GLYCOSYLATION MUTANTS OF LEISHMANIA

超出所提供的空间。利什曼原虫是一种重要的热带原生动物寄生虫，在全世界造成1000多万人患病。通过了解这种寄生虫生存和致病的机制，将有可能设计和开发改进的化疗和疫苗接种策略。这里我们关注寄生虫的表面，它被一个致密的主要由脂磷酸聚糖（LPG）组成的糖萼所覆盖。这种gpi锚定的多磷酸，以及结构相关的糖缀合物，在寄生虫的生存和毒力中起重要作用。通过产生LPG突变体，然后分离挽救这种缺陷的基因，我们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LPG生物合成途径，并了解特定LPG基因和糖缀合物如何导致疾病。这些研究将侧重于皮肤利什曼病的病原体大利什曼原虫，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，并且很快就可以获得完整的基因组序列。我们实验室的初步数据表明，这些对L. major LPG的研究也适用于L. donovani，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突变体来识别相关的LPG生物合成基因；通常，这些是“新”基因，其活性在任何生物体中都没有被研究过；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，确定候选基因，并确定其在LPG生物合成中的作用；3)在完全毒力的寄生虫背景（包括相关对照）中创建LPG基因的零“敲除”突变体，然后利用这些突变体绘制出它们对LPG家族糖结合物的合成和结构的影响，以及它们在涉及小鼠、巨噬细胞和沙蝇感染的测试中对寄生虫毒力的影响；4)剖析LPG和相关糖缀合物的关键结构域损害毒力的分子机制。作为一个例子，我们将研究一种遗传模型（磷酸甘聚糖缺乏），它将急性毒力与持久性分开，并探索其在疫苗策略中的效用。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================