CELL AND DEVELOPMENTAL BIOLOGY OF TRYPANOSOMATID PARASITES

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。在过去的一年里，我们的研究已经阐明了来自L .的独特的锥虫表面膜3'-核苷酸酶/核酸酶（3' nt /Nu）家族的几个不同成员的基因结构和染色体位点。donovani， L. mexicana（与P.A. Bates合作），L. major（与A. Ivans合作）和Crithidia luciliae。这些3'NT/Nu基因的各种突变表达构建体和gfp嵌合体被用来鉴定这些蛋白质的特定区域，这些区域负责它们的膜锚定，酶活性以及它们对内质网和寄生虫细胞表面的适当靶向。这些3'NT/Nu突变的表达构建体也被证明对这些通常以可溶性形式结合的膜结合寄生虫酶的生产和纯化是有用的。后者已被证明对鉴定这些酶的新的合成核苷酸抑制剂有用（与R. Johnson合作）。删除这些3'NT/Nu基因的尝试一致导致它们在寄生虫基因组的其他地方重复，这表明它们可能对寄生虫的生存至关重要。相反，过表达一个反义结构会导致C. luciliae中3'NT/Nu活性完全消失。类似的利什曼反义突变体将在白蛉和小鼠身上进行生存能力测试。对独特的利什曼表面膜酸性磷酸酶的类似研究正在进行中。在其他研究中（与H. Nakhasi），我们证明了L. donovani calreticulin （Ld-Cal）蛋白在促进寄生虫内质网中糖蛋白（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。目前正在测试Ld-Cal的反义和过表达结构，以进一步阐明这种明显必需的蛋白质在寄生虫生长和存活中的作用。这些研究的结果应该证明由这些不同基因编码的蛋白质是否实际上是这些病原体在其昆虫媒介和/或哺乳动物宿主内生存所必需的。因此，目前的研究旨在确定这些生物的内在需求和确定它们的临界性质。我们最近和正在进行的研究结果继续为理解这些生物体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. 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 (3'NT/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 3'NT/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 3'NT/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 3'NT/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 3'NT/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 parasite's 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.