Cell And Developmental Biology Of Trypanosomatid Parasit

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，几种多诺多菌酒石酸盐敏感、组氨酸分泌- (LdSAcPs)基因和一种独特的抗酒石酸盐表面膜- (LdMAcP)酸性磷酸酶基因被用作分子研究的探针。生化和分子结合的研究结果表明，该酶家族在所有被检测的致病性利什曼原虫以及其他与人类有较远亲缘关系的锥虫寄生虫中都具有功能保守性。这些ldsacp和LdMAcP同源物在所有致病性利什曼原虫中的保守性。提示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。与P.A. Bates合作，我们证明了独特的锥虫表面膜酶，一种3'-核苷酸酶/核酸酶在墨西哥利什曼原虫中是保守的，并且它在这种寄生虫中是差异和发育表达的。此外，我们的分子分析表明，这种利什曼酶是唯一的表面膜锚定的i类外显核酸酶家族成员。我们的综合研究结果表明，3′nt /NU在所有致病性利什曼原虫中普遍存在，并且具有结构上的保守性。在平行的生化和分子研究中，我们最近在L. donovani中发现了一种新的硫醇敏感的表面膜外核糖核酸酶。这一发现提供了另一种独特的假定必需的表面膜核苷挽救酶作为潜在的化疗靶点在这种病原体。在其他研究中，我们证明了L. donovani calreticulin （LdCal）蛋白在促进寄生虫内质网内糖蛋白（例如利什曼分泌酸性磷酸酶）的适当折叠和加工中发挥凝集素样伴侣的作用。利用LdCal的反义和过表达结构获得的结果表明，这种伴侣蛋白对寄生虫的生长和生存至关重要。我们最近的研究结果继续为理解这些生物独特的病理生理提供相关信息。此外，这些研究对于证明这些寄生虫酶和调节蛋白是否是1)设计新的化疗药物的逻辑靶点，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具有实际意义。

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. For example, the genes for several L. donovani tartrate-sensitive, histidine, secretory- (LdSAcPs) and a unique tartrate-resistant, surface membrane- (LdMAcP) acid phosphatases were used as probes in molecular studies. Results of combined biochemical and molecular studies showed that this family of enzymes was functionally conserved among all pathogenic leishmanial species examined as well as, other more distantly related trypanosomatid parasites of humans. The conservation of such LdSAcPs and LdMAcP homologs amongst all pathogenic Leishmania sps. suggests that they must play significant functional roles in the growth, development and survival of all members of this important group of human pathogens. In collaboration with P.A. Bates, we demonstrated that the unique trypanosomatid surface membrane enzyme, a 3'-nucleotidase/nuclease was conserved in Leishmania mexicana and that it was differentially and developmentally expressed in this parasite. Further, our molecular analyses showed that this leishmanial enzyme is unique being the only surface membrane-anchored member of the Class-I family of exo-nucleases. Our combined results of these studies have demonstrated both the ubiquity and structural conservation of the 3'NT/NU among all pathogenic Leishmania parasites. In parallel biochemical and molecular studies, we recently identified in a new thiol-sensitive, surface membrane, exo-ribonuclease in L. donovani. This finding provides yet another unique putatively essential surface membrane nucleoside-salvage enzyme as potential chemotherapeutic target in this pathogen. In other studies, we demonstrated that the L. donovani calreticulin (LdCal) 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 parasite's endoplasmic reticulum. Results obtained from the use of both anti-sense and over-expression constructs of the LdCal demonstrated that this chaperone-protein is essential for parasite growth and survival. Results of our recent 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.