MECHANISMS OF E HISTOLYTICA ADHERENCE AND CYTOLYSIS

溶组织内阿米巴是一种侵袭性肠道病原体，可引起 世界范围内的发病率和死亡率很高。目前的证据表明 阿米巴黏附和细胞溶解事件在本病的发病机制中起关键作用 疾病。这项提案的目标是定义生化和 阿米巴黏附和细胞溶解机制的细胞基础。中的阿米巴 N-乙酰-D-氨基半乳糖(GalNAc)介导的体外黏附事件 可抑制的粘附素凝集素。对目标细胞的坚持启动了一种 需要阿米巴微丝功能的细胞溶解事件，磷脂酶 A(PLA)活性、Ca~(2+)离子通量和酸性pH的维持 细胞内的小泡。这项建议的具体目的和方法 1)细胞功能鉴定和阿米巴GalNAc的纯化 可抑制粘附素(凝集素)分子。体外黏附机制的研究进展 阿米巴对人结肠粘膜的作用将被定义。绑定研究 I125-asialofetuin(ASF)将列举和表征GalNAc 可抑制的表面粘附素分子，包括细胞控制及其 与毒力的关系。结合于细胞表面的鼠单抗 粘附素分子就会产生。可溶性GalNAc抑制凝集素 用ASF亲和层析或单抗抗体亲和层析进行纯化。 用SDS-PAGE、免疫沉淀等方法对纯化后的凝集素进行了鉴定 与单抗免疫球蛋白结合，通过竞争结合实现碳水化合物特异性 I125-ASF试剂盒检测。单抗对阿米巴黏附的影响 将描述纯化凝集素的多克隆免疫球蛋白。2)定义角色 GalNAc可抑制凝集素、钙离子、阿米巴聚乳酸酶和酸 阿米巴细胞溶解事件中的胞内小泡。细胞毒作用的研究 将描述纯化的GalNAc可抑制凝集素。阿米巴 胞质内游离钙离子将通过使用钙离子进行测量和可视化 激活的光蛋白，Aequorin。阿米巴聚乳酸与游离钙离子的关系 表面粘附素分子将被定义。解放军的活动将是 在靶细胞溶解过程中以阿米巴为单位进行测量。阿米巴聚乳酸酶 将通过离子交换层析纯化，并检测 细胞毒性。将研究抗聚乳酸多克隆抗体的作用。 囊泡pH值与磷脂酶A活性、游离钙离子、表面粘附素的关系 分子分布、囊泡胞吐和酶的掺入 将被定义为囊泡。我们的长期目标是将这些 发展一种免疫学或药理学手段的发现 预防侵袭性阿米巴病。

Entamoeba histolytica is an invasive enteric pathogen which causes substantial worldwide morbidity and mortality. Present evidence indicates that amebic adherence and cytolytic events are crucial in pathogenesis of disease. The objective of this proposal is to define the biochemical and cellular basis of amebic adherence and cytolytic mechanisms. The amebic in vitro adherence event is mediated by an N-acetyl-D-galactosamine (GalNAc) inhibitable adhesin lectin. Adherence to target cells initiates a cytolytic event which requires amebic microfilament function, phospholipase A (PLA) activity, Ca2+ ion flux, and maintenance of an acid pH in intracellular vesicles. The specific aims and methods of this proposal are: 1) To characterize the cellular function and purify the amebic GalNAc inhibitable adhesin (lectin) molecule. Mechanisms of in vitro adherence of amebae to human colonic mucosa will be defined. Binding studies with I125-asialofetuin (ASF) will enumerate and characterize the GalNAc inhibitable surface adhesin molecules, including cellular control and its relation to virulence. Mouse monoclonal antibodies which bind to the adhesin molecule will be produced. The soluble GalNAc inhibitable lectin will be purified by affinity chromatography with ASF or monoclonal IgG. The purified lectin will be characterized by SDS-PAGE, immunoprecipitation with monoclonal IgG, and carbohydrate specificity by competitive binding assays with I125-ASF. The effect on amebic adherence of monoclonal or polyclonal IgG to purified lectin will be described. 2) To define the role of the GalNAc inhibitable lectin, Ca2+ ions, amebic PLA enzymes, and acid pH intracellular vesicles in the amebic cytolytic event. Cytotoxicity of the purified GalNAc inhibitable lectin will be described. Amebic cytoplasmic free Ca2+ will be measured and visualized by use of the calcium activated photoprotein, aequorin. The relation of amebic PLA to free Ca2+ and surface adhesin molecules will be defined. PLA activity will be measured in amebae during cytolysis of target cells. Amebic PLA enzymes will be purified by ion exchange chromatography and assayed for cytotoxicity. Effects of anti-PLA-polyclonal antibodies will be studied. The relation of vesicle pH to PLA activity, free Ca2+, surface adhesin molecules distribution, vesicle exocytosis, and incorporation of enzymes into vesicles will be defined. Our long term goal is to apply these findings to the development of an immunologic or pharmacologic means of prophylaxis against invasive amebiasis.