Chitosan in Cryptococcus

描述(申请人提供)：新生隐球菌是一种在世界范围内发现的致病真菌，可引起脑膜脑炎，特别是在免疫功能低下的人中。除非得到治疗，否则它总是致命的，而且由于固有的毒性或无法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，世界上每年有100多万新的隐球菌病病例，导致60多万人死亡。需要新的药物来治疗隐球菌，而真菌细胞壁是一个有吸引力的目标，因为它是独一无二的 对真菌和人类都不存在。我们已经证明了壳聚糖，甲壳素的脱乙酰化形式，是隐球菌细胞壁的关键成分，并且是使用 隐球菌病小鼠模型。这对小鼠的持久性也是必要的--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要快得多。我们的研究进一步确定了在该真菌中生产壳聚糖所需的关键酶。由于隐球菌是一种可操控的生物，具有强大的遗传系统和可靠的动物模型，是研究壳聚糖生物合成和壳聚糖在疾病进展中作用的良好模型系统。在这一新的应用中，我们建议扩大我们对壳聚糖生物合成和壳聚糖在发病机制中作用的研究，长期目标是描述壳聚糖生物合成所必需的成分及其相互作用。在三个具体目标中，我们将解决以下问题：目标1.如何实现对由单一几丁质合成酶(八个)和单一几丁质合成酶调节剂(三个)产生的甲壳素进行脱乙酰化？目的2.壳聚糖的生产需要哪些蛋白质，它们是如何相互作用的？我们已经鉴定了一些蛋白质，并将测试它们是否具有限制性，以及它们是否共定位并形成复合体。我们还将探索是否有更多的蛋白质是壳聚糖生产所必需的。目的3.为什么壳聚糖缺乏的突变体清除得如此之快？宿主对壳聚糖缺陷突变体的反应是否与野生型隐球菌的宿主反应不同，以及/或者壳聚糖缺陷细胞更容易被宿主杀死？

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?