GLYCOSYLATION MUTANTS OF LEISHMANIA

利什曼原虫是重要的热带原生动物寄生虫，导致1000多万人患病。 全世界。通过了解这种寄生虫生存和致病的机制，它将是 有可能设计和开发改进的化疗和疫苗接种战略。在这里，我们重点关注 寄生虫表面，被主要由脂磷聚糖(LPG)组成的致密的糖萼覆盖。 这种GPI锚定的多聚磷酸糖，以及结构上相关的糖共轭化合物，发挥着重要的作用 在寄生虫生存和毒力方面的作用。通过产生液化石油气突变体，然后分离出拯救 这个缺陷，我们以前已经建立了一个强大的基因系统来探测寄生虫的毒力。我们会 使用这种方法对液化石油气生物合成途径有一个全面的了解，并 了解特定的LPG基因和糖偶联物是如何导致疾病的。这些研究将集中在 大利什曼原虫，皮肤利什曼病的病原体，因为它提供了许多实验优势 包括在定义明确的动物和昆虫模型中检查完整的感染周期的能力，以及 很快，完整的基因组序列就会出来。我们实验室的初步数据表明 这些主要液化石油气的研究也适用于致死性内脏利什曼病的病原体杜氏乳杆菌。 我们的具体目标是1)使用液化石油气突变体在正向遗传方法中鉴定相关的液化石油气 生物合成基因；典型地，这些是“新的”基因，其活性以前从未在任何 生物体；2)结合利什曼原虫基因组计划，使用“反向遗传”方法和 生物信息学策略，以确定候选基因，并确定它们在液化石油气生物合成中的作用；3)创造 在完全致病的寄生虫背景下，LPG基因的零‘敲除’突变，包括相关的对照，以及 然后用它们来描绘它们对LPG家族糖共轭化合物的合成和结构的影响，以及 在小鼠、巨噬细胞和沙蝇感染试验中，它们对寄生虫毒力的影响；以及4) 剖析LPG和LPG的关键结构域损害毒力的分子机制 相关的糖共轭化合物。作为一个例子，我们将研究一种遗传模型(磷酸多糖缺乏)，它 将急性毒力与持久性分开，并探索其在疫苗策略中的用途。

Leishmania are important tropical protozoan parasites, causing disease in more than 10 million people worldwide. By understanding the mechanisms used by this parasite to survive and cause disease, it will be possible to design and develop improved chemotherapy and vaccination strategies. Here we focus on the parasite surface, which is covered by a dense glycocalyx consisting primarily of lipophosphoglycan (LPG). This GPI-anchored polyphosphosaccharide, along with structurally related glycoconjugates, play important roles in parasite survival and virulence. By generating LPG mutants and then isolating genes which rescue this defect, we have previously established a powerful genetic system to probe parasite virulence. We will use this methodology to develop a comprehensive understanding of the LPG biosynthetic pathway, and to understand how specific LPG genes and glycoconjugates contribute to disease. These studies will focus on Leishmania major, the agent of cutaneous leishmaniasis, as it offers many experimental advantages including the ability to examine the complete infectious cycle in well defined animal and insect models, and soon the complete genome sequence will be available. Preliminary data from our laboratory suggest that these studies of L. major LPG are applicable to L. donovani as well, the agent of fatal visceral leishmaniasis. Our specific aims are 1) to use LPG mutants in a forward genetic approach to identify the relevant LPG biosynthetic genes; typically these are 'new' genes whose activity has not been studied previously in any organism; 2) to use 'reverse genetic' approaches, in combination with the Leishmania genome project and bioinformatic strategies, to identify candidate genes, and establish their role in LPG biosynthesis; 3) to create null 'knockout' mutants in LPG genes in a fully virulent parasite background, including relevant controls, and then to use these to map out their effects on the synthesis and structure of LPG family glycoconjugates, and their effect on parasite virulence in tests involving infections of mice, macrophages and sand flies; and 4) to dissect the molecular mechanisms by which virulence is compromised by key structural domains of LPG and related glycoconjugates. As one example, we will study a genetic model (phosphoglycan-deficiency) which separates acute virulence from persistence, and explore its utility in vaccine strategies.