Rab GTPases of Entamoeba histolytica

溶组织内阿米巴的 Rab GTP 酶

• 批准号: 6497294
• 负责人: LESLY A TEMESVARI
• 金额: $ 17.5万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497294
• 关键词: CHO cells; Entamoeba histolytica; SCID mouse; affinity chromatography; guanosinetriphosphatases; immunoelectron microscopy; immunofluorescence technique; parasitism; pathologic process; protein structure function; yeast two hybrid system

DESCRIPTION: The enteric protozoan parasite, Entamoeba histolytica, infects 10 percent of the world's population, leading to 50 million cases of invasive amebiasis and 100,000 deaths annually. Vaccines or chemoprophylactic agents, which can protect residents of endemic areas or travelers, are not available. Infection is acquired by ingestion of the cyst form, followed by excystation of amoeboid trophozoites, which migrate to and colonize the bowel lumen. The endosomal and lysosomal (endo-lysosomal (EL)) system of Entamoeba appears to play a role in its pathogenesis as (I) uptake and digestion of nutrients, (ii) invasion of the intestinal epithelium, and (iii) dissemination and establishment of extra-intestinal infections, including liver abscess, rely on endocytosis and the action of hydrolytic enzymes and pore-forming proteins secreted from the pathogen. Despite its importance, little is known about the molecular factors goveming the Entamoeba EL system, including associated proteins which may regulate EL functions. Such proteins may be candidates for vaccine development. Three genes have been isolated from an E. histo!ytica cDNA library encoding a protein (EhRabl 1) that is 56 percent identical in amino acid sequence to human Rabi 1, a protein (EhRab7) that is 56 percent identical in amino acid sequence to human Rab7, and a protein that is a novel member (EhRabA) of the Rab family of GlPases; Rab GTPases are known to regulate vesicular trafficking. EhRabl 1 is enriched in magnetically purified early endosomes of Entamoeba and EhRabA and EhRab7are enriched in magnetically purified early and late endosomes of Enfamoeba. The subcellular localization of these Rab GTPases suggests that they play a role in EL function of E. histolytica. To test this hypothesis, the following aims are proposed. In Specific Aim I the subcetlular location of the EhRabs will be refined using immunofluorescence and immunoelectron microscopy of Entamoeba trophozoites. In Specific Aim 2 the role of the EhRabs in EL function and pathogenicity will be addressed. Genetically engineered Entamoeba cell lines overexpressing dominant inhibitory and constitutively active versions of the EhRabs will be generated. In addition, Entamoeba cell lines expressing anisense transcripts of the EhRabs (to reduce the cellular levels of the EhRab) will be generated. EL processes will be examined in these strains, including pinocytosis of fluid phase and phagocytosis of large particles, maintenance of intra-endosomal pH, and secretion of hydrolases. In addiion, the virulence of these genetically altered strains will be assessed by measuring their ability to (i) carry out contact-mediated cell lysis of Chinese Hamster Ovary cells, (ii) release pore-forming peptides responsible for the disintegration of host cell membranes (iii) correctly localize an important adherence molecule to the cell surface and, (iv) establish liver abscess in the SCID mouse model. To gain further insight into how EhRabs function, in Specific Aim 3, Entamoeba proteins that interact with the EhRabs will be identified by yeast two-hybrid screening and affinity chromatography. These studies represent the first examination of the role of Rab GiPases of Entamoeba in EL function and pathogenicity and will significantly advance the field by contributing to the understanding of how vesicles and proteins are trafficked in this pathogen.

描述：肠道原生动物寄生虫，Entamoeba Histolictica，感染10 全球人口的百分比，导致5000万例入侵病例 Amebiasis和100,000例死亡。疫苗或化学预防剂， 可以保护流行地区或旅行者的居民。 感染是通过摄入囊肿形式获得的，随后进行了 变形虫滋养体，迁移到肠腔并定居。这 Entamoeba的内体和溶酶体（溶酶体（EL））系统似乎 作为（i）摄取和消化营养物质的发病机理，（ii）发挥作用 肠上皮的入侵，以及（iii）传播和 建立包括肝脓肿在内的肠外感染依靠 内吞作用以及水解酶和孔形成蛋白的作用 从病原体分泌。尽管它很重要，但对 分子因素goveming the entamoeba el系统，包括相关的 可能调节EL功能的蛋白质。这种蛋白质可能是候选 疫苗开发。 已经从编码A的E. histo！ytica cDNA库中分离出三个基因 蛋白质（Ehrabl 1）在氨基酸序列中与人类相同56％ RABI 1，一种蛋白质（Ehrab7），在氨基酸序列中相同56％ 对人类rab7和一种新型成员（ehraba）的蛋白质 Glpases;已知Rab GTPases调节囊泡贩运。 Ehrabl 1 富含Entamoeba和Ehraba的磁纯化的早期内体 Ehrab7are富含磁性纯化的早期和晚期内体 Enfamoeba。这些RAB GTPases的亚细胞定位表明它们 在E. histolictica的EL功能中起作用。为了检验这一假设， 提出了以下目标。在特定目的中 Ehrab将使用免疫荧光和免疫电子显微镜进行完善 entamoeba滋养体。在特定的目标2中，ehrabs在El中的作用 功能和致病性将被解决。基因工程的Entamoeba 过表达优势抑制和组成性活性的细胞系 将生成Ehrabs的版本。另外，entamoeba细胞系 表达ehrabs的动态转录本（降低细胞水平 Ehrab）将生成。 EL过程将在这些菌株中检查， 包括液相的生细胞增多和大颗粒的吞噬作用， 维持内体pH值和水解酶的分泌。加上 这些遗传改变的菌株的毒力将通过测量来评估 他们的能力（i）进行中国仓鼠的接触介导的细胞裂解 卵巢细胞，（ii）释放负责孔形成的肽 宿主细胞膜（III）的分解正确定位了重要的 遵守细胞表面的分子，（iv）在 SCID鼠标模型。为了进一步了解ehrabs的运作方式， AIM 3，与Ehrab相互作用的Entamoeba蛋白将通过 酵母双杂交筛选和亲和力色谱法。这些研究代表 首次检查了Entamoeba的Rab Gipase在EL功能中的作用 和致病性，并将通过贡献 对囊泡和蛋白质如何在这种病原体中运输的理解。