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，首次确定其在寄生虫生长，发育和分化过程中的表达和调控。例如，在过去的一年中，我们的研究已经阐明了酶的活性，基因结构和染色体位点的L。墨西哥几丁质酶此外，使用各种嵌合构建体，我们表明，在寄生虫的发展和分化，这种酶的表达在转录后水平的调节。此外，还对几种独特的L. donovani组氨酸分泌型（LdSAcPs）和膜型（LdMAcP）酸性磷酸酶在分子研究中被用作探针。这些生化和分子研究的结果表明，这个家族的酶是功能保守的所有致病性利什曼原虫物种以及其他更远的人类锥虫寄生虫。这些LdSAcP和LdMAcP同源物在所有致病性利什曼原虫中的保守性。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。此外，我们将这些基因中的几个与绿色荧光蛋白（-GFP）串联的嵌合构建体，以确定负责将这些酶靶向进入并通过寄生虫分泌途径的独特分子信号。此外，我们的研究涉及基因缺失，突变，和过表达的L。donovani钙网蛋白证明，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。此外，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 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.