Pathogenesis of Acanthamoeba Keratitis

描述（由申请方提供）：阿米巴角膜炎（AK）是由阿米巴属寄生虫引起的严重、使人虚弱和剧烈疼痛的角膜感染。目前，这种疾病的诊断并不简单，治疗要求很高。在目前的资助期间，我们已经确定了阿米巴的主要毒力蛋白，甘露糖结合蛋白（MBP）介导的寄生虫粘附到宿主细胞。具体来说，我们已经克隆和表征了阿米巴MBP，并已表明，它是一个典型的细胞表面受体的特征的跨膜蛋白。最近的研究表明，正常人泪液含有抗MBP伊加抗体，其抑制寄生虫粘附到宿主细胞，并且与正常个体的泪液相比，AK患者的泪液含有显著降低水平的针对MBP的N-末端结构域的抗体，在此称为截短的MBP（T-MBP）。总的来说，这些数据使我们假设：（i）正常个体泪液中的抗MBP伊加通过抑制寄生虫粘附于宿主细胞提供了第一道防线，以及（ii）T-MBP内缺乏足够量的抗粘附抑制和/或可能的其他保护性表位的抗体，会造成感染的风险。在目的1中，我们将表征T-MBP表位的结构和功能，针对T-MBP表位的抗体存在于正常泪液中，但在AK患者的泪液中不存在或以减少的量存在。由于阿米巴原虫通过MBP介导的基于甘露糖的识别与宿主细胞结合，我们假设泪液中特异性针对MBP的碳水化合物识别结构域（CRD）的抗体的存在应该是保护性的。在目标2中，使用MBP的缺失突变体，我们将鉴定和表征编码MBP的CRD的序列，并将确定CRD是否包含目标1中提出的保护性表位。在目标3中，我们将检验一个假设，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生。具体而言，在这个目标中，使用缺失突变体，我们将确定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的糖蛋白，在目的4中，我们将确定MBP的CRD是否通过影响人补体系统的功能来调节先天免疫系统。所提出的研究对两方面都有意义：（a）对许多眼部感染的基本机制的基本理解，（B）对患者的研究有意义的翻译。我们希望拟议的研究将有助于开发新的，合理设计的策略，以管理和保护：（i）短期内的AK，和（ii）从长远来看由其他生物体产生的角膜炎。角膜感染是失明的主要原因，影响全球2500多万人。在所提出的应用中，使用Acathamoeba角膜炎（AK）作为角膜感染的模型，我们提出了旨在有助于我们理解角膜感染性疾病的致病机制的研究。我们希望，拟议的研究将最终有助于开发新的，合理设计的策略，以管理致盲性角膜感染，并防止他们。

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.