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（一组人类原生动物病原体）的细胞生物学、生物化学和分子生物学。所有利什曼原虫寄生虫都经历二态性生命周期：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无鞭毛体形式繁殖，最终破坏这些细胞；2) 在其昆虫载体（吸血白蛉）内，它们以细胞外前鞭毛体在消化道内形成的形式分化和繁殖，并最终迁移到口部进行传播。据世界卫生组织估计，利什曼原虫寄生虫每年在全世界引起超过 1200 万例人类疾病（利什曼病）。在受感染的人类中，这些寄生虫会破坏皮肤或内脏器官（即脾、肝和骨髓）内的巨噬细胞，导致大面积毁容的恶性皮肤溃疡（例如由墨西哥乳杆菌引起）或退行性且最常见致命的内脏疾病（例如由杜诺瓦尼乳杆菌引起）。我们实验室之前的研究已经证实，利什曼原虫可以持续分泌 40 多种不同的可溶性蛋白质、糖蛋白和碳水化合物成分。这些分泌产物可以很容易地渗透到利什曼原虫所在的宿主微环境中，并可能改变利什曼原虫所在的宿主微环境。因此，了解这些寄生虫产品的性质似乎至关重要。为此，对寄生虫分泌酶进行了研究，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，以确定它们在寄生虫生长、发育和分化过程中的表达和调节。 在这种情况下，我们之前鉴定并表征了编码独特的利什曼原虫分泌几丁质酶家族的基因。在 2008-09 财年，我们使用生化和分子相结合的方法来证明该酶家族在利什曼原虫的所有致病物种中是保守的。这表明它们必须在这组人类病原体的所有成员的生长、发育和生存中发挥重要的功能作用。在这方面，在 2008-09 财年的持续合作研究中，我们检查了墨西哥乳杆菌几丁质酶过度表达寄生虫突变体在允许的白蛉载体宿主中生存和生长的能力。我们的观察表明，这些几丁质酶过度表达转染子：1）能够以更快的速度逃离蝇宿主的围食膜区室； 2）在白蛉肠道中产生相当高的寄生虫负担； 3) 对白蛉食道瓣造成更大的损害，4) 与对照寄生虫相比，在受感染的小鼠中产生更大的损伤。总的来说，这些数据表明，寄生虫几丁质酶通过促进其在白蛉载体中的生存和传播，充当墨西哥乳杆菌的多功能毒力因子。 在其他正在进行的合作研究中，正在尝试从功能上删除墨西哥乳杆菌几丁质酶的基因，并检查这些突变体在哺乳动物（小鼠）宿主和白蛉载体中的活力和生存能力。 此外，之前我们证明，几乎所有利什曼原虫，像其他锥虫寄生虫一样，都是嘌呤营养缺陷型，因此完全依赖于从其昆虫媒介和哺乳动物宿主中回收这些必需化合物。因此，在 2008-09 财年，我们鉴定并表征了墨西哥乳杆菌中一种独特的新型 35 kDa 分泌性核酸酶的生化和功能特性。我们的研究表明，这种酶是由这种寄生虫的无鞭毛体和前鞭毛体发育形式组成型释放/分泌的。使用分子方法，我们鉴定并表征了基因 LmexNucS，该基因编码来自这些生物体的这种新的 I 类核酸酶家族成员。序列分析表明，LmexNucS 具有 P1/S1 真菌/植物分泌核酸酶家族的信号肽和五个结构基序特征。 Northern 印迹和蛋白质分析证实，LmexNucS 在寄生虫生命周期（无鞭毛体>>前鞭毛体）中进行转录和差异翻译。蛋白质印迹和酶活性分析证实 LmexNucS 是由 L.mex Pro 和无鞭毛体发育形式组成型分泌/释放的。为了描述 LmexNucS 的功能特性，该基因在 LmexNucS-HA 转染子中过量表达。抗 HA 免疫沉淀/酶活性联合测定结果表明，LmexNucS 是 N 连接糖基化的，它可以轻松降解 RNA、单链 DNA、双链 DNA 以及各种合成多核苷酸底物（即聚 A、聚 I 和聚 U）。我们进一步证明，LmexNucS 会被巯基还原剂（例如硫醇）不可逆地失活。数码地面电视。总的来说，我们的结果表明 L.mexNucs 在促进这种重要人类病原体的生长、发育和生存方面必须发挥重要作用。在这方面，这种利什曼原虫分泌性核酸酶可用于诊断或治疗目的。 2008-09 财年，我们还参与了一个有关杜氏乳杆菌分泌蛋白组蛋白质组分析的合作项目。在这些研究中，定量质谱法用于分析杜氏乳杆菌的各种发育形式在体外生长过程中释放/分泌的蛋白质。分析结果表明，这些寄生虫显然释放/分泌了超过 151 种蛋白质。计算机分析表明，大多数外泌体蛋白不具有氨基末端信号肽，该基序通常与经典真核生物内质网靶向分泌途径相关。总的来说，这项研究的结果表明，利什曼原虫的蛋白质分泌是一个异质过程，由经典信号肽途径以及多种非经典分泌途径机制（包括外泌体样微泡的释放）介导。 总而言之，我们最近和正在进行的研究结果继续为了解这些寄生虫的独特病理生理学提供相关且重要的信息。此外，这些研究对于证明特定/独特的寄生虫酶和调节蛋白是否是 1）新化疗药物的设计，2）新诊断工具的开发和/或 3）可用作针对这些人类病原体的潜在疫苗的逻辑目标具有实际意义。

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 amastigote forms 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 promastigote forms 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, 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 have been identified and characterized toward defining their expression and regulation during parasite growth, development and differentiation. In that context, previously we identified and characterized the genes encoding the unique Leishmania secretory chitinase enzyme family. In FY 2008-09, we used combined biochemical and molecular approaches to demonstrate that this enzyme family was conserved among all pathogenic species of Leishmania. This suggested that they must play significant functional roles in the growth, development and survival of all members of this group of human pathogens. In that regard, in continuing collaborative studies, in FY 2008-09, we examined the ability of L. mexicana chitinase over-expressor parasite mutants to survive and grow within a permissive sand fly vector host. Our observations showed that these chitinase over-expressor transfectants were:1) able to escape from the fly-host peritrophic membrane compartment at a faster rate; 2) produce considerably higher parasite burdens in the sandfly gut; 3) cause greater damage to the sand fly stomodeal valve and 4)to produce larger lesions in infected mice than control parasites. Cumulatively, these data showed that the parasite chitinase acts as a multifunctional virulence factor for L. mexicana by facilitating its survival and