GLYCOSYLATION MUTANTS OF LEISHMANIA

描述(申请人提供)：利什曼原虫是重要的热带原生动物寄生虫，全世界有1000多万人患病。通过了解这种寄生虫用于生存和致病的机制，将有可能设计和开发改进的化疗和疫苗接种策略。在这里，我们主要关注寄生虫的表面，它被一种主要由脂磷脂多糖(LPG)组成的致密的糖萼所覆盖。这种GPI锚定的多聚磷酸糖，以及结构上相关的糖共轭，在寄生虫的生存和毒力方面发挥着重要的作用。通过产生LPG突变体，然后分离修复这一缺陷的基因，我们以前已经建立了一个强大的遗传系统来探测寄生虫的毒力。我们将使用这种方法来全面了解液化石油气的生物合成途径，并了解特定的液化石油气基因和糖结合物是如何导致疾病的。这些研究将集中在皮肤利什曼病的病原体大利什曼原虫上，因为它提供了许多实验优势，包括能够在明确定义的动物和昆虫模型中检查完整的感染周期，很快就会有完整的基因组序列。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利什曼原虫。 我们的具体目标是1)利用LPG突变体以正向遗传的方法识别相关的LPG生物合成基因；通常这些是以前在任何生物体中都没有研究过活性的‘新’基因；2)使用‘反向遗传’方法，结合利什曼原虫基因组计划和生物信息学策略，识别候选基因，并确定它们在LPG生物合成中的作用；3)在全毒力寄生虫背景下创建LPG基因的零基因敲除突变体，包括相关对照，然后利用这些突变体来绘制它们对LPG家族糖结合物的合成和结构的影响，以及它们在小鼠、巨噬细胞和沙蝇感染试验中对寄生虫毒力的影响；以及4)剖析LPG及其相关糖结合物的关键结构域损害毒力的分子机制。作为一个例子，我们将研究一种将急性毒力与持久性分开的遗传模型(磷酸多糖缺乏)，并探索其在疫苗策略中的应用。

DESCRIPTION (provided by the applicant): 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.