BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ENZYMES SECRETED BY LEISHMANIA

利什曼原虫分泌的酶的生物化学和分子特征

• 批准号: 6431659
• 负责人: DENNIS DWYER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431659
• 关键词: Leishmania; affinity chromatography; enzyme activity; enzyme structure; gastrointestinal disorder; genetic mapping; host organism interaction; human tissue; isozymes; life cycle; microorganism genetics; microorganism metabolism; parasitism; protein structure function; secretory protein; tissue /cell culture

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 sandfies), 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. Since such secretory products can readily permeate and presumably alter the host micro-environments in which Leishmania reside, an understanding of the nature of these parasite products seems essential. To that end, several parasite secretory enzymes 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. During the past year, our studies have elucidated the enzymatic activity, gene structure and chromosomal locus of the leishmanial chitinase in both L. donovani and L. mexicana. Further, we showed that all pathogenic Leishmania possess a highly conserved locus for this gene and all of these organisms express structurally related secretory chitinase activities. Similarly, the genes for several new members of the unique leishmanial secretory acid phosphatase (SAcP) family were identified and characterized from L. donovani. Our biochemical and molecular studies showed that this family of enzymes was functionally conserved among all pathogenic leishmanial species including L. major as well as, other more distantly related trypanosomatid parasites of humans. Further, we demonstrated that these conserved SAcPs are synthesized by amastigote forms of all pathogenic Leishmania species during the course of both human cutaneous and visceral disease. Moreover, we made chimeric constructs of these genes in tandem with the green fluorescent protein (-GFP) to determine the unique molecular signals responsible for targeting these enzymes into the parasite secretory pathway. Experiments involving gene-deletion, -mutation, and -over expression are in progress toward demonstrating that these secretory enzymes are, in fact, essential to the survival of these human pathogens. Further, physical/structural analyses of the L. donovani SAcP, were also carried out in collaboration with Dr. Olafson's lab. Results of these mass spectrometry studies showed that the C-terminal serine residues of SAcP were heavily decorated with O-linked carbohydrate side chains composed of highly negatively charged repeating units (n=32) of phospho-disaccharides. These [-PO4-Gal-Man-] repeat units are the same as those which constitute the parasite's major surface membrane glycolipid, the lipophosphoglcan. This is the first physical proof for the existence of this unusual phospho-disaccharide moeity in both a secretory glycoprotein and a glycolipid. Moreover, we found that these [-PO4-Gal-Man-] repeat units accounted, at least in part, for the more than 700 moles of hexose present in the SAcP per mole of protein and for the extreme resistance of this enzyme to host proteolytic-degradation . 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 an pathway and functions to regulate protein over-expression of these organisms.

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。所有利什曼原虫都经历一个二型生命周期：1）在哺乳动物（人类）中，它们在巨噬细胞的溶酶体系统中以专性细胞内形式[无鞭毛体]繁殖，最终破坏这些细胞，2）在其昆虫载体中（吸血沙飞），它们分化和繁殖，在消化道内形成细胞外形式[前鞭毛体]，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.我们实验室的先前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。由于这种分泌产物可以很容易地渗透，并可能改变宿主的微环境中，利什曼原虫居住，这些寄生虫产品的性质的理解似乎是必不可少的。为此，正在研究几种寄生虫分泌酶，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。在过去的一年中，我们对利什曼原虫几丁质酶的酶活性、基因结构和染色体定位进行了研究。donovani和L.墨西哥。此外，我们发现，所有致病性利什曼原虫具有高度保守的基因座，所有这些生物体表达结构相关的分泌几丁质酶活性。同样地，从利什曼原虫中鉴定并鉴定了几个独特的利什曼原虫分泌型酸性磷酸酶（SAcP）家族新成员的基因。donovani。我们的生物化学和分子生物学研究表明，该酶家族在所有致病性利什曼原虫物种包括L。主要以及其他更远的人类锥虫寄生虫。此外，我们证明，这些保守的SAcPs合成的无鞭毛体形式的所有致病性利什曼原虫物种在人类皮肤和内脏疾病的过程中。此外，我们将这些基因与绿色荧光蛋白（-GFP）串联嵌合构建，以确定负责将这些酶靶向进入寄生虫分泌途径的独特分子信号。实验涉及基因缺失，突变和过度表达的进展，以证明这些分泌酶，事实上，这些人类病原体的生存是必不可少的。此外，还对L. donovani SAcP，也与Olafson博士的实验室合作进行。这些质谱研究的结果表明，SAcP的C-末端丝氨酸残基被大量修饰的O-连接的碳水化合物侧链组成的高度带负电荷的重复单元（n=32）的磷酸二糖。这些[-PO 4-Gal-Man-]重复单元与构成寄生虫主要表面膜糖脂（脂磷酸聚糖）的重复单元相同。这是第一个物理证据，证明在分泌糖蛋白和糖脂中都存在这种不寻常的磷酸二糖部分。此外，我们发现，这些[-PO 4-Gal-Man-]重复单元至少部分地解释了SAcP中每摩尔蛋白质中存在的超过700摩尔的己糖以及这种酶对宿主蛋白水解降解的极端抗性。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，该途径与分泌途径相互作用，并起到调节这些生物体的蛋白质过表达的作用。