Rab GTPases of Entamoeba histolytica

描述：肠道原虫寄生虫，溶组织内阿米巴，感染10 占世界人口的10%，导致5000万例侵入性 阿米巴病每年造成100,000人死亡。疫苗或化学避孕药， 没有能够保护疫区居民或旅行者的设备。 感染是通过摄入囊状病毒，然后排出尿液 阿米巴滋养体，迁徙到肠腔并在肠腔内定居。这个 内阿米巴的内体和溶酶体(EL)系统似乎 在其发病机制中发挥作用：(I)吸收和消化营养物质，(Ii) 侵袭肠上皮，以及(Iii)播散和 肠外感染的建立，包括肝脓肿，依赖于 内吞作用与水解酶和造孔蛋白的作用 从病原体分泌出来的。尽管它很重要，但人们对它知之甚少 调控内阿米巴EL系统的分子因素，包括相关的 可能调节EL功能的蛋白质。这些蛋白质可能是 疫苗研发。 从编码组埃希氏菌的c DNA文库中分离到3个基因 氨基酸序列与人类有56%同源性的蛋白质(EhRabl 1 Rabi 1，氨基酸序列有56%同源性的蛋白质(EhRab7) 与人Rab7结合，以及Rab家族的一个新成员(EhRabA) 已知Rab GTP酶调节囊泡运输。EhRabl 1 富含磁性纯化的内阿米巴和EhRabA的早期内涵体 和EhRab7在磁提纯的早期和晚期内吞体内富含 阿法米巴。这些Rab GTP酶的亚细胞定位表明它们 在溶组织乳杆菌的EL功能中发挥作用。为了检验这一假设， 提出了以下目标。在特定的目标I中，中央以下的位置 EhRabs将使用免疫荧光和免疫电子显微镜进行提纯 内阿米巴滋养体。在特定目标2中，EhRabs在EL中的作用 将讨论其功能和致病性。转基因内阿米巴 过表达显性抑制性和结构性活性的细胞系 将生成不同版本的EhRabs。此外，内阿米巴细胞株 表达EhRabs的八角转录本(以降低细胞水平 EhRab)将被生成。电致发光过程将在这些菌株中进行检测， 包括液体的吞噬和大颗粒的吞噬， 维持内含体内的pH值，并分泌水解酶。此外， 这些转基因菌株的毒力将通过测量 它们(I)对中国仓鼠进行接触介导的细胞裂解 卵巢细胞，(Ii)释放造孔肽，负责 宿主细胞膜的解体(III)正确定位重要的 黏附分子到细胞表面，(Iv)建立肝脓肿。 SCID小鼠模型。要进一步深入了解EhRabs的工作原理，尤其是 目的3，与EhRabs相互作用的内阿米巴蛋白将通过以下方式鉴定 酵母双杂交筛选及亲和层析。这些研究代表了 内阿米巴Rab-GiPase在EL功能中作用的初步研究 和致病性，并将显著促进该领域的发展 对囊泡和蛋白质如何在这种病原体中运输的理解。

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.