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，溶组织内阿米巴，感染 10 占世界人口的 5000 万例，导致 5000 万例侵入性感染病例 阿米巴病每年导致 10 万人死亡。疫苗或化学预防剂， 无法保护流行地区的居民或旅行者。 通过摄入包囊形式，然后排出包囊而获得感染。 变形虫滋养体，迁移到肠腔并定居。这 内阿米巴的内体和溶酶体（内溶酶体（EL））系统似乎 在其发病机制中发挥作用，因为（I）营养物质的吸收和消化，（ii） 肠上皮的侵袭，以及（iii）传播和 肠外感染（包括肝脓肿）的建立依赖于 内吞作用以及水解酶和成孔蛋白的作用 由病原体分泌。尽管它很重要，但人们对它知之甚少 控制内阿米巴 EL 系统的分子因素，包括相关的 可能调节 EL 功能的蛋白质。此类蛋白质可能是候选蛋白 疫苗开发。 已从溶组织内阿米巴 cDNA 文库中分离出三个基因，编码 蛋白质 (EhRabl 1) 的氨基酸序列与人类有 56% 相同 Rabi 1，一种氨基酸序列 56% 相同的蛋白质 (EhRab7) 人类 Rab7，以及 Rab 家族新成员 (EhRabA) 的蛋白质 GLP酶； Rab GTPases 已知可调节囊泡运输。埃拉布尔1号 富含磁纯化的内阿米巴和 EhRabA 早期内体 和 EhRab7 富含磁性纯化的早期和晚期内体 恩法阿米巴。这些 Rab GTPases 的亚细胞定位表明它们 在溶组织内阿米巴的 EL 功能中发挥作用。为了检验这个假设， 提出了以下目标。在特定目标 I 的细胞下位置 EhRab 将使用免疫荧光和免疫电子显微镜进行精制 内阿米巴滋养体。在具体目标 2 中，EhRab 在 EL 中的作用 功能和致病性将得到解决。基因改造的内阿米巴 过度表达显性抑制和组成型活性的细胞系 将生成 EhRab 的版本。此外，内阿米巴细胞系 表达 EhRab 的茴香转录本（以降低细胞水平 EhRab）将被生成。将在这些菌株中检查 EL 过程， 包括液相的胞饮作用和大颗粒的吞噬作用， 维持内体 pH 值和水解酶的分泌。此外， 这些转基因菌株的毒力将通过测量来评估 他们的能力（i）对中国仓鼠进行接触介导的细胞裂解 卵巢细胞，(ii) 释放成孔肽，负责 宿主细胞膜的崩解 (iii) 正确定位重要的 将分子粘附到细胞表面，并且（iv）在细胞内建立肝脓肿 SCID小鼠模型。为了进一步了解 EhRabs 的功能，请参阅 Specific 目标 3，与 EhRab 相互作用的内阿米巴蛋白将被鉴定 酵母双杂交筛选和亲和层析。这些研究代表 首次检查内阿米巴 Rab GiPases 在 EL 功能中的作用 和致病性，并将通过贡献显着推进该领域 了解囊泡和蛋白质如何在这种病原体中运输。