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，利什曼原虫是一组人类原生动物病原体。所有利什曼原虫寄生虫都经历二型生命周期：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细胞内无鞭毛体形式繁殖，最终破坏这些细胞，以及2）在它们的昆虫载体（吸血白蛉）中，它们在消化道内分化和繁殖为细胞外前鞭毛体形式，并最终迁移到口腔部位进行传播。据世界卫生组织估计，利什曼原虫每年在全世界造成超过1200万例人类疾病（利什曼病）。在受感染的人中，这些寄生虫破坏皮肤或内部器官（即脾、肝和骨髓）内的巨噬细胞，导致大的和毁容的恶性皮肤溃疡（例如由L. mexicana）或退行性和最常见的致命性内脏疾病（例如由L. Donovani）。我们实验室以前的研究已经确定，利什曼原虫组成性分泌超过40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整个宿主体内，并可能改变利什曼原虫所处的宿主微环境。因此，了解这些寄生虫产品的性质似乎至关重要。为此，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体的生存、维持、生长和传播中的功能作用。此外，编码这些蛋白质的基因已被鉴定和表征，以确定它们在寄生虫生长、发育和分化期间的表达和调节。例如，最近我们鉴定和表征了编码独特的利什曼原虫分泌几丁质酶家族的基因。使用生物化学和分子生物学相结合的方法，我们发现，这个家庭的酶是功能保守的所有致病性种利什曼原虫检查。这表明它们必须在这一重要的人类病原体组的所有成员的生长、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、表征和表达了编码L。墨西哥几丁质酶此外，我们还研究了L. mexicana几丁质酶过表达的转染子。我们的观察结果表明，这些生物体能够从主机围食膜室和感染白蛉主机在显着升高的水平与控制。此外，这样的转染子对苍蝇气孔阀系统造成更大的损害，这导致更大数量的寄生虫被传播到它们的宿主。这些结果表明，这种分泌酶在该寄生虫的发育和传播中起着重要的作用。 在其他正在进行的研究中，正在尝试从功能上删除L。mexicana几丁质酶，并检查这些突变体在哺乳动物（小鼠）宿主和白蛉载体中的生存力。在其他研究中，我们正在评估是否接种小鼠利什曼原虫几丁质酶的cDNA将导致改变寄生虫的病理挑战感染后，与有毒的寄生虫。此外，之前，我们证明了几乎所有的利什曼原虫，与其它锥虫一样，寄生虫是嘌呤营养缺陷型，因此完全依赖于从其昆虫载体和哺乳动物宿主中回收这些必需化合物。在这方面，在平行进行的研究中，我们最近确定并表征了杜氏利什曼原虫的一种独特的新的35 kDa核酸酶的生化和功能特性。我们的研究表明，这种酶组成型释放/分泌的无鞭毛体和前鞭毛体发育形式的寄生虫。使用分子方法，我们确定，表征和附加型表达的基因，LdNucS编码这种新的I类核酸酶家族成员从这些生物体。生物化学和酶促表征表明，LdNucS核酸酶能够切割各种合成的多核苷酸底物，以及有效地水解天然RNA、单链和双链DNA底物。我们假设，这种利什曼原虫分泌核酸酶可以采取行动，在远离寄生虫，水解宿主衍生的核酸，以满足这些生物体的基本嘌呤需求。这表明它必须在促进这种重要的人类病原体在其昆虫载体和哺乳动物宿主中的存活、生长和发育方面发挥重要作用。为了支持后者，我们最近已经证明，来自多个不同地理地方病疫源地的患者似乎都能产生与利什曼原虫LdNucS核酸酶反应并免疫沉淀的抗体。这些观察结果表明，这种酶在人类感染过程中由寄生虫合成和释放。在这方面，这种利什曼原虫分泌核酸酶可用于诊断或治疗目的。 总之，我们最近和正在进行的研究的结果继续提供相关的和重要的信息，了解这些寄生虫的独特病理生理学。此外，这些研究对于证明特异性/独特的寄生虫酶和调节蛋白是否是1）新化疗药物的设计，2）新诊断工具的开发和/或3）可用作针对这些人类病原体的潜在疫苗的逻辑靶标具有实际意义。

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.