Rab GTPases of Entamoeba histolytica

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50%，导致5000万例侵入性 每年有10万人死于阿米巴病。疫苗或化学预防剂， 可以保护流行区居民或旅行者的疫苗。 感染是通过摄入囊肿形式获得的，随后是 阿米巴滋养体，迁移到肠腔并定殖。的 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，如（I）营养物质的吸收和消化，（ii） 肠上皮的侵袭，和（iii）传播和 包括肝脓肿在内的肠外感染的建立依赖于 内吞作用以及水解酶和成孔蛋白的作用 从病原体中分泌出来尽管它的重要性，很少有人知道， 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。这些蛋白质可能是候选者， 疫苗研发。 从一株E. histo！一个编码 蛋白质（EhRabl 1），其氨基酸序列与人类相同56% Rabi 1，一种氨基酸序列有56%相同的蛋白质（EhRab 7） 人Rab 7，以及Rab家族的新成员（EhRabA）的蛋白质 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的早期内体 和EhRab 7富集在磁性纯化的早期和晚期内体中， 阿米巴原虫这些Rab GTP酶的亚细胞定位表明， 在E.溶组织剂为了验证这一假设， 提出了以下目标。在特定目标I中， 将使用免疫荧光和免疫电子显微镜对EhRab进行精制 内阿米巴滋养体在具体目标2中，EhRabs在EL中的作用 功能和致病性。基因工程内阿米巴 过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的异义转录物（以降低EhRab的细胞水平）。 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和分泌水解酶。此外， 这些基因改变的菌株的毒力将通过测量 它们（i）进行接触介导的中国仓鼠细胞裂解的能力 卵巢细胞，（ii）释放孔形成肽负责 宿主细胞膜的解体（iii）正确定位重要的 粘附分子的细胞表面和，（iv）建立肝脓肿， SCID小鼠模型。为了进一步了解EhRabs的功能，具体来说， 目的3、通过免疫荧光法鉴定与EhRab相互作用的内阿米巴蛋白。 酵母双杂交筛选和亲和层析。这些研究代表了 内阿米巴Rab Gipases在EL功能中作用的初步研究 和致病性，并将大大推进该领域的贡献， 了解囊泡和蛋白质如何在这种病原体中运输。

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.