Cell And Developmental Biology Of Trypanosomatid Parasit

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，了解它们的表膜和其他功能蛋白的组成和功能似乎是必不可少的。为此，对寄生虫独特的表面膜、分泌酶和调节蛋白进行了鉴定和生化特征，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。例如，我们已经鉴定并鉴定了编码几种独特的杜氏乳杆菌酒石酸敏感、组氨酸、分泌型酸性磷酸酶(LdSAcps)的基因。最近，我们用生物化学和分子生物学相结合的方法证明了这个酶家族在所有被研究的利什曼原虫致病物种中是功能保守的。这表明它们必须在这一重要的人类病原体群体的所有成员的生长、发育和生存中发挥重要的功能作用。为了在宿主内发挥作用，这些LdSAcp通常必须对内源性宿主蛋白酶具有抵抗力。在这方面，最近的结果表明，LdSAcps几乎完全抵抗各种不同的丝氨酸酶、硫醇金属酶和混合蛋白水解酶的降解。这些结果进一步强调了这些酶在功能上促进寄生虫在其受感染宿主内生存的重要性。同时，我们还证明了所有致病利什曼原虫的前鞭毛体都释放了非常高水平的锌金属蛋白水解酶(GP-63S)的可溶性、分泌型同工酶。我们的结果表明，这种分泌型蛋白水解酶有助于促进寄生虫在不同宿主环境中的生长。在其他生化和分子研究中，我们证明了一种独特的杜氏钩端螺旋体ARF1蛋白的功能是维持跨高尔基体池网络结构的完整性，并促进这些寄生虫的分泌和表面膜蛋白通过高尔基体的适当运输和转运。此外，利用定点定向突变，我们证明了LdARF1-蛋白对于这些生物的生存是必不可少的。此外，我们最近还成功地开发了一种体外培养系统，用于大量产生和繁殖具有感染性的形式(即无菌无鞭毛体)。这种材料的提供现在应该极大地促进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。总体而言，我们最近和正在进行的研究结果继续为了解这些寄生虫的独特病理生理学提供相关和重要的信息。此外，这些研究对于证明特定/独特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)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 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.