Biochemical And Molecular Characterization Of Enzymes Se

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以专性细胞内形式[无鞭毛体]繁殖，最终破坏这些细胞，2）在其昆虫载体（吸血白蛉）中，它们在消化道内分化和繁殖为细胞外形式[前鞭毛体]，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，我们的研究阐明了L.墨西哥几丁质酶该基因的表达构建体正被用于研究该酶在白蛉载体和哺乳动物宿主内寄生虫生长、发育和分化中的作用。此外，我们确定并表征了一个新的基因编码一个独特的寄生虫转化酶[蔗糖酶]酶。生化和分子研究的结果表明，这种酶是高度发育上表达的前鞭毛体，这表明它可能提供了一个独特的营养优势，寄生虫在其昆虫载体宿主。最近，我们还证明，所有致病性利什曼原虫物种的前鞭毛体在体外生长期间都会向其培养上清液中释放非常高水平的蛋白酶活性。生物化学和免疫化学标记的研究结果表明，这种蛋白酶活性是一种可溶性的，分泌型的GP-63，这些生物体的主要表面膜锌金属蛋白酶的异构体。我们的研究结果表明，这种分泌型蛋白酶亚型艾滋病促进寄生虫在其各种宿主环境中的生长。在其他涉及L. donovani钙网蛋白，我们证明了这种常驻ER分子伴侣折叠蛋白实际上是这些人类病原体生存所必需的。同时，使用几种不同的截短表面膜蛋白的GFP构建体，我们定义了利什曼原虫中独特的内体运输途径，其与分泌途径和功能相互作用，至少部分地调节这些生物体的蛋白质过表达。

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 forms [amastigotes] 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 forms [promastigotes] 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, our studies have elucidated the enzymatic activity, gene structure and chromosomal locus of the L. mexicana chitinase. Expression constructs of this gene are being used to examine the role of this enzyme in parasite growth, development and differentiation within their sandfly vector and mammalian hosts. In addition, we identified and characterized a new gene encoding a unique parasite invertase [sucrase] enzyme. Results of both biochemical and molecular studies showed that this enzyme was highly developmentally upexpressed in promastigotes suggesting that it presumably affords a unique nutritional advantage to the parasite within its insect vector host. Recently, we also demonstrated that promastigotes of all pathogenic Leishmania species released very high levels of protease enzyme activity into their culture supernatants during their growth in vitro. Results of biochemical and immunochemical labeling studies showed that this protease activity was a soluble, secretory isoform of GP-63 , the major surface membrane zinc-matallo-protease of these organisms. Our results suggest that this secretory protease isoform aids in facilitating parasite growth in its various host environments. In other studies involving gene-deletion, -mutation, and -over expression of the L. donovani calreticulin, we demonstrated that this resident ER chaperone-folding protein is in fact, essential to the survival of these human pathogens. In parallel, using GFP constructs of several different truncated surface membrane proteins, we defined a unique endosomal trafficking pathway in Leishmania which interfaces with the secretory pathway and functions, at least in part, to regulate protein over-expression of these organisms.