Chitosan in Cryptococcus

描述（由申请方提供）：新型隐球菌是一种致病真菌，在世界范围内发现，可引起脑膜脑炎，尤其是在免疫功能低下的个体中。除非治疗，否则它总是致命的，并且由于固有的毒性或不能杀死真菌并防止复发，目前的抗真菌药物不足以有效治愈这种疾病。最近的研究表明，全世界每年有超过1，000，000例新的隐球菌病病例，导致超过600，000人死亡。需要新的药物来治疗隐球菌，真菌细胞壁是一个有吸引力的目标，因为它是独特的 而在人类中则不存在。我们已经证明，壳聚糖，脱乙酰化形式的几丁质，是一个关键组成部分的隐球菌细胞壁，是绝对需要的毒力，使用 隐球菌病小鼠模型。它也是在小鼠中持续存在所必需的-缺乏壳聚糖的菌株在48小时内被清除，比其他主要毒力因子快得多。我们的研究进一步确定了该真菌生产壳聚糖所需的关键酶。由于隐球菌是一种可操纵的生物体，具有强大的遗传系统和可靠的动物模型，因此它是研究壳聚糖生物合成和壳聚糖在疾病进展中的作用的极好模型系统。在这个更新的应用程序中，我们建议扩展我们的研究壳聚糖的生物合成和壳聚糖的发病机制中的作用，与长期目标划定壳聚糖生物合成的必要组件和它们之间的相互作用。在三个具体目标中，我们将解决以下问题：目标1。如何实现特异性以使由单一几丁质合成酶（八个中的）和单一几丁质合成酶调节剂（三个中的）产生的几丁质脱乙酰化？目标二。哪些蛋白质是壳聚糖生产所必需的，它们如何相互作用？我们已经鉴定了一些蛋白质，并将测试它们中是否有限制性的，以及它们是否共定位并形成复合物。我们还将探讨是否有其他蛋白质是壳聚糖生产所必需的。目标3：为什么壳聚糖缺乏突变体清除得这么快？宿主对壳聚糖缺陷突变体的反应是否不同于宿主对野生型隐球菌的反应和/或壳聚糖缺陷细胞是否更容易被宿主杀死？

DESCRIPTION (provided by applicant): Cryptococcus neoformans is a pathogenic fungus that is found world-wide and causes meningioencephalitis, particularly in immunocompromised individuals. It is invariably fatal unless treated, and the current antifungals are inadequate to effectively cure this disease, due to inherent toxicities or the inability to kill the fungus and prevent relapse. Recent studies have indicated that there are over 1,000,000 new cases of cryptococcosis in the world each year, which results in over 600,000 deaths. New agents to treat Cryptococcus are needed, and the fungal cell wall is an attractive target, since it is unique to fungi and absent in humans. We have shown that chitosan, the deacetylated form of chitin, is a critical component of the Cryptococcal cell wall and is absolutely required for virulence using a mouse model of cryptococcosis. It is also necessary for persistence in the mouse - strains lacking chitosan were cleared within 48 hrs, much more rapidly than other major virulence factors. Our studies have further identified the key enzymes required for chitosan production in this fungus. Because Cryptococcus is a manipulable organism, with a robust genetic system and solid animal models, it is an excellent model system to study chitosan biosynthesis and the role of chitosan in disease progression. In this renewal application, we propose to extend our studies on chitosan biosynthesis and chitosan's role in pathogenesis, with the long-term goal of delineating the necessary components and their interactions for chitosan biosynthesis. In three specific aims, we will address the following questions: Aim 1. How is specificity achieved to deacetylate the chitin produced by the single chitin synthase (out of eight) and the single chitin synthase regulator (out of three)? Aim 2. Which proteins are necessary for chitosan production, and how do they interact? We have already identified some of the proteins, and will test whether any of them are limiting, and whether they are co-localized and form complexes. We will also explore whether there are additional proteins that are necessary for chitosan production. Aim 3. Why are chitosan deficient mutants cleared so quickly? Is the host response to the chitosan deficient mutants different than the host response to wild type Cryptococcus and/or are the chitosan deficient cells more susceptible to killing by the host?