Acanthamoeba keratitis (AK) is a serious, debilitating, and intensely painful infection of the cornea caused by parasites of the genus Acanthamoeba. At present, diagnosis of the disease is not straightforward and treatment is very demanding. During the current funding period, we have characterized a major virulence protein of Acanthamoeba, the mannose-binding protein (MBP), that mediates the adhesion of parasites to host cells. Specifically, we have cloned and characterized the Acanthamoeba MBP and have shown that it is a transmembrane protein with characteristics of a typical cell surface receptor. Recent studies have revealed that normal human tear fluid contains anti-MBP IgA antibodies that inhibit the adhesion of parasites to host cells and that, compared to tear fluid of normal individuals, tear fluid of AK patients contain significantly reduced levels of antibodies against the N-terminal domain of MBP, referred to here in as truncated MBP (T-MBP). Collectively, these data lead us to hypothesize that: (i) anti-MBP IgA in tears of normal individuals provide the first line of defense by inhibiting the adhesion of parasites to host cells and that (ii) the absence of sufficient quantities of antibodies against adherence-inhibiting and/or possibly other protective epitopes within the T-MBP, poses risk of infection.
In Aim 1, we will characterize the structure and the function of the T-MBP epitopes, against which, the antibodies are present in normal tears, but are absent or present in reduced amounts in tears of AK patients. Because amoebae bind to host cells via a mannose-based recognition mediated by the MBP, we hypothesize that the presence of antibodies in tear fluid specifically against the carbohydrate recognition domain (CRD) of MBP should be protective.
In Aim 2, using deletion mutants of the MBP, we shall identify and characterize the sequence encoding the CRD of MBP and will determine whether the CRD encompasses the protective epitope addressed in Aim 1.
In Aim 3, we will test a hypothesis that subsequent to the adhesion of parasites to the host cells via the CRD of the extracellular domain of the lectin, a cascade of signal transduction events begins via the intracellular cytoplasmic (CT) domain of MBP, leading to the expression of cytotoxic proteinases that ultimately lead to the development of cytopathic effect. Specifically, in this Aim, using deletion mutants we will determine the function of the CT domain of MBP in the pathogenesis of AK. Since many of the complement proteins are mannose-containing glycoproteins, in Aim 4, we will determine whether the CRD of MBP modulates the innate immune system by influencing the function of the human complement system. The studies proposed have implications for both: (a) a basic understanding of the fundamental mechanisms of many ocular infections in general, and (b) meaningful translation to studies on patients. We are hopeful that the proposed studies will help develop novel, rationally designed strategies to manage and protect against: (i) AK in the short run, and (ii) keratitis produced by other organisms in the long run.
Infections of the cornea are a major cause of blindness affecting more than 25 million individuals worldwide. In the proposed application, using Acathamoeba keratitis (AK) as a model of corneal infection, we propose studies that are designed to contribute to our understanding of the pathogenic mechanisms of the infectious diseases of the cornea. We are hopeful that the proposed studies will ultimately help develop new, rationally designed strategies to manage blinding corneal infections and protect against them.
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