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，利什曼原虫是人类的一组原生动物病原体。所有利什曼原虫都经历了一个二态生活周期：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的细胞内形式[无鞭毛体]繁殖，最终摧毁这些细胞；2)在它们的昆虫媒介(吸血沙虫)内，它们在消化道内以细胞外形式[前鞭毛体]的形式分化和繁殖，最终迁移到口腔部位进行传播。据世界卫生组织估计，利什曼原虫每年导致全世界超过1200万例人类疾病(利什曼病)。在受感染的人类中，这些寄生虫破坏皮肤或内脏(即脾、肝和骨髓)内的巨噬细胞，导致大的、毁容的、恶性的皮肤溃疡(例如由墨西哥利什曼原虫引起)或退行性的、通常是致命的内脏疾病(例如由多诺瓦尼利什曼原虫引起)。我们实验室以前的研究已经证实，利什曼原虫结构性地分泌40多种不同的可溶性蛋白质、糖蛋白和碳水化合物成分。由于这种分泌产物很容易渗透并可能改变利什曼原虫所处的宿主微环境，因此对这些寄生虫产物的性质的了解似乎是至关重要的。为此，人们正在研究几种寄生虫分泌酶，以确定它们在这些生物的生存、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鉴定，以确定它们在寄生虫生长、发育和分化过程中的表达和调控。在过去的一年里，我们的研究已经阐明了杜氏利什曼原虫和墨西哥利什曼原虫几丁质酶的酶活性、基因结构和染色体位置。此外，我们发现所有致病的利什曼原虫都具有高度保守的几丁质酶基因，并且所有这些生物都表达与结构相关的分泌性几丁质酶活性。类似地，从杜氏利什曼原虫中鉴定了独特的利什曼分泌型酸性磷酸酶(SACP)家族的几个新成员的基因。我们的生化和分子研究表明，这个酶家族在所有致病利什曼原虫中都是功能保守的，包括大利什曼原虫以及其他更远缘的人类锥虫寄生虫。进一步，我们证明了这些保守的SACP是由所有致病利什曼原虫的无鞭毛体在人类皮肤和内脏疾病过程中合成的。此外，我们将这些基因与绿色荧光蛋白(-GFP)串联构建，以确定负责将这些酶靶向寄生虫分泌途径的独特分子信号。涉及基因缺失、突变和过度表达的实验正在进行中，以证明这些分泌酶实际上对这些人类病原体的生存是必不可少的。此外，还与Olafson博士的实验室合作进行了对L.donovani SACP的物理/结构分析。这些质谱学研究的结果表明，SACP的C-末端丝氨酸残基被由高负电荷重复单元(n=32)的磷酸二糖组成的O-连接碳水化合物侧链严重修饰。这些[-PO4-Gal-Man-]重复单位与构成寄生虫主要表膜糖脂-脂磷脂的那些单位相同。这是首次在分泌型糖蛋白和糖脂中都存在这种不寻常的磷酸二糖基团的物理证据。此外，我们发现，这些[-PO4-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700多摩尔己糖，以及这种酶对宿主蛋白降解的极端抵抗。此外，利用几种不同截短的表膜蛋白的GFP结构，我们在利什曼原虫中定义了一条独特的内体运输途径，它与分泌途径A相连，并具有调节这些生物蛋白质过度表达的功能。

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.