MECHANISMS OF E HISTOLYTICA ADHERENCE AND CYTOLYSIS

肠道原生动物溶组织内阿米巴引起疾病 寄生虫粘附和细胞溶解事件在全球范围内至关重要， 侵袭性阿米巴病的发病机制。 我们的目标是定义 阿米巴粘附和溶细胞的生物化学和分子基础 申请开发疫苗保护的活动 对抗侵袭性阿米巴病 寄生虫体外粘附，介导 通过170 Kdalton半乳糖/N-乙酰基-D-半乳糖胺（Gal/GalNAc） 可降解的表面凝集素，启动靶细胞的细胞溶解。 阿米巴细胞溶解活性是由佛波酯刺激的， 阿米巴磷脂酶A活性，内吞囊泡中的酸性pH- 靶细胞内游离Ca++（（CA++））的致死性增加。 疫苗的候选物包括Gal/GalNAc凝集素、细胞溶解性凝集素、细胞溶解性凝集素和细胞溶解性凝集素。 寄生虫蛋白和主要阿米巴抗原。 具体目标 本建议的方法是：（1）测定体内 纯化的Gal/GalNAc凝集素在沙鼠中的疫苗效力！模型 肠定植和肝脓肿;（2）描述 蛋白激酶C（PKC）介导的调节和刺激 通过测定pborbol酯诱导的PKC胞质溶胶的细胞溶解活性， 膜转位和蛋白质底物磷酸化;（3） 为了表征纯化的Gal/GalNAc凝集素的细胞毒性， 包括凝集素结合，对（Ca++）的影响，以及 （4）确定靶细胞死亡的机制， 包括Ca++ DNA降解，磷酸脂酶， 蛋白酶和膜“孔”; 5）表征阿米巴蛋白 参与细胞溶解活性（Cyt蛋白）， 突变和选择缺乏 细胞溶解活性（Cyt-），Cyt-与Cyt+阿米巴的比较 双向凝胶电泳和免疫印迹鉴定 Cyt-突变体中改变的Cyt+蛋白，部分氨基酸 这些蛋白质的测序为合成 寡核苷酸探针筛选E.溶bistolytica Cyt+ cDNA和 基因组文库，并通过测定Cyt蛋白的功能， 从基因组中克隆的全长DNA的序列分析 抗Cyt抗体库及其对阿米巴细胞溶解的影响 活性;和（6）确定免疫原性和疫苗 Cyt蛋白和公认的主要阿米巴抗原的效力 通过用纯化的B- 半乳糖苷酶融合蛋白。 这项研究将扩大我们的 E的知识。 并有助于消除它 人类疾病的原因

The enteric protozoan Entamoeba histolytica causes disease worldwide; parasite adherence and cytolytic events are crucial in pathogenesis of invasive amebiasis. Our objective is to define the biochemical and molecular basis of amebic adherence and cytolytic activities with application for development of a vaccine protective against invasive amebiasis. Parasite in vitro adherence, mediated by a 170 Kdalton galactose/N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable surface lectin, initiates cytolysis of target cells. Amebic cytolytic activity is stimulated by phorbol esters requires amebic phospholipase A activity, an acid pH in endocytic vesicles- lethal increase in target cell free intracellular Ca++ ((CA++)). Candidates for a vaccine include the Gal/GalNAc lectin, cytolytic parasite proteins, and major amebic antigens. The specific aims and methods of this proposal are: (1) to determine the in vivo vaccine efficacy of purified Gal/GalNAc lectin in gerbil! models of intestinal colonization and liver abscess; (2) to describe protein kinase C (PKC) mediated regulation and stimulation of cytolytic activity by determining pborbol esterinduced PKC cytosol- membrane translocation and protein substrate phosphorylation; (3) to characterize the cytotoxicity of the purified Gal/GalNAc lectin, including lectin binding, effects on (Ca++), and uptake by the target cell; (4) to determine the mechanism of target cell death, including the role of Ca++ DNA degradation, phorpholipases, proteases, and membrane "pores"; 5) to characterize amebic proteins which participate in cytolytic activity (Cyt proteins) by mutagenesis and selection of histolytica clones deficient in cytolytic activity (Cyt-), comparison of Cyt- to Cyt+ amoebae by two-dimensional gel electrophoresis and immunoblotting to identify Cyt+ proteins altered in Cyt- mutants, partial amino acid sequencing of such proteins providing information for synthesis of oligonucleotide probes to screen E. bistolytica Cyt+ cDNA and genomic libraries, and determining function(s) of Cyt proteins by sequence analysis of full length DNA cloned from the genomic library and effects of anti-Cyt antibodies on amebic cytolytic activity; and (6) to determine the immunogenicity and vaccine efficacy of Cyt proteins and the major amebic antigens recognized by human immune sera by immunization of gerbils with purified B- galactosidase fusion proteins. This research will expand our knowledge of E. histolytica and contribute to eliminating it as a cause of human disease.