CELL AND DEVELOPMENTAL BIOLOGY OF TRYPANOSOMATID PARASITES

锥虫寄生虫的细胞和发育生物学

• 批准号: 6288799
• 负责人: DENNIS DWYER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6288799
• 关键词: Leishmania; Trypanosoma; calreticulin; developmental genetics; enzyme activity; gene deletion mutation; gene expression; genetic mapping; host organism interaction; human tissue; in situ hybridization; intracellular parasitism; microorganism culture; microorganism genetics; microorganism growth; protein structure; transfection

Leishmania are obligate intracellular protozoan pathogens of humans. Within infected patients, various species of this organism inhabit and destroy macrophages within the skin or internal organs (i.e., spleen, liver and bone marrow). Thus, they cause either ulcerative, non- healing, disfiguring malignant skin lesions (e.g. L. mexicana) or degenerative and most often fatal visceral disease (e.g. L. donovani). According to World Health Organization estimates, these diseases afflict over 12 million patients annually in the Tropics and Neo- tropics worldwide. Our studies are aimed at defining the mechanisms involved in the pathophysiology of these organisms. In that regard, the basic cell, molecular and developmental biology of Leishmania and related trypanosomatid protozoa are investigated toward identifying and characterizing parasite molecules which are essential for the survival of these human pathogens. How these parasites are able to survive, access nutrients, multiply and differentiate within their insect vector and mammalian hosts are questions central to understanding the basic parasitic nature and evolutionary adaptations of these organisms. Since these parasites interact directly with their hosts, knowledge of the composition and functions of their surface membranes seems essential. To that end, unique parasite surface membrane enzymes and regulatory proteins are identified and biochemically characterized to determine their functional roles in the survival of these organisms. Further, the genes encoding such proteins are being isolated and characterized for the first time, toward defining their expression and regulation during course of parasite growth, differentiation and development. During the past year, our studies have elucidated the gene structure and chromosomal loci of several different members of the unique trypanosomatid surface membrane 3-nucleotidase/nuclease (3NT/Nu) family from L .donovani, L. mexicana (with P.A. Bates), L. major (with A. Ivans) and Crithidia luciliae. Various mutated-expression constructs and GFP-chimeras of these 3NT/Nu genes were made and used to identify the specific regions of these proteins responsible for their membrane anchors, enzymatic activities and for their proper targeting to the endoplasmic reticulum and parasite cell surface. Several of these 3NT/Nu mutated expression-constructs also proved useful for the production and purification of these normally membrane-bound parasite enzymes in a soluble form. The latter have proven useful for identifying new synthetic nucleotide inhibitors of these enzymes (collaboration with R. Johnson). Attempts to delete these 3NT/Nu genes has uniformly resulted in their duplication elsewhere in the parasite genome indicating that they are probably essential for parasite survival. In contrast, overexpression of an anti-sense construct resulted in the total ablation of 3NT/Nu activity in C. luciliae. Similar leishmanial anti-sense mutants will be tested for their viability both in sandflies and mice. Similar studies are in progress with the unique leishmanial surface membrane acid phosphatase. In other studies (with H. Nakhasi), we demonstrated that the L. donovani calreticulin (Ld-Cal) protein functions as a lectin-like chaperone in facilitating the proper folding and processing of glycoproteins (e.g., the leishmanial secretory acid phosphatase) within the parasites endoplasmic reticulum. Both anti-sense and overexpression constructs of the Ld-Cal are being tested to further elucidate the roles of this apparently essential protein in parasite growth and survival. Results of these studies should demonstrate whether the proteins encoded by these various genes are, in fact, essential for the survival of these pathogens within their insect-vector and/or mammalian hosts. Thus, the current studies are aimed at defining the intrinsic needs of these organisms and determining their critical nature. Results of our recent and ongoing studies continue to provide pertinent information toward understanding the unique pathophysiology of these organisms. In addition, these studies are of practical relevance toward demonstrating whether such parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs, 2) the development of new diagnostic tools and/or 3) useful as potential vaccines against these human pathogens.

