Cell And Developmental Biology Of Trypanosomatid Parasit

锥虫寄生虫的细胞和发育生物学

• 批准号: 6984869
• 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/6984869
• 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 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 course of parasite growth, differentiation and development. For example, we have identified and characterized the genes that encode several unique L. donovani tartrate-sensitive, histidine, secretory-acid phosphatase enzymes (LdSAcPs). Recently, we showed using combined biochemical and molecular approaches 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. In order to function within their hosts, these LdSAcPs would have to be generally resistant to endogenous host proteases. In that regard, recent results showed that the LdSAcPs were virtually totally resistant to hydrolytic-degradation by various different serine-, thiol- metallo- and mixed proteases. These results further underscore the significance of these enzymes in functionally facilitating parasite survival within their infected hosts. In parallel, we also demonstrated that promastigotes of all pathogenic Leishmania species released very high levels of a soluble, secretory isoform of a zinc-metallo-protease enzyme (GP-63S). Our results suggest that this secretory protease helps facilitate parasite growth in its various host environments. 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 to the foregoing, recently we succeeded in developing an in vitro system for generating and propagating large quantities of infectious forms (i.e. axenic amastigotes) of L. donovani. The availability of such material should now greatly facilitate studies in general concerning the basic cell and molecular biology of this human parasite. 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。在受感染的患者体内，这种生物的各种物种栖息并破坏皮肤或内脏器官（即脾、肝和骨髓）内的巨噬细胞。因此，它们引起溃疡性、不愈合、毁容的恶性皮肤病变（如墨西哥乳杆菌）或退行性和最常见的致命内脏疾病（如多诺瓦氏乳杆菌）。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1200多万患者。我们的研究旨在确定这些生物的病理生理机制。在这方面，研究利什曼原虫和相关的锥虫原虫的基本细胞、分子和发育生物学，以鉴定和表征这些人类病原体生存所必需的寄生虫分子。这些寄生虫如何能够在其昆虫媒介和哺乳动物宿主中生存、获取营养、繁殖和分化，是了解这些生物的基本寄生性质和进化适应的核心问题。由于这些寄生虫直接与宿主相互作用，因此了解其表面膜和其他功能蛋白的组成和功能似乎是必不可少的。为此，鉴定了独特的寄生虫表面膜、分泌酶和调节蛋白，并对其进行了生化表征，以确定它们在这些生物体生存中的功能作用。此外，编码这些蛋白的基因首次被分离和表征，旨在确定它们在寄生虫生长、分化和发育过程中的表达和调控。例如，我们已经鉴定并鉴定了编码几种独特的L. donovani酒石酸敏感、组氨酸、分泌酸磷酸酶（LdSAcPs）的基因。最近，我们使用生化和分子结合的方法表明，该酶家族在所有检测的利什曼原虫致病性物种中功能保守。这表明它们必须在这一重要人类病原体群的所有成员的生长、发育和生存中发挥重要的功能作用。为了在宿主体内发挥作用，这些LdSAcPs通常必须抵抗内源性宿主蛋白酶。在这方面，最近的研究结果表明，LdSAcPs几乎完全抵抗各种不同的丝氨酸、硫醇-金属和混合蛋白酶的水解降解。这些结果进一步强调了这些酶在功能上促进寄生虫在其感染宿主内存活的重要性。同时，我们还证明了所有致病性利什曼原虫的原鞭毛菌释放出非常高水平的锌金属蛋白酶（GP-63S）的可溶性分泌异构体。我们的研究结果表明，这种分泌蛋白酶有助于促进寄生虫在各种宿主环境中的生长。在其他生化和分子研究中，我们证明了一种独特的L. donovani arf1蛋白的功能是维持反式高尔基池网络的结构完整性，并促进分泌蛋白和表面膜蛋白通过这些寄生虫的高尔基腔室的适当运输和运输。此外，利用位点特异性靶向突变，我们发现ldarf1蛋白对这些生物的生存至关重要。除此之外，最近我们成功地开发了一种体外系统，用于产生和繁殖大量的多诺瓦氏乳杆菌感染形式（即无性系无性系）。这些材料的可用性现在将极大地促进有关这种人类寄生虫的基本细胞和分子生物学的一般研究。总的来说，我们最近和正在进行的研究结果继续为理解这些寄生虫独特的病理生理提供相关和重要的信息。此外，这些研究对于证明特定/独特的寄生虫酶和调节蛋白是否是1)设计新的化疗药物，2)开发新的诊断工具和/或3)作为针对这些人类病原体的潜在疫苗有用的逻辑靶点具有实际意义。