BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ENZYMES SECRETED BY LEISHMANIA

这个项目中正在进行的研究涉及基本细胞 利什曼原虫的生物学、生物化学和分子生物学 人类的原生动物病原体。所有利什曼原虫 经历一个周期的发育周期：1)在哺乳动物中，它们 转化为专性胞内[无鞭毛体]并繁殖为专性胞内[无鞭毛体] 宿主巨噬细胞的吞噬-溶酶体系统内的形式和 2)在其嗜血的沙蝇媒介宿主中，它们分化 转化为胞外[前鞭毛体]，并以胞外[前鞭毛体]形式在 昆虫？S的消化道。这种有机体的不同物种导致 全世界有超过1200万例人类疾病。在受感染的地方 人类，这些寄生虫破坏皮肤内的巨噬细胞或 内脏(脾、肝和骨髓)引起 毁容的溃疡性皮肤损害(例如，由L. 墨西哥)或退行性和致命的内脏疾病(例如，由 L.donovani)。我们实验室的研究已经证实 利什曼原虫是顶端极化的分泌细胞， 它们结构性地分泌超过40种不同的可溶性蛋白质， 糖蛋白和碳水化合物组分进入其生长介质 在试管中。这种可溶性的、细胞外分泌的产品很容易 从这些寄生虫身上扩散开来，渗透到它们的环境中。 在自然界中，这些生物居住和繁殖在不同的 在它们的几个宿主中的水微环境。因为这些 生物体主动分泌各种不同的酶，这些酶 可以改变这样的宿主微环境，理解 这些寄生虫酶的性质似乎是必不可少的。为此， 几种寄生虫分泌酶的研究旨在确定 它们在生存、维持、生长和发育中的功能作用 这些生物的传播。此外，编码这些的基因 首次对蛋白质进行鉴定和表征 确定它们在寄生虫过程中的表达和调控 生长发育和分化。在过去的一年里，我们 研究已经阐明了它的酶活性、基因结构和 杜氏利什曼原虫几丁质酶的染色体定位我们 还表明，所有致病性利什曼原虫都具有高度的 该基因的保守位点并表达抗原性相关 分泌型几丁质酶活性。同样，几个人的基因 独特的利什曼分泌酸的不同成员 多诺瓦尼乳杆菌磷酸酶家族的特征和显示 在其他相关利什曼物种中功能保守。 这些基因与绿色荧光的融合构建 制备蛋白质(-GFP)，并用其描绘靶向 这些酶中编码的信号。涉及到的实验 基因缺失、基因突变、基因过度表达和基因重组 在确定它们的编码酶是否是， 事实上，对这些人类病原体的生存至关重要。

Ongoing studies in this project concern the basic cell biology, biochemistry and molecular biology of Leishmania, a group of protozoan pathogens of humans. All Leishmania parasites undergo a cyclic developmental cycle: 1) in mammals, they transform into and multiply as obligate intracellular [amastigote] forms within the phago-lysosomal system of host macrophages and 2) in their hematophagous sandfly-vector hosts, they differentiate into, and multiply as, extracellular [promastigote] forms within the insect?s alimentary tract. Various species of this organism cause over 12 million cases of human disease worldwide. In infected humans, these parasites destroy macrophages within the skin or internal organs (spleen, liver and bone marrow) causing either disfiguring ulcerative cutaneous lesions (e.g., caused by L. mexicana) or degenerative and fatal visceral disease (e.g., caused by L. donovani). Studies from our laboratory have established that Leishmania parasites are apically polarized secretory cells and that they constitutively secrete over 40 different soluble protein, glycoprotein and carbohydrate constituents into their growth media in vitro. Such soluble, extracellularly secreted products can readily diffuse away from these parasites and permeate their environments. In nature, these organisms reside and multiply within various aqueous micro-environments in their several hosts. Since these organisms actively secrete a variety of different enzymes which could alter such host microenvironments, an understanding of the nature of these parasite enzymes seems essential. To that end, several parasite secretory enzymes are investigated toward defining their functional roles in the survival, maintenance, growth and transmission of these organisms. Further, genes encoding these proteins are being 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 L. donovani. We also showed that all pathogenic Leishmania possess a highly conserved locus for this gene and express antigenically related secretory chitinase activities. Similarly, the genes for several different members of the unique leishmanial secretory acid phosphatase family were characterized from L. donovani and shown to be functionally conserved in other related leishmanial species. Fusion constructs of these genes in tandem with green fluorescent protein (-GFP) were made and used to delineate the targeting signals encoded in these enzymes. Experiments involving gene-deletion, -mutation, -over expression and - reconstitution are in progress toward determining whether their encoded enzymes are, in fact, essential to the survival of these human pathogens.