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。

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 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. In light of the foregoing, for example, in FY 2008, we identified, characterized and expressed the full length gene that encodes a unique L. donovani LdK39B-kinesin protein. Kinesins are a superfamily of motor proteins that are important components of the microtubule cytoskeletons of most eukaryotic cells. Given the importance and extensive nature of the microtubule cytoskeleton in Leishmania parasites, kinesins are also likely to play central roles in the growth and differentiation of these organisms. In support of that concept, during the course of human visceral leishmaniasis, patients produce a very significant antibody response against the LdK39-kinesins. Despite its clinical relevance, the molecular functions of the LdK39 kinesin proteins have remained unknown. However, in the current studies, a variety of cell and molecular biology approaches were used to identify the subcellular localization of both the endogenous LdK39B kinesin protein and to assess the behavior of several separate LdK39B expressed chimeric gene constucts in transfected parasites. Results of these studies demonstrated that this large (>261 kDa) motor protein was constitutively expressed in both the insect vector and mammalian developmental forms of this organism. Moreover, results of confocal fluorescence, immunoelectron microscopy and subcellular fractionation studies demonstrated that the LdK39-kinesin protein was localized and tightly/integrally bound to the apical poles of the parasite kinetoplast, a unique mitochondrial-DNA containing organelle which is found only in these organisms. It is significance to note that this is the first kinesin to be found associated with this unique parasite organelle. Based on its motor functions and its unique subcellular structural association with the parasite kinetoplast, the LdK39 kinesin might represent a logical target for chemotherapeutic intervention against these pathogens. Leishmania and other related trypanosomatid parasites are all obligate purine auxotrophs i.e. they are incapable of synthesizing purines and therefore must salvage these compounds from their hosts in order to survive and multiply. Thus, these organisms have evolved a number of enzymes to salvage essential purines from their hosts. Therefore, to address this issue, in other parallel studies in FY 2008, we used variety of anti-sense RNA methods, to completely abrogate the expression and functional activity of a unique, bi-functional trypanosomatid parasite purine salvage enzyme, i.e. the 3-nucleotidase/nuclease. Results of these studies demonstrated for the first time that this unique parasite purine-salvage enzyme was essential for the survival, growth and development of these organisms. Thus, these results indicated that this critical enzyme might represent a logical target for potential therapeutic intervention against these auxotropic parasitic organisms. In higher eukaryotes, it has been shown that protein translocation across the endoplasmic reticulum (ER) is often mediated by the Signal Recognition Particle (SRP). Thus, in FY 2008 this SRP hypothesis was investigated in collaborative studies, using several different trypanosomatids. In these studies, we down-regulated the SRP using two approaches, i.e. RNAi silencing of genes encoding SRP proteins and the over-expression of dominant-negative mutants of 7SL RNA. These studies showed that down-expression of the SRP was not detrimental to the expression of proteins containing endogenous signal peptides. However, the overall results of these studies demonstrated that, as in bacteria, but in contrast to mammalian cells, the trypanosomatid SRP are generally essential/necessary for biogenesis of surface membrane targeted 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.