are needed. In this study, we investigated the effect of the Acanthamoeba proteinase on the host's defense-oriented or regulatory proteins.

The ocular surface is regularly exposed to the external environment and potential pathogens, however, is protected by vigorous defense mechanisms that would promptly eliminate the pathogens. These defense mechanisms are largely provided by tear film that forms the first line of defense between the external environment and the ocular surface. Tears contain variety of humoral defense components such as lactoferrin, lysozyme, and lactoperoxidase. In addition to innate humoral defense mechanisms, tears also contain various other components that act as defense-oriented or regulatory molecules (Hazlett and Berk, 1989; Fullard and Snyder, 1990).

One of them is immunoglobulins that may play a major role as a host defense mechanism to prevent or modulate microbial adhesion, invasion, and infectivity (Allansmith et al., 1979; Wells and Hazlett, 1985). In the present study, the effect of A. castellanii proteinase on immunoglobulins was investigated. The purified enzyme of A. castellanii degraded slgA. slgA is the major immunoglobulin isotype present in normal tears where it constitutes a front line of defense against microorganisms primarily as an immunologic barrier by preventing adherence and adsorption of microbes (Gachon et al., 1982; Gregory and Allansmith, 1986; Mestecky et al., 1986; Masinick et al., 1997). It may also prevent colonization by neutralizing microbial pathogens within corneal epithelial cells. Inhibition of microorganism's adherence to the ocular surface would be advantageous, because this might diminish the host inflammatory cell response to eliminate microorganisms from the cornea, in turn leading to less innocent bystander destruction of the cornea tissue. Similar to most other ocular pathogens, the first step of Acanthamoeba infection is adhesion of trophozoites to corneal epithelial surfaces (Niderkorn et al., 1992; Yang et al., 1997). Inhibiting the binding of Acanthamoeba trophozoites to the corneal epithelium may be a highly effective mechanism for preventing corneal infection. In fact, the orally immunized animals produced IgA antibodies specific for Acanthamoeba antigens, and they inhibited the adhesion of trophozoites to corneal epithelial cells in vitro (Leher et al., 1998b; Leher et al., 1999). Therefore, cleavage of slgA by proteinases would presumably enhance the ability of the organism to survive on corneal surfaces. The proteinase of A. castellanii was also able to degrade IgG and IgM. IgG and IgM are involved in the complement-dependent killing mechanism such as opsonization and phagocytosis, in microbial infections. Although repeated intramuscular immunizations of Acanthamoeba antigens induced Acanthamoeba-specific serum IgG and delayed-type hypersensitivity (DTH) responses, they failed to protect against an ocular challenge by Acanthamoeba trophozoites (Alizadeh et al., 1995; van Klink et al., 1997). Moreover, repeated ocular infections do not induce protective immunity (Alizadeh et al., 1995; van Klink et al., 1997). Failure of protective immunity against Acanthamoeba infection may be partially explained by the fact that Acanthamoeba proteinase degraded immunoglobulins. Degradation of immunoglobulins by the Acanthamoeba proteinase may result in a decrease of host immune activity to Acanthamoeba and may render hosts more vulnerable to subsequent opportunistic infections.

We also investigated the effect of A. castellanii proteinase on several protease inhibitors. Protease inhibitors have been identified in tears, and their amounts are increased in the case of infections or in corneal ulcerations (Stanifer et al., 1983). These endogenous protease inhibitors are involved in critical regulation of various cascade systems, including the complement-dependent defense system, the kallikrein-kinin system, the fibrinolytic and clotting system, and inflammation reactions. These cascades may be highly enhanced when the inhibitors are inactivated. Furthermore, protease inhibitors of hosts may act as defense-oriented molecules that inhibit proteinases of pathogens. As shown in this study, the endogenous protease inhibitors, such as α2-macroglobulin, α2-antiplasmin, and α1-antitrypsin, did not inhibit the activity of A. castellanii proteinase, and the inhibitors themselves were completely degraded. Acanthamoeba proteinases are known to be important virulence factors that induce tissue damage and facilitate invasion of the amoeba into the corneal stroma (He et al., 1990; Mitro et al., 1994; Na et al., 2001). Therefore, degradation of various protease inhibitors may not only enhance the pathogenicity of Acanthamoeba, but also may affect the activity of endogenous proteinases in the cornea, resulting in more severe pathological consequences, such as tissue destruction, edema formation, and pain. Actually, when the purified enzyme was injected intrastromally to rabbit cornea, characteristic ring infiltration and corneal lesions that clinically resembled those in Acanthamoeba keratitis were observed (Na et al., 2001). These lesions were significantly inhibited by the protease inhibitor, phenylmethyl sulfonylfluoride. This further lends support to this hypothesis.

The inability of corneal infections to induce primary cell-mediated immune responses is due to the absence of resident antigen presenting cells (APCs) in the central cornea. Langerhans cells (LCs), known to be major APCs in the cornea, are uniquely distributed in the peripheral region of the cornea. Peripheral LCs migrate into the central cornea either by intracorneal injection of IL-1 or by instillation of sterile latex beads prior to infection with Acanthamoeba trophozoites, and they produce a remarkable resistance to infection (van Klink et al., 1993; van Klink et al., 1997). Although a definite mechanism of the protective effect is not clear yet, this suggests that LCs play important roles in immunological protection against Acanthamoeba infection. It is well known that potential pathogens and antigens would be phagocytosed by corneal epithelial cells, which in turn would elaborate increased quantities of IL-1, a molecule which plays a central role in the induction, progression, and maintenance of an inflammatory response (Dinarello, 1994). The soluble IL-1 would then act as a potent chemoattractant for peripheral LCs residing in the limbus. LCs would migrate in a unidirectional manner and accumulate at the site of antigen deposition and IL-1 synthesis, where they encounter the activated corneal epithelial cells. The corneal epithelial cells may display the partially processed antigen on their cell membranes for delivery to the infiltrating LCs. Although LCs itself did not provoke primary cell-mediated immunity, further antigen processing occurred within the LCs, which would then migrate to regional lymphoid tissues for the final stage of antigen presentation to T and B lymphocytes.

The enzyme of Acanthamoeba degraded both IL-lα and IL-lβ. Degradation of IL-1 by the enzyme might not induce migration of LCs into the central cornea, thereby resulting in failure of primary cell-mediated immunity. Consequently, it might enhance the survival of Acanthamoeba against the hosts' systemic immune response and make symptoms more severe.

In conclusion, in the present study, we investigated a potent proteolytic action of Acanthamoeba proteinase on the host's defense-oriented or regulatory proteins, including immunoglobulins, interleukin-1, and several protease inhibitors. Acanthamoeba proteinase could degrade these proteins. These results suggest that Acanthamoeba proteinase is a potential virulence factor that could enhance the ability of Acanthamoeba to survive against the host's defense mechanism and render hosts more vulnerable to subsequent opportunistic infections.