Cell And Developmental Biology Of Trypanosomatid Parasit

锥虫寄生虫的细胞和发育生物学

• 批准号: 7299898
• 负责人: DENNIS DWYER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7299898
• 关键词: Cell; Developmental; Biology; Trypanosomatid; Parasit

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 secretory chitinase family. Using combined biochemical and molecular approaches we showed 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 addition, in previous studies, we demonstrated that virtually all Leishmania sp., like other trypanosomatid parasites are purine auxotrophs and therefore are, totally dependent upon salvaging these essential compounds from their insect vector and mammalian hosts. In that regard, in ongoing studies,we recently identified and characterized the biochemical and functional properties of a unique new, 35 kDa, nuclease from these organisms. Our studies demonstrated that this enzyme was constitutively released/secreted by both developmental forms of the parasite. Using a molecular approach, we identified, characterized and episomally-expressed the gene, LdNuc[S] which encodes this new Class I nuclease family member from these organisms. Results of both structural and functional analyses showed that the LdNuc[S] expressed-enzyme had properties identical to the native, wild-type, parasite released/secreted nuclease. Further, both LdNuc[S] mRNA and secretory nuclease activity were differentially up-expressed by amastigotes >> promastigotes. Biochemical analyses showed that the LdNuc[S] secreted nuclease could hydrolyze a variety of synthetic polynucleotide substrates as well as, both single- and double-stranded DNAs and RNAs. Based on these observations, we hypothesize that this leishmanial ?secretory? nuclease could act, at a distance away from the parasite, to hydrolyze host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, LdNuc[S] must have essential roles in facilitating the survival, growth and development of this important human pathogen. In other biochemical and molecular studies, we demonstrated that a 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 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, we showed that the Ld Rab5b was essential for the normal endocytic trafficing in these organisms. In addition, in collaborative studies we succeeded in identifying and characterizing a gene encoding a specific blood-induced chitinolytic enzyme system in the midgut of Phlebotomus papatasi and Lutzomyia longipalpis which are the sandfly vectors for Leishmania major and L. (donovani) chagasi, respectively. A recombinant expressed protein from this gene demonstrated high chitinolytic enzyme activity against a variety of chitin substrates. This is the first chitinase gene to be described from these important vectors for human parasites. 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.

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，因此了解它们的表膜、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，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，编码这些蛋白质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，最近我们鉴定并鉴定了编码独特的利什曼原虫分泌型几丁质酶家族的基因。结合生物化学和分子生物学方法，我们发现该酶家族在所研究的所有病原利什曼原虫中是功能保守的。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。此外，在以前的研究中，我们证明了几乎所有的利什曼原虫，像其他锥虫寄生虫一样，都是嘌呤营养缺乏症，因此完全依赖于从它们的昆虫媒介和哺乳动物宿主中抢救这些必要的化合物。在这方面，在正在进行的研究中，我们最近从这些生物中鉴定并表征了一种独特的新的35 kDa核酸酶的生化和功能特性。我们的研究表明，这种酶是由两种发育形式的寄生虫结构性地释放/分泌的。利用分子生物学方法，我们从这些生物中鉴定了编码这个新的I类核酸酶家族成员的基因LdNuc[S]，并对其进行了表型表达。结构和功能分析结果表明，LdNuc[S]表达的酶具有与天然、野生型、寄生虫释放/分泌核酸酶相同的性质。此外，LdNuc[S]基因和分泌核酸酶活性在无鞭毛体&gt；和gt；前鞭毛体中均有差异表达。生化分析表明，S分泌的核酸酶可以降解多种人工合成的多核苷酸底物以及单链和双链DNA和RNA。基于这些观察，我们假设这种利什曼？分泌？在远离寄生虫的地方，核酸酶可以作用于水解宿主来源的核酸，以满足这些生物体对嘌呤的基本需求。因此，LdNuc[S]在促进这种重要的人类病原体的生存、生长和发展方面必须发挥重要作用。在其他生化和分子研究中，我们证明了一种独特的杜诺瓦尼乳杆菌Rab5b蛋白在鉴定和维持这种寄生虫早期内体隔室的结构特性和完整性方面发挥了作用。我们发现，这种蛋白有助于促进内分泌性和表面膜蛋白在这些寄生虫独特的内细胞室中的适当运输和转运。此外，使用位点特异的GFP标记构建物，我们表明LD Rab5b对于这些生物的正常内皮运输是必不可少的。此外，在合作研究中，我们成功地在帕帕塔西白头翁和长纹夜蛾的中肠中找到了编码特定血液诱导的几丁质分解酶系统的基因，这两个基因分别是大利什曼原虫和查加西的沙蝇载体。该基因的重组表达蛋白对多种甲壳素底物具有较高的几丁质酶活性。这是从这些重要的人类寄生虫载体中描述的第一个几丁质酶基因。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。