Cell And Developmental Biology Of Trypanosomatid Parasit

锥虫寄生虫的细胞和发育生物学

• 批准号: 7189407
• 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/7189407
• 关键词: Leishmania; Trypanosoma; biochemical evolution; enzyme activity; gene expression; genetic mapping; intracellular parasitism; intracellular transport; macrophage; membrane proteins; metalloendopeptidases; microorganism culture; microorganism growth; microorganism mass culture; microorganism reproduction; northern blottings; parasite infection mechanism; protein structure function; protein transport; protozoal antigen; protozoal genetics; recombinant proteins; southern blotting; transfection; western blottings

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 membrane and secretory enzymes and other functional proteins seems essential. To that end, unique parasite surface membrane, secreted 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 the course of parasite growth, differentiation and development. For example, previously we have identified and characterized the genes that encode several unique L. donovani tartrate-sensitive, histidine, secretory-acid phosphatase enzymes (LdSAcPs). Using combined biochemical and molecular approaches we also showed that this family of enzymes was functionally conserved among all pathogenic species of Leishmania examined. This suggested that they must play significant functional roles in the growth, development and survival of all members of this important group of human pathogens. Further, we recently demonstrated that pathogenic Leishmania mexicana parasites released high levels of a soluble, secretory chitinase enzyme activity (LmCht1). Using a combined biochemical and molecular approach, we identified and characterized the gene encoding this unique enzyme. We showed that transfected parasites overexpressing this enzyme survived better in monocyte-derived macrophages in vitro and produced significantly larger lesions in mice than controls. Our results suggest that this parasite secretory enzyme functionally facilitates parasite survival and growth within both their sandfly vector and mammalian hosts. In other biochemical and molecular studies, we demonstrated that a unique L. donovani ARF1-protein functions to maintain the structural integrity of the trans-Golgi cisternal network and facilitates the proper trafficking and transit of both secretory and surface membrane proteins through the Golgi compartment in these parasites. Further, using site specific targeted-mutations, we showed that the LdARF1-protein was essential for the survival of these organisms. In addition, in collaborative studies we succeeded in identifying and characterizing a gene encoding a specific blood-induced chitinolytic enzyme system in the midgut of P. papatasi and L. longipalpis which are the sandfly vectors for Leishmania major and L. (donovani) chagasi, respectively. A recombinant expressed protein from this gene demonstrated high chitinolytic enzyme activity against a variety of chitin substrates. This is the first chitinase gene to be described from these important vectors for human parasites. Cumulatively, the results of our recent and ongoing studies continue to provide pertinent and significant information toward understanding the unique pathophysiology of these parasites. In addition, these studies are of practical relevance toward demonstrating whether specific /unique 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.

利什曼原虫是人类的专性细胞内原生动物病原体。在感染的患者体内，该生物体的各种物种栖息并破坏皮肤或内脏内的巨噬细胞（即，脾、肝和骨髓）。因此，它们引起溃疡性、不愈合、毁容的恶性皮肤病变（例如L. mexicana）或退行性和最常见的致命性内脏疾病（例如L. Donovani）。据世界卫生组织估计，这些疾病每年在全世界热带和新热带地区折磨着1 200多万患者。我们的研究旨在确定这些生物体的病理生理学机制。在这方面，利什曼原虫和相关锥虫原虫的基本细胞，分子和发育生物学的研究，以确定和表征这些人类病原体的生存所必需的寄生虫分子。这些寄生虫如何能够生存，获取营养，繁殖和分化的昆虫载体和哺乳动物宿主是核心问题，了解这些生物体的基本寄生性质和进化适应。由于这些寄生虫直接与其宿主相互作用，因此了解其表面膜和分泌酶以及其他功能蛋白的组成和功能似乎至关重要。为此，独特的寄生虫表面膜，分泌的酶和调节蛋白的鉴定和生化特性，以确定其在这些生物体的生存功能的作用。此外，编码这些蛋白质的基因正在首次被分离和表征，以确定它们在寄生虫生长、分化和发育过程中的表达和调节。例如，以前我们已经鉴定和表征了编码几种独特的L. donovani酒石酸盐敏感的组氨酸分泌型酸性磷酸酶（LdSAcPs）。使用生物化学和分子生物学相结合的方法，我们还表明，这个家庭的酶是功能保守的所有致病性种利什曼原虫检查。这表明它们必须在这一重要的人类病原体组的所有成员的生长、发育和生存中发挥重要的功能作用。此外，我们最近证明，致病性墨西哥利什曼原虫寄生虫释放高水平的可溶性分泌几丁质酶活性（LmCht 1）。使用生物化学和分子生物学相结合的方法，我们确定并表征了编码这种独特酶的基因。我们发现，转染过表达这种酶的寄生虫在体外单核细胞衍生的巨噬细胞中存活得更好，并在小鼠中产生比对照组明显更大的病变。我们的研究结果表明，这种寄生虫分泌酶功能上有利于寄生虫的生存和生长在他们的白蛉载体和哺乳动物宿主。在其他生化和分子生物学研究中，我们证明了一个独特的L。donovani ARF 1蛋白的功能是维持trans-Golgi池网络的结构完整性，并促进分泌和表面膜蛋白通过这些寄生虫中的高尔基体区室的适当运输和转运。此外，使用位点特异性靶向突变，我们表明LdARF 1蛋白对这些生物体的生存至关重要。此外，在合作研究中，我们成功地确定和表征了一个基因编码的特定血液诱导几丁质酶系统中的P。papatasi和L。longipalpis是主要利什曼原虫和L.（donovani）chagasi，分别。从该基因的重组表达蛋白表现出高几丁质酶活性对各种几丁质底物。这是第一个从这些重要的人类寄生虫载体中描述的几丁质酶基因。累积起来，我们最近和正在进行的研究的结果继续提供相关的和重要的信息，了解这些寄生虫的独特病理生理学。此外，这些研究对于证明特异性/独特的寄生虫酶和调节蛋白是否是1）新化疗药物的设计，2）新诊断工具的开发和/或3）可用作针对这些人类病原体的潜在疫苗的逻辑靶标具有实际意义。