Pathogenesis of Acanthamoeba Keratitis

描述（由申请人提供）：棘阿米巴角膜炎（AK）是由棘阿米巴属寄生虫引起的一种严重的、使人衰弱的、剧烈疼痛的角膜感染。目前，该病的诊断并不直截了当，治疗要求很高。在目前的资助期内，我们已经确定了棘阿米巴的主要毒力蛋白，甘露糖结合蛋白（MBP），介导寄生虫对宿主细胞的粘附。具体来说，我们已经克隆并表征了棘阿米巴MBP，并表明它是一种具有典型细胞表面受体特征的跨膜蛋白。最近的研究表明，正常人泪液中含有抗MBP的IgA抗体，可以抑制寄生虫对宿主细胞的粘附，与正常人的泪液相比，AK患者的泪液中含有抗MBP n端结构域的抗体水平显著降低，这里称为截断的MBP （T-MBP）。总的来说，这些数据使我们假设：(i)正常个体眼泪中的抗mbp IgA通过抑制寄生虫对宿主细胞的粘附提供了第一道防线；（ii） T-MBP中缺乏足够数量的抗粘附抑制和/或可能的其他保护性表位的抗体，增加了感染的风险。在Aim 1中，我们将描述T-MBP表位的结构和功能，针对T-MBP表位，抗体存在于正常泪液中，但在AK患者泪液中不存在或存在量减少。由于变形虫通过MBP介导的甘露糖识别与宿主细胞结合，我们假设泪液中存在特异性针对MBP碳水化合物识别结构域（CRD）的抗体可能具有保护作用。在Aim 2中，使用MBP的缺失突变体，我们将识别和表征编码MBP CRD的序列，并确定CRD是否包含Aim 1中定位的保护性表位。在Aim 3中，我们将验证一个假设，即寄生虫通过凝集素胞外结构域的CRD粘附到宿主细胞后，一系列信号转导事件通过MBP的胞内细胞质（CT）结构域开始，导致细胞毒性蛋白酶的表达，最终导致细胞病变效应的发展。具体来说，在本研究中，我们将利用缺失突变体确定MBP的CT结构域在AK发病机制中的功能。由于许多补体蛋白是含甘露糖糖蛋白，在Aim 4中，我们将确定MBP的CRD是否通过影响人类补体系统的功能来调节先天免疫系统。提出的研究具有以下两方面的意义：(a)对许多眼部感染的基本机制的基本理解，以及(b)对患者研究的有意义的转化。我们希望拟议的研究将有助于开发新的，合理设计的策略来管理和保护：(i)短期内AK，以及（ii）长期内由其他生物产生的角膜炎。角膜感染是全球超过2500万人失明的主要原因。在申请中，我们使用棘阿米巴角膜炎（AK）作为角膜感染的模型，我们提出的研究旨在帮助我们了解角膜感染性疾病的致病机制。我们希望提出的研究最终将有助于开发新的，合理设计的策略来管理致盲性角膜感染并预防它们。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE 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.