Biochemical And Molecular Characterization Of Enzymes Secreted By Leishmania

该项目正在进行的研究涉及利什曼原虫的细胞生物学、生物化学和分子生物学，利什曼原虫是人类的一组原生动物病原体。所有利什曼原虫都经历了二相性生活史：1)在哺乳动物(人类)中，它们在巨噬细胞的溶酶体系统内作为专性的细胞内无鞭毛体形式繁殖，最终摧毁这些细胞；2)在它们的昆虫媒介(吸血沙蝇)中，它们以细胞外前鞭毛体的形式在消化道内分化繁殖，并最终迁移到口腔部位进行传播。据世界卫生组织估计，利什曼原虫每年导致全世界超过1200万例人类疾病(利什曼病)。在受感染的人类中，这些寄生虫破坏皮肤或内脏(即脾、肝和骨髓)内的巨噬细胞，导致大的、毁容的、恶性的皮肤溃疡(例如由墨西哥乳杆菌引起)或退行性的、通常是致命的内脏疾病(例如由多诺瓦尼乳杆菌引起)。我们实验室以前的研究已经证实，利什曼原虫结构性地分泌40多种不同的可溶性蛋白质、糖蛋白和碳水化合物成分。这种分泌产物可以很容易地渗透到利什曼原虫的整个宿主微环境中，并可能改变宿主的微环境。因此，了解这些寄生虫产品的性质似乎是至关重要的。为此，人们正在研究几种寄生虫分泌酶和调节蛋白，以确定它们在这些生物的生存、维持、生长和传播中的功能作用。此外，编码这些蛋白的基因已被鉴定和鉴定，以确定它们在寄生虫生长、发育和分化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。在这方面，在正在进行的合作研究中，我们还分离、鉴定和表达了墨西哥乳杆菌几丁质酶的编码基因。此外，我们还检测了墨西哥乳杆菌几丁质酶高表达载体在其沙蝇载体中的表型。我们的观察表明，与对照相比，这些微生物能够从寄主的周转膜室中排出，感染沙蝇的寄主。此外，这种转基因对苍蝇的气门系统造成了更大的破坏，导致更多的寄生虫被传播给它们的宿主。这些结果表明，这种分泌酶在该寄生虫及其桑蝇媒介的发育和传播中起着至关重要的作用。在其他正在进行的研究中，人们正在尝试从功能上删除墨西哥乳杆菌几丁质酶基因，并检查这些突变体在哺乳动物(小鼠)宿主和沙蝇载体中的生存能力。在其他研究中，我们正在评估给小鼠接种利什曼几丁质酶基因是否会导致强毒寄生虫攻击感染后寄生虫病理的改变。此外，在此之前，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，在平行的正在进行的研究中，我们最近鉴定并表征了杜氏利什曼原虫一种独特的新的35 kDa核酸酶的生化和功能性质。我们的研究表明，这种酶是由该寄生虫的无鞭毛体和前鞭毛体发育的形式结构性地释放/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LdNucS，并对其进行了表型表达。生化和酶学特性表明，LdNucS核酸酶能够裂解多种人工合成的多核苷酸底物，并能有效地降解天然RNA、单链和双链DNA底物。我们推测，这种利什曼分泌核酸酶可以在远离寄生虫的地方作用，水解宿主来源的核酸，以满足这些生物对嘌呤的基本需求。这表明它必须在促进这种重要的人类病原体在其昆虫媒介和哺乳动物宿主中的生存、生长和发展方面发挥重要作用(S)。为了支持后者，我们最近证明，来自多个不同地理流行区的患者似乎都具有与利什曼原虫LdNucS核酸酶反应和免疫沉淀的抗体。这些观察表明，这种酶是在人类感染过程中由寄生虫合成和释放的。在这方面，这种利什曼分泌核酸酶可用于诊断或治疗目的。 综上所述，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。

Ongoing studies in this project concern the cell biology, biochemistry and molecular biology of Leishmania, a group of protozoan pathogens of humans. All Leishmania parasites undergo a dimorphic life cycle: 1) in mammals (humans), they multiply as obligate intracellular amastigote forms within the lysosomal system of macrophages, eventually destroying these cells and 2) within their insect vectors (blood-sucking sandflies), they differentiate and multiply as, extracellular promastigote forms within the alimentary tract and eventually migrate to the mouth parts for transmission. By World Health Organization estimates, Leishmania parasites annually cause well-over 12 million cases of human disease (leishmaniasis) worldwide. In infected humans, these parasites destroy macrophages within the skin or internal organs (i.e. spleen, liver and bone marrow) causing either large and disfiguring, malignant skin ulcers (e.g. caused by L. mexicana) or degenerative and most often fatal visceral disease (e.g. caused by L. donovani). Previous studies from our laboratory have established that Leishmania parasites constitutively secrete over 40 different soluble protein, glycoprotein and carbohydrate constituents. Such secretory products can readily permeate throughout and presumably alter the host micro-environments in which Leishmania reside. Thus, an understanding of the nature of these parasite products seems essential. To that end, several parasite secretory enzymes and regulatory proteins are being investigated toward defining their functional roles in the survival, maintenance, growth and transmission of these organisms. Further, genes encoding these proteins have been identified and characterized toward defining their expression and regulation during parasite growth, development and differentiation. 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 that regard, in on going collaborative studies, we have also isolated, characterized and expressed the gene encoding the L. mexicana chitinase. Further, we have examined the phenotype of L. mexicana chitinase over-expressor transfectants in their sandfly vectors. Our observations indicate that these organisms are able to egress from the host peritrophic membrane compartment and infect the sandfly host at significantly elevated levels vs controls. In addition, such transfectants cause greater damage to the fly stomadeal valve system which results in greater numbers of parasites being transmitted to their host. These results indicate that this secretory enzyme plays essential roles in the development and transmission of this parasite it its sanfly vector. In other on going studies, attempts are being made to functionally delete the gene for the L. mexicana chitinase and to examine the viability of these mutants in both the mammalian (mouse) host and sandfly vectors. In other studies, we are evaluating whether vaccination of mice with cDNA of the Leishmanial chitinase will result in alteration of parasite pathology following challenge infections with virulent parasites. In addition, previously, 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 parallel ongoing studies, we recently identified and characterized the biochemical and functional properties of a unique new, 35 kDa, nuclease from Leishmania donovani. Our studies demonstrated that this enzyme was constitutively released/secreted by both amastigotes and promastigote developmental forms of this parasite. Using a molecular approach, we identified, characterized and episomally-expressed the gene, LdNucS which encodes this new Class I nuclease family member from these organisms. Biochemical and enzymatic characterization showed that the LdNucS nuclease was capable of cleaving a variety of synthetic polynucleotide substrates as well as effectively hydrolyzing natural RNA, single stranded and double stranded DNA substrates. 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. This suggests that it must have essential role(s) in facilitating the survival, growth and development of this important human pathogen in both its insect vector and mammalian hosts. In support of the latter, we have recently demonstrated that patients from multiple diverse geographic endemic foci all appear to posses antibodies which react with and immunoprecipitate the Leishmania LdNucS nuclease. These observations suggest that this enzyme is synthesized and released by the parasite during the course of human infections. In that regard, this leishmanial secretory nuclease might be exploited for diagnostic or for therapeutic purposes. Taken together, 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.