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，全球热带和新热带地区每年有超过1200万患者患有这些疾病。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，因此了解其表面膜和其他功能蛋白的组成和功能似乎至关重要。为此，独特的寄生虫表面膜，分泌的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。例如，我们已经鉴定和表征了编码几种独特的L. donovani酒石酸盐敏感的组氨酸分泌型酸性磷酸酶（LdSAcPs）。最近，我们使用生物化学和分子生物学相结合的方法表明，这个家族的酶在所有致病性利什曼原虫检查物种的功能保守。这表明它们必须在这一重要的人类病原体组的所有成员的生长、发育和生存中发挥重要的功能作用。为了在其宿主内发挥功能，这些LdSAcP通常必须对内源性宿主蛋白酶具有抗性。在这方面，最近的结果表明，LdSAcP几乎完全耐受各种不同的丝氨酸-、巯基-金属-和混合蛋白酶的水解降解。这些结果进一步强调了这些酶在功能上促进寄生虫在其感染宿主中存活的重要性。与此同时，我们还证明了所有致病性利什曼原虫物种的前鞭毛体释放非常高水平的可溶性分泌型锌金属蛋白酶（GP-63 S）。我们的研究结果表明，这种分泌性蛋白酶有助于促进寄生虫在其各种宿主环境中的生长。在其他生化和分子生物学研究中，我们证明了一个独特的L。donovani ARF 1蛋白的功能是维持trans-Golgi池网络的结构完整性，并促进分泌和表面膜蛋白通过这些寄生虫中的高尔基体区室的适当运输和转运。此外，使用位点特异性靶向突变，我们表明LdARF 1蛋白对这些生物体的生存至关重要。除上述外，最近我们成功地开发了一种体外系统，用于产生和繁殖大量的感染性形式（即无鞭毛体）的L。donovani。这种材料的可用性现在应该大大促进了关于这种人类寄生虫的基本细胞和分子生物学的一般研究。累积起来，我们最近和正在进行的研究的结果继续提供相关的和重要的信息，了解这些寄生虫的独特病理生理学。此外，这些研究对于证明特异性/独特的寄生虫酶和调节蛋白是否是1）新化疗药物的设计，2）新诊断工具的开发和/或3）可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 membrane and other functional proteins seems essential. To that end, unique parasite surface membrane, secreted 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, we have identified and characterized the genes that encode several unique L. donovani tartrate-sensitive, histidine, secretory-acid phosphatase enzymes (LdSAcPs). Recently, we showed using combined biochemical and molecular approaches that this family of enzymes was functionally conserved among all pathogenic species of Leishmania examined. This suggested that they must play significant functional roles in the growth, development and survival of all members of this important group of human pathogens. In order to function within their hosts, these LdSAcPs would have to be generally resistant to endogenous host proteases. In that regard, recent results showed that the LdSAcPs were virtually totally resistant to hydrolytic-degradation by various different serine-, thiol- metallo- and mixed proteases. These results further underscore the significance of these enzymes in functionally facilitating parasite survival within their infected hosts. In parallel, we also demonstrated that promastigotes of all pathogenic Leishmania species released very high levels of a soluble, secretory isoform of a zinc-metallo-protease enzyme (GP-63S). Our results suggest that this secretory protease helps facilitate parasite growth in its various host environments. In other biochemical and molecular studies, we demonstrated that a unique L. donovani ARF1-protein functions to maintain the structural integrity of the trans-Golgi cisternal network and facilitates the proper trafficking and transit of both secretory and surface membrane proteins through the Golgi compartment in these parasites. Further, using site specific targeted-mutations, we showed that the LdARF1-protein was essential for the survival of these organisms. In addition to the foregoing, recently we succeeded in developing an in vitro system for generating and propagating large quantities of infectious forms (i.e. axenic amastigotes) of L. donovani. The availability of such material should now greatly facilitate studies in general concerning the basic cell and molecular biology of this human parasite. Cumulatively, the results of our recent and ongoing studies continue to provide pertinent and significant information toward understanding the unique pathophysiology of these parasites. In addition, these studies are of practical relevance toward demonstrating whether specific /unique 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.