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的原生动物病原体。所有利什曼原虫都经历一个二态的生命周期：1)在哺乳动物（人类）中，它们在巨噬细胞溶酶体系统中以细胞内形式（无尾虫）繁殖，最终破坏这些细胞；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形式（原生虫），并最终迁移到口部传播。据世界卫生组织估计，利什曼原虫每年在全世界造成远远超过1200万例人类疾病（利什曼病）。在受感染的人体内，这些寄生虫破坏皮肤或内脏器官（即脾脏、肝脏和骨髓）内的巨噬细胞，造成大面积毁容的恶性皮肤溃疡（例如由墨西哥乳杆菌引起）或退行性且最常见的致命内脏疾病（例如由多诺瓦氏乳杆菌引起）。我们实验室以前的研究已经确定利什曼原虫组成性地分泌超过40种不同的可溶性蛋白、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并可能改变利什曼原虫居住的宿主微环境。因此，了解这些寄生虫产物的性质似乎是必不可少的。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，编码这些蛋白的基因首次被鉴定和表征，从而确定了它们在寄生虫生长、发育和分化过程中的表达和调控。例如，在过去的一年里，我们的研究已经阐明了L. mexicana几丁质酶的酶活性、基因结构和染色体位点。此外，利用各种嵌合结构，我们发现在寄生虫发育和分化过程中，该酶的表达在转录后水平受到调节。此外，还利用多诺瓦菌几种独特的组氨酸分泌酶（LdSAcPs）和膜酸性磷酸酶（LdMAcP）基因作为探针进行了分子研究。这些生化和分子研究结果表明，该酶家族在所有检测的致病性利什曼原虫物种以及其他与人类关系较远的锥虫寄生虫中都具有功能保守性。这些ldsacp和LdMAcP同源物在所有致病性利什曼原虫中的保守性。提示它们必须在这一重要人类病原体群的所有成员的生长、发育和生存中发挥重要的功能作用。此外，我们将这些基因中的几个与绿色荧光蛋白（-GFP）串联构建嵌合结构，以确定负责将这些酶靶向进入和通过寄生虫分泌途径的独特分子信号。此外，我们对多诺瓦氏乳杆菌钙网蛋白的基因缺失、突变和过表达的研究表明，这种常驻内质网伴侣折叠蛋白实际上对这些人类病原体的生存至关重要。此外，利用几种不同的截断表面膜蛋白的GFP构建，我们在利什曼原虫中定义了一条独特的内体运输途径，该途径与分泌途径相结合，并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 for the first time toward defining their expression and regulation during parasite growth, development and differentiation. For example, during the past year, our studies have elucidated the enzymatic activity, gene structure and chromosomal locus of the L. mexicana chitinase. Further, using various chimeric constructs, we showed that during parasite development and differentiation, the expression of this enzyme is regulated at the post-transcriptional level. In addition, the genes for the several unique L. donovani histidine secretory- (LdSAcPs) and membrane- (LdMAcP) acid phosphatases were used as probes in molecular studies. Results of those biochemical and molecular studies showed that this family of enzymes was functionally conserved among all pathogenic leishmanial species examined as well as, other more distantly related trypanosomatid parasites of humans. The conservation of such LdSAcPs and LdMAcP homologs amongst all pathogenic Leishmania sps. suggests that they must play significant functional roles in the growth, development and survival of all members of this important group of human pathogens. Moreover, we made chimeric constructs of several of these genes in tandem with the green fluorescent protein (-GFP) to determine the unique molecular signals responsible for targeting these enzymes into and through the parasite secretory pathway. Further, our studies involving gene-deletion, -mutation, and -over expression of the L. donovani calreticulin demonstrated that this resident ER chaperone-folding protein is in fact, essential to the survival of these human pathogens. In addition, 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.