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。在受感染的患者体内，这种生物的各种物种栖息并破坏皮肤或内脏器官（即脾、肝和骨髓）内的巨噬细胞。因此，它们引起溃疡性、不愈合、毁容的恶性皮肤病变（如墨西哥乳杆菌）或退行性和最常见的致命内脏疾病（如多诺瓦氏乳杆菌）。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1200多万患者。我们的研究旨在确定这些生物的病理生理机制。在这方面，研究利什曼原虫和相关的锥虫原虫的基本细胞、分子和发育生物学，以鉴定和表征这些人类病原体生存所必需的寄生虫分子。这些寄生虫如何能够在其昆虫媒介和哺乳动物宿主中生存、获取营养、繁殖和分化，是了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，了解它们的表面膜、分泌酶和其他功能蛋白的组成和功能似乎是必不可少的。为此，鉴定了独特的寄生虫表面膜、分泌酶和调节蛋白，并对其进行了生化表征，以确定它们在这些生物体生存中的功能作用。此外，编码这些蛋白的基因首次被分离和表征，旨在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，最近我们鉴定并鉴定了编码独特利什曼原虫rab5b蛋白的基因。在这些生化和分子研究中，我们证明了这种独特的L. donovani rab5b蛋白在识别和维持这种寄生虫早期内体室的结构特性和完整性方面起作用。我们发现这种蛋白还有助于促进分泌蛋白和表面膜蛋白进入和通过这些寄生虫独特的内吞区室的适当内体运输和运输。此外，利用位点特异性GFP标记构建体结合共聚焦荧光显微镜定位研究，我们发现Ld Rab5b对于这些生物的正常内噬运输是必不可少的。运动蛋白是一个运动蛋白超家族，是大多数真核细胞微管细胞骨架的重要组成部分。鉴于利什曼原虫微管细胞骨架的重要性，驱动蛋白也可能在这些生物体的生长和分化中发挥核心作用。例如，一种利什曼原虫K39-驱动蛋白在人类内脏利什曼病过程中产生，并且感染的患者对K39表位产生强烈的体液反应。因此，K39驱动蛋白已被用作内脏利什曼病的血清诊断检测，但尽管其临床相关性，K39驱动蛋白的分子功能仍然未知。在这方面，在正在进行的细胞和分子生物学研究中，我们最近从多诺瓦利什曼原虫中鉴定、克隆、鉴定和分析了全长K39激酶开放阅读框。此外，我们研究了内源性LdK39蛋白在寄生虫中的定位，并评估了几个独立表达的LdK39蛋白嵌合结构域的行为。利用分子生物学和细胞生物学方法的结合，我们发现内源性LdK39激酶和截断的GFP-K39嵌合融合蛋白以atp依赖的方式结合并沿着寄生虫的细胞骨架移动，并且它们在这些人类寄生虫的后细胞极积累。这些研究结果表明，LdK39-kinesin是一种独特的运动蛋白，参与细胞内运输和沿细胞骨架微管的运输，并参与这些寄生虫的纺锤体极分裂。基于其功能，LdK39激酶可能是治疗这些寄生虫的合理靶点。值得注意的是，这是第一个从这些重要的人类病原体中描述的激酶基因。在其他平行研究中，我们使用各种反义RNA方法，完全废除了一种独特的双功能锥虫表面膜酶的表达和功能活性，即3-核苷酸酶/核酸酶。此外，这些研究结果首次证明了这种独特的寄生虫酶对这些寄生虫的生存、生长和发育至关重要。因此，这些结果表明，这种关键酶可能代表了潜在的治疗干预这些营养不良生物的逻辑目标。先前的研究表明，蛋白质在内质网（ER）上的易位通常是由信号识别颗粒（SRP）介导的。因此，在合作研究中，我们研究了几种不同锥虫的SRP。我们通过RNAi沉默编码SRP蛋白的基因和过表达7SL RNA的显性阴性突变体两种方法下调SRP。这些研究的总体结果表明，与细菌一样，但与哺乳动物细胞不同，锥虫虫的SRP通常对表面膜蛋白的生物发生至关重要。

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 secretory enzymes 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 the course of parasite growth, differentiation and development. For example, recently we identified and characterized the genes that encode the unique Leishmania Rab5b-protein. In these biochemical and molecular studies, we demonstrated that this unique L. donovani Rab5b-protein functions in identifying and maintaining the structural identity and integrity of early endosomal compartments in this parasite. We found that this protein also helps to facilitate the proper endosomal trafficking and transit of both secretory and surface membrane proteins into and through the unique endocytic compartments of these parasites. Further, using site-specific GFP tagged-constructs in conjunction with confocal fluorescence microscopy localization studies, we showed that the Ld Rab5b was essential for the normal endocytic trafficking in these organisms. Kinesins are a superfamily of motor proteins that are important components of the microtubule cytoskeletons of most eukaryotic cells. Given the importance of the microtubule cytoskeleton in Leishmania parasites, kinesins are also likely to play central roles in the growth and differentiation of these organisms. For example, a Leishmania K39-kinesin was previously shown to be produced during the course of human visceral leishmaniasis and that infected patients mounted a strong humoral response to a K39 epitope. Thus, the K39 kinesin has been used as a serodiagnostic assays for visceral leishmaniasis but despite its clinical relevance, the molecular function of the K39 kinesin protein remained unknown. In that regard, in ongoing cell and molecular biology studies, we recently identified, cloned, characterized and analyzed the full length K39 kinesin open reading frame from Leishmania donovani. Further, we investigated the localization of the endogenous LdK39 protein in the parasite and assessed the behavior of several separate expressed LdK39 protein chimeric domains. Using a combination of molecular and cell biological approaches, we showed that the endogenous LdK39 kinesin and a truncated GFP-K39 chimeric fusion protein bind to and move along the parasite cytoskeleton in an ATP-dependant manner and that they accumulate at the posterior cell pole of these human parasites. Results of these studies demonstrated that the LdK39-kinesin was a unique motor protein involved in intracellular transport and trafficking along cytoskeletal microtubules and that it was also involved spindle pole division in these parasites. Based on its functions, the LdK39 kinesin might represent a logical target for therapeutic intervention against these parasites. It is significance to note that this is the first kinesin gene to be described from these important human pathogens. In other parallel studies, we used various anti-sense RNA methods, to completely abrogate the expression and functional activity of a unique, bi-functional trypanosomatid surface membrane enzyme, i.e. the 3-nucleotidase/nuclease. Further, results of these studies demonstrated for the first time that this unique parasite enzyme was essential for the survival, growth and development of these parasites. Thus, these results indicated that this critical enzyme might represent a logical target for potential therapeutic intervention against these auxotropic organisms. Previously, it has been shown that protein translocation across the endoplasmic reticulum (ER) is often mediated by the Signal Recognition Particle (SRP). Thus,in collaborative studies, we investigated the SRP in several different trypanosomatids. We down-regulated the SRP using two approaches, i.e. RNAi silencing of genes encoding SRP proteins and over-expression of dominant-negative mutants of 7SL RNA. The overall results of these studies demonstrated that, as in bacteria, but unlike mammalian cells, the trypanosomatid SRP are generally essential for biogenesis of surface membrane proteins. 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.