CELL AND DEVELOPMENTAL BIOLOGY OF TRYPANOSOMATID PARASITES

利什曼原虫是专性细胞内原生动物 人类的病原体在受感染的患者中， 这种微生物栖息并破坏皮肤内的巨噬细胞， 内部器官（即，脾、肝和骨髓）。所以他们 导致溃疡性、不愈合、毁容的皮肤损伤 (e.g., L.墨西哥）或退化和最常见的致命内脏 疾病（例如，L. Donovani）。这些疾病折磨着1200多万人 全球热带和新热带地区的患者。我们的研究是 旨在确定病理生理学中涉及的机制， 这个有机体。在这方面，基本的细胞、分子和 利什曼原虫及其相关锥虫的发育生物学 原生动物的研究是为了识别和表征 这些寄生虫分子对于它们的生存至关重要， 人类病原体这些寄生虫是如何生存的 营养素，在其昆虫载体内繁殖和分化， 哺乳动物宿主是理解 这些生物体的寄生性和进化适应性。 由于这些寄生虫直接与宿主相互作用， 它们表面膜的组成和功能似乎 具有本质意义为此，独特的寄生虫表面膜酶 和调节蛋白被鉴定出来， 以确定其在生存中的功能作用， 这些有机体。此外，编码这些蛋白质的基因是 第一次被分离出来， 它们在寄生虫生长过程中的表达和调节， 分化与发展。在过去的一年里，我们的研究 已经阐明了几个基因结构和染色体位点 独特的锥虫表面膜的不同成员 三个？来自杜氏乳杆菌（L .donovani）、L. mexicana及 光皮蛛这些基因的各种突变表达构建体 基因被制造出来，并用来识别这些基因的特定区域。 蛋白质负责其酶活性和 正确地瞄准寄生虫细胞表面。几个这样 突变的表达构建体也被证明可用于 这些化合物实验室规模生产和纯化通常 可溶形式的膜结合寄生虫酶。类似 利什曼原虫的伴侣蛋白 蛋白、L d-钙网蛋白和表面膜酸性磷酸酶。 此外，涉及这些基因的靶向缺失的实验 和功能无效突变寄生虫的生产是在 中求进工作总这种突变寄生虫的生存能力将在 原地。这些研究的结果应表明， 事实上，由这些基因编码的蛋白质是 这些病原体在其昆虫载体内的存活，和/或 哺乳动物宿主因此，目前的研究旨在定义 这些生物体的内在需求，并确定其关键 自然我们最近和正在进行的研究结果继续 提供相关信息，以了解独特的 这些生物体的病理生理学。此外，这些研究还 对于证明这种寄生虫 酶和调节蛋白是1）设计的逻辑目标， 新的化疗药物和2）开发新的 诊断工具。它们也与确定是否 它们可用作对抗这些人类病原体的潜在疫苗。

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 cutaneous lesions (e.g., L. mexicana) or degenerative and most often fatal visceral disease (e.g., L. donovani ). These diseases afflict over 12 million patients in the Tropics and Neo-tropics worldwide. Our studies are aimed at defining the mechanisms involved in the pathophysiology of this organism. 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 family from L .donovani, L. mexicana and Crithidia luciliae. Various mutated-expression constructs of these genes were made and used to identify the specific regions of these proteins responsible for their enzymatic activities and for their proper targeting to the parasite cell surface. Several of these mutated expression-constructs have also proven useful for the laboratory-scale production and purification of these normally membrane-bound parasite enzymes in a soluble form. Similar studies are in progress with the unique leishmanial chaperone protein, Ld-calreticulin and surface membrane acid phosphatase. Further, experiments involving the targeted deletion of these genes and the production of functionally null mutant parasites are in progress. The viability of such mutant parasites will be tested in situ. Results of such studies should demonstrate whether the proteins encoded by these 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 and 2) the development of new diagnostic tools. They are also relevant for determining whether they are useful as potential vaccines against these human pathogens.