transmission in its sandfly vector. In other on going collaborative studies, attempts are being made to functionally delete the gene for the L. mexicana chitinase and to examine the viability and survivability of these mutants in both a mammalian (mouse) host and sandfly vectors. In addition, previously, we demonstrated that virtually all Leishmania sp., like other trypanosomatid parasites are purine auxotrophs and therefore are, totally dependent upon salvaging these essential compounds from their insect vector and mammalian hosts. Thus in FY 2008-09, we identified and characterized the biochemical and functional properties of a unique new, 35 kDa,secretory nuclease from L. mexicana. Our studies showed that this enzyme was constitutively released/secreted by both amastigotes and promastigote developmental forms of this parasite. Using a molecular approach, we identified, characterized the gene, LmexNucS that encodes this new Class I nuclease family member from these organisms. Sequence analysis revealed that LmexNucS possesses a signal peptide and five structural motifs characteristic of the P1/S1 fungal/plant secretory nuclease family. Northern blot and protein analyses confirmed that LmexNucS was transcribed and differentially translated through the parasites life cycle (Amastigotes>>Promastigotes). Western blot and enzyme activity analyses verified that LmexNucS was constitutively secreted/released by both L.mex Pro- and Amastigote developmental forms. In order to delineate the functional properties of the LmexNucS, the gene was episomally over-expressed in LmexNucS-HA transfectants. Results of combined anti-HA immunoprecipitation/ enzyme activity assays showed that LmexNucS was N-linked glycosylated and that it could readily degrade RNA, single stranded DNA, double stranded DNA as well as various synthetic polynucleotide substrates (i.e. poly-A, -I, and -U). Further we demonstrated that LmexNucS was irreversibly inactivated by sulfhydryl reducing agents e.g. DTT. Cumulatively, our results indicate that the L.mexNucs must play important roles in facilitating the growth, development and survival of this important human pathogen. In that regard, this leishmanial secretory nuclease might be exploited for diagnostic or for therapeutic purposes. In FY2008-09 we were also involved in a collaborative project concerning the proteomic analysis of the secretome of L. donovani. In these studies, quantitative mass spectrometry was used to analyze the proteins released/secreted by various developmental forms of L. donovani during their growth in vitro. Analyses resulted in the identification that more that 151 proteins that are apparently released/secreted by these parasites. In silico analyses showed that most of these exosomal proteins did not possess an amino-terminal signal peptide a motif typically associated with the classical eukaryotic ER-targeted secretory pathway. Cumulatively, results of this study indicated that protein secretion in Leishmania is a heterogeneous process, which is mediated by both classical signal peptide pathways as well as by multiple non-classical secretion pathway mechanisms including the release of exosome-like microvesicles. Taken together, the results of our recent and ongoing studies continue to provide pertinent and significant information toward understanding the unique pathophysiology of these parasites. In addition, these studies are of practical relevance toward demonstrating whether specific /unique parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs, 2) the development of new diagnostic tools and/or 3) useful as potential vaccines against these human pathogens.