GLYCOSYLATION MUTANTS OF LEISHMANIA

利什曼原虫是重要的热带原生动物寄生虫，导致超过1000万人患病 国际吧通过了解这种寄生虫生存和致病的机制， 可以设计和开发改进的化疗和疫苗接种策略。在这里，我们将重点放在 寄生虫表面，其被主要由脂磷酸聚糖（LPG）组成的致密糖萼覆盖。 这种GPI锚定的多聚磷酸糖，沿着结构上相关的糖缀合物， 在寄生虫存活和毒力中的作用。通过产生液化石油气突变体，然后分离基因， 由于这一缺陷，我们先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 使用这种方法来发展LPG生物合成途径的全面了解，并 了解特定的LPG基因和糖复合物如何导致疾病。这些研究将侧重于 主要利什曼原虫，皮肤利什曼病的代理人，因为它提供了许多实验优势 包括在明确定义的动物和昆虫模型中检查完整感染周期的能力， 不久之后，完整的基因组序列就可以得到。我们实验室的初步数据表明， L.主要液化石油气适用于L。也是致命的内脏利什曼病的病原体 我们的具体目标是：1）在正向遗传方法中使用LPG突变体来鉴定相关的LPG 生物合成基因;通常这些是“新的”基因，其活性以前没有在任何研究中研究过。 2）使用“反向遗传”方法，结合利什曼原虫基因组计划， 生物信息学策略，以确定候选基因，并建立其在LPG生物合成中的作用; 3）创建 在完全毒力寄生虫背景中LPG基因的无效“敲除”突变体，包括相关对照，和 然后利用这些来绘制它们对LPG家族糖缀合物的合成和结构的影响， 它们在涉及小鼠、巨噬细胞和白蛉感染的试验中对寄生虫毒力的影响;以及4） 剖析LPG关键结构域损害毒力的分子机制， 相关的糖缀合物。作为一个例子，我们将研究一种遗传模型（磷酸聚糖缺乏症）， 分离急性毒力和持久性，并探索其在疫苗策略中的效用。

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.