Rab GTPases of Entamoeba histolytica

溶组织内阿米巴的 Rab GTP 酶

• 批准号: 6628012
• 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/6628012
• 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，溶组织内阿米巴原虫，感染10