Chitosan in Cryptococcus

描述(申请人提供)：真菌病原体新生隐孢子菌的细胞壁是一种重要的细胞器，它将甲壳素作为一种刚性的、不溶于水的多糖聚合物结合在一起。脱乙酰化将甲壳素转化为壳聚糖，后者是一种更灵活、更易溶的聚合物。虽然甲壳素是真菌细胞壁的重要组成部分，但壳聚糖对细胞壁完整性的重要性尚不清楚。该项目的总体目标是确定隐球菌细胞壁生物合成的关键步骤，这些步骤将成为抗真菌药物的有吸引力的靶点。我们的初步数据表明，无论是在营养生长过程中还是在小鼠肺部感染过程中，壳聚糖都是隐球菌细胞壁中丰富的成分。我们已经探索了影响壳聚糖生产的基因，并表明只有8个推定的甲壳素合成酶中的一个和3个推定的甲壳素合成酶调节因子中的一个显著影响壳聚糖的水平。此外，我们还证明了四种潜在的多糖脱乙酰酶中有三种可以产生壳聚糖。在壳聚糖生物合成途径中，缺失基因的菌株具有共同的表型，包括细胞完整性缺陷和黑色素泄漏，表明这些表型是壳聚糖大幅减少的结果。从该项目获得的数据将确定壳聚糖在新生梭菌细胞壁完整性和毒力中的作用，壳聚糖是如何附着在细胞壁上的，并确定这一方面的关键步骤，可以作为抗真菌的靶点。这个项目有三个具体目标。首先，我们将确定壳聚糖的组成以及在哺乳动物宿主的生长过程中它是如何与细胞壁相连的。在第二部分中，我们将测试这三种甲壳素脱乙酰酶在壳聚糖生物合成、细胞壁完整性和毒力方面的作用。在第三个目标中，我们将测试我们目前的模型，即一个甲壳素合成酶及其调控因子共同合成被三个甲壳素脱乙酰酶脱乙酰化的甲壳素，以生成壳聚糖，并确定这些蛋白质之间的相互作用，这些蛋白质对壳聚糖的生产至关重要。这些研究的结果将有助于更好地了解壳聚糖在新生隐球菌中的生物合成和功能，并确定壳聚糖的生产是否可以作为新的抗隐球菌药的可行的抗真菌靶点。近年来，由于艾滋病、器官移植和化疗导致的免疫功能低下患者数量的增加，真菌感染变得更加普遍。系统性真菌感染是严重的健康威胁，目前还没有安全、高效的抗真菌疗法。真菌细胞壁的生物合成是抗真菌治疗的一个有吸引力的靶点，因为细胞壁是人类宿主中不存在的重要细胞器，本项目将描述真菌病原体新生隐球菌细胞壁的生物合成，并确定其成分之一壳聚糖是否是潜在的抗真菌靶点。

DESCRIPTION (provided by applicant): The cell wall of the fungal pathogen C. neoformans is an essential organelle that incorporates chitin as a rigid, insoluble, polysaccharide polymer. De-acetylation converts chitin to chitosan, a more flexible and soluble polymer. While chitin is an essential component of fungal cell walls, the importance of chitosan to cell wall integrity is unclear. The overall goal of this project is to identify key steps in Cryptococcus cell wall biosynthesis that will make attractive targets for antifungal drugs. Our preliminary data indicate that chitosan is an abundant component of Cryptococcus cell walls, both during vegetative growth and during infection of mouse lungs. We have explored genes that contribute to chitosan production, and shown that only one of eight putative chitin synthases, and one of three putative chitin synthase regulators substantially impact chitosan levels. In addition, we have demonstrated that three of four potential polysaccharide deacetylases produce chitosan. The shared phenotypes among strains deleted of genes in the chitosan biosynthetic pathway, include defects in cell integrity and leaky melanin, and suggest that these phenotypes are the result of substantial reduction of chitosan. Data obtained from this project will establish the role of chitosan in C. neoformans cell wall integrity and virulence, how chitosan is attached to the cell wall, and identify critical steps in this aspect of cell wall biosynthesis that could serve as antifungal targets. This project has three specific aims. In the first, we will determine the composition of chitosan and how it is linked to the cell wall during growth in the mammalian host. In the second, we will test the roles of the three chitin deacetylases in chitosan biosynthesis, cell wall integrity and virulence. In the third aim, we will test our current model that one chitin synthase and its regulator together synthesize the chitin that is deacetylated by the three chitin deacetylases to generate chitosan and determine the interactions among the these proteins that are critical for chitosan production. The results of these studies should provide a better understanding of chitosan biosynthesis and function in C. neoformans, and determine if chitosan production would be a viable antifungal target for novel anti-cryptococcal therapy. Fungal infections have become more prevalent in recent years due to the increase in the immunocompromised patient population from AIDS, organ transplants and chemotherapies. Systemic fungal infections are serious health threats, and safe, highly effective antifungal therapies are not available. Biosynthesis of the fungal cell wall is an attractive target for antifungal therapies because the cell wall is an essential organelle that is not present in the human host, and this project will delineate the biosynthesis of the cell wall of a fungal pathogen, Cryptococcus neoformans, and determine if one of its components, chitosan, is a potential antifungal target.