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。在过去的一年中，我们的研究已经阐明了杜氏锥虫、杜氏锥虫和杜氏锥虫的表面膜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。墨西哥（与P.A. Bates），L.大（A。Ivans）和Crithidia luciliae。制备了这些3 'NT/Nu基因的各种突变表达构建体和GFP嵌合体，并用于鉴定这些蛋白质的负责其膜锚定、酶活性和其适当靶向内质网和寄生虫细胞表面的特定区域。这些3 'NT/Nu突变的表达构建体中的几种也证明可用于以可溶形式产生和纯化这些正常膜结合的寄生虫酶。后者已被证明可用于鉴定这些酶的新的合成核苷酸抑制剂（与R。约翰逊）。删除这些3 'NT/Nu基因的尝试一致地导致它们在寄生虫基因组的其他地方复制，表明它们可能是寄生虫生存所必需的。相反，反义构建体的过表达导致C. Luciliae。将测试类似的利什曼原虫反义突变体在白蛉和小鼠中的生存力。类似的研究正在进行中的独特利什曼原虫表面膜酸性磷酸酶。在其他研究中（与H. Nakhasi），证明了L. donovani钙网蛋白（Ld-Cal）蛋白作为凝集素样伴侣蛋白在促进糖蛋白的正确折叠和加工中起作用（例如，利什曼原虫分泌型酸性磷酸酶）。Ld-Cal的反义和过表达构建体都正在进行测试，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。这些研究的结果应证明这些不同的基因编码的蛋白质是否，事实上，这些病原体在其昆虫载体和/或哺乳动物宿主的生存是必不可少的。因此，目前的研究旨在确定这些生物的内在需求，并确定其关键性质。我们最近和正在进行的研究结果继续提供相关信息，了解这些生物体的独特病理生理学。此外，这些研究对于证明这样的寄生虫酶和调节蛋白是否是1）新的化疗药物的设计、2）新的诊断工具的开发和/或3）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相关性。

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.