Genome-wide investigations in fungal pathogens utilizing an invertebrate model ho

描述（由申请人提供）：迫切需要开发新的方法来研究真菌的发病机制。然而，一些毒力性状仅在宿主中诱导，因此这些毒力性状的鉴定可能需要体内检测。该提案的主要目标是开发和实施基于整个动物秀丽隐杆线虫的全基因组测定，以研究真菌毒力。梭Elegans模型在蠕虫的存活/死亡方面提供了明确的测定终点，允许使用机器人填充测定板，并允许使用板读取器和成像显微镜进行自动读出。本研究的详细内容有望开发出基于C. elegans系统的真菌发病机制，并实施这些测定鉴定的毒力性状。这些研究将拓展C. elegans的真菌发病机制模型，并确定基因的基本相关的发病机制，独立的模型系统使用。这项建议有两个目的：1。建立大量的新生隐球菌随机突变株，并实施C。线虫测定以鉴定毒力相关基因，以及，2.全动物C。elegans试验来鉴定与白色念珠菌和光滑念珠菌感染相关的毒力性状。基于我们之前的C。elegans检测，包括C.利用与目标1中提出的文库相似的文库和我们对念珠菌文库的研究，我们预期鉴定总共800- 1，000个低毒性突变体。根据我们对该模型的经验，我们预计我们将能够鉴定出大多数突变体中的破坏基因，并且这些基因中的大多数将参与哺乳动物感染。重要的是，毒力基因将得到确认、研究和优先排序，突变体的所有表型数据将通过公共数据库提供，所有菌株都将公开提供。我们预计，这些收藏品将成为研究真菌致病机理的工作者的重要资源。C.线虫是一种令人兴奋的、伦理上有利的、快速的和廉价的替代宿主，用于研究真菌致病机制。C.秀丽隐杆线虫测定法使哺乳动物的痛苦最小化，并且使得能够在病原体和宿主都可以被遗传操纵的系统中研究真菌发病机理。我们预计，这个模型系统将扩大我们的理解进化保守的因素真菌致病机制，并提供了一个新的和令人兴奋的方式来研究真菌毒力。此外，这些研究将作为其他病原体的未来研究的范例，可以利用相对简单的模型宿主在体内全基因组范围内研究感染过程。公共卫生相关性：迫切需要开发新的方法来研究真菌的发病机制。该提案的主要目的是开发高通量的、基于整只动物秀丽隐杆线虫的检测方法，该方法可用于研究真菌的发病机制。该模型可以最大限度地减少哺乳动物的痛苦，并有助于确定真菌致病机理和宿主反应的一些基本方面。

DESCRIPTION (provided by applicant): There is an urgent need for the development of novel approaches to study fungal pathogenesis. However, some virulence traits are induced only in the host and therefore the identification of these virulence traits may require detection in vivo. The main objective of this proposal is the development and implementation of whole animal Caenorhabditis elegans-based, genome-wide assays to study fungal virulence. The C. elegans model provides an unambiguous assay endpoint in the survival/death of the worms, allows the use of robots for filling assay plates and permits automated readouts using plate readers and imaging microscopes. The studies detailed in this proposal are expected to develop high-throughput pathogenesis assays that are based on the C. elegans systems of fungal pathogenesis, and implement these assays for the identification of virulence traits. These studies will expand the C. elegans models of fungal pathogenesis and identify genes of fundamental relevance to pathogenesis, independent of the model system used. There are two Aims in this proposal: 1. Develop a large collection of random Cryptococcus neoformans mutants and implement C. elegans assays to identify virulence associated genes, and, 2. Implement whole animal C. elegans assays to identify virulence traits associated with Candida albicans and Candida glabrata infection. Based on our previous C. elegans assays, including C. neoformans assay that utilized a library similar to the one proposed in Aim 1 and our studies of the Candida libraries, we expect to identify a total of 800-1,000 hypovirulent mutants. Based on our experience with this model, we anticipate that we shall be able to identify the disrupted genes in the majority of these mutants and most of these genes will be involved in mammalian infection. Importantly, virulence genes will be confirmed, studied and prioritized and all phenotypic data from the mutants will be accessible via a public database and all strains will be publicly available. We anticipate that these collections will be an important resource for workers that study fungal pathogenesis. C. elegans is an exciting, ethically expedient, rapid and inexpensive substitute host to study fungal pathogenesis. C. elegans assays minimize mammalian suffering and enable the study of fungal pathogenesis in a system where both the pathogen and the host can be genetically manipulated. We anticipate that this model system will expand our understanding of evolutionarily conserved elements of fungal pathogenesis and provide a new and exciting way to study fungal virulence. Moreover, these studies will serve as a paradigm of future studies in other pathogens that the infectious process can be studied in vivo in genome-wide scale utilizing a relatively simple model host. PUBLIC HEALTH RELEVANCE: There is an urgent need for the development of new approaches to study fungal pathogenesis. The main objective of this proposal is the development of high throughput, whole animal Caenorhabditis elegans-based assays that can be used to study fungal pathogenesis. This model can minimize mammalian suffering and help identify some basic aspects of fungal pathogenesis and host response.