利什曼原虫是人类专性细胞内原生动物病原体。 在受感染的患者体内，存在着不同种类的这种生物体并 破坏皮肤或内脏器官（即脾脏、 肝脏和骨髓）。因此，它们会导致溃疡性、非 愈合、毁容恶性皮肤损伤（例如 L. mexicana）或 退行性且最常致命的内脏疾病（例如杜诺瓦尼乳杆菌）。 据世界卫生组织估计，这些疾病 热带和新热带地区每年有超过 1200 万患者受其折磨 全世界的热带地区。我们的研究旨在确定机制 参与这些生物体的病理生理学。在这方面， 利什曼原虫的基础细胞、分子和发育生物学 研究相关的锥虫原生动物以识别和 表征对生存至关重要的寄生虫分子 这些人类病原体。这些寄生虫如何能够生存， 获取营养，在其昆虫载体内繁殖和分化 和哺乳动物宿主是理解基本知识的核心问题 这些生物体的寄生性质和进化适应。自从 这些寄生虫直接与其宿主相互作用，了解 它们表面膜的组成和功能似乎至关重要。 为此，独特的寄生虫表面膜酶和调节 蛋白质被鉴定和生化表征以确定 它们在这些生物体的生存中发挥的功能作用。此外， 编码此类蛋白质的基因正在被分离和表征 第一次，旨在定义它们的表达和调节 寄生虫生长、分化和发育的过程。期间 去年，我们的研究阐明了基因结构和 几个不同成员的染色体位点是独一无二的 锥虫表面膜 3-核苷酸酶/核酸酶 (3NT/Nu) 家族 来自 L.donovani、L.mexicana（与 P.A. Bates）、L.major（与 A. Ivans）和 Crithidia luciliae。各种突变表达结构 制作这些 3NT/Nu 基因的 GFP 嵌合体并用于鉴定 这些蛋白质负责其膜的特定区域 锚、酶活性及其正确靶向 内质网和寄生虫细胞表面。其中几个 3NT/Nu 突变表达结构也被证明对 这些通常膜结合的寄生虫的产生和纯化 可溶形式的酶。后者已被证明有用 鉴定这些酶的新合成核苷酸抑制剂 （与 R. 约翰逊合作）。尝试删除这些 3NT/Nu 基因 一致导致它们在寄生虫的其他地方重复 基因组表明它们可能对寄生虫至关重要 生存。相反，反义构建体的过度表达 导致 C. luciliae 中 3NT/Nu 活性的完全消除。 类似的利什曼原虫反义突变体将进行测试 白蛉和小鼠的生存能力。类似的研究正在进行中 具有独特的利什曼原虫表面膜酸性磷酸酶。在其他方面 研究（与 H. Nakhasi），我们证明了 L. donovani 钙网蛋白 (Ld-Cal) 蛋白作为凝集素样伴侣在 促进糖蛋白的正确折叠和加工（例如， 寄生虫内的利什曼原虫分泌酸性磷酸酶） 内质网。反义和过表达构建体 Ld-Cal 正在接受测试，以进一步阐明其作用 显然是寄生虫生长和生存所必需的蛋白质。结果 这些研究应该证明编码的蛋白质是否 事实上，这些不同的基因对于这些物种的生存至关重要。 昆虫媒介和/或哺乳动物宿主内的病原体。因此， 目前的研究旨在确定这些人的内在需求 有机体并确定其临界性质。我们最近的结果 正在进行的研究继续提供相关信息 了解这些生物体独特的病理生理学。在 此外，这些研究对于证明 这些寄生虫酶和调节蛋白是否符合逻辑 1）新化疗药物的设计，2） 开发新的诊断工具和/或 3) 具有潜在用途 针对这些人类病原体的疫苗。