CELL AND DEVELOPMENTAL BIOLOGY OF TRYPANOSOMATID PARASITES

利什曼原虫是人类特有的细胞内原生动物病原体。在受感染的患者体内，这种微生物的各种物种栖息并破坏皮肤或内脏(即脾、肝脏和骨髓)内的巨噬细胞。因此，它们要么导致溃烂、无法愈合、毁容的恶性皮肤损害(例如墨西哥乳杆菌)，要么导致退行性和最常见的致命内脏疾病(例如多诺瓦尼乳杆菌)。根据世界卫生组织的估计，这些疾病每年在全球热带和新热带地区折磨着1200万以上的患者。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，对利什曼原虫和相关锥虫原虫的基本细胞、分子和发育生物学进行了研究，以鉴定和表征对这些人类病原体的生存至关重要的寄生虫分子。这些寄生虫如何能够在它们的昆虫媒介和哺乳动物宿主中生存、获取营养、繁殖和分化，是理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，因此了解它们表面膜的组成和功能似乎是必不可少的。为此，确定了独特的寄生虫表膜酶和调节蛋白，并对其进行了生化特征分析，以确定它们在这些生物生存中的功能作用。此外，编码这些蛋白的基因正在首次被分离和鉴定，以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L、墨西哥锥虫、伊凡斯锥虫和露斑锥虫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置。构建了这些3‘NT/NU基因的各种突变表达载体和GFP嵌合体，并用它们来鉴定这些蛋白的特定区域，这些区域负责它们的膜锚定、酶活性以及它们正确地靶向内质网和寄生虫细胞表面。这些3‘Nt/Nu突变的表达构建体中的几个也被证明对以可溶性形式生产和纯化这些正常膜结合的寄生虫酶很有用。后者已被证明对识别这些酶的新的合成核苷酸抑制剂很有用(与R.Johnson合作)。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，这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了3‘NT/NU活性的完全消融。类似的利什曼反义突变体将在沙蝇和小鼠身上进行生存测试。类似的研究正在进行中，研究对象是独特的利什曼表面膜酸性磷酸酶。在与H.Nakhasi的其他研究中，我们证明了杜氏利什曼氏菌钙网蛋白(LD-Cal)蛋白作为凝集素样伴蛋白在寄生虫内质网中促进糖蛋白(例如利什曼分泌酸性磷酸酶)的正确折叠和加工。目前正在测试LD-Cal的反义和过表达结构，以进一步阐明这种表面上必不可少的蛋白质在寄生虫生长和生存中的作用。这些研究的结果应该证明，这些不同基因编码的蛋白质实际上是否对这些病原体在昆虫媒介和/或哺乳动物宿主中的生存至关重要。因此，目前的研究旨在确定这些生物体的内在需求并确定它们的关键性质。我们最近和正在进行的研究结果继续为理解这些生物的独特病理生理学提供相关信息。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，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的合乎逻辑的目标具有实际意义。

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.