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体内诱导，因此鉴定这些毒力性状可能需要在体内检测。本提案的主要目的是开发和实施基于秀丽隐杆线虫的全动物基因组分析，以研究真菌毒力。秀丽隐杆线虫模型提供了蠕虫存活/死亡的明确分析终点，允许使用机器人填充分析板，并允许使用板阅读器和成像显微镜自动读取数据。本提案中详细的研究有望开发基于秀丽隐杆线虫真菌发病机制系统的高通量发病机制分析，并将这些分析用于鉴定毒力性状。这些研究将扩展秀丽隐杆线虫的真菌发病机制模型，并确定与发病机制基本相关的基因，而不依赖于所使用的模型系统。这一提议有两个目的：1.；开发大量随机的新型隐球菌突变体，并实施秀丽隐球菌检测，以鉴定毒力相关基因；实施全动物秀丽隐杆线虫试验，以鉴定与白色念珠菌和光秃念珠菌感染相关的毒力特征。基于我们之前的秀丽隐杆线虫实验，包括利用类似于Aim 1中提出的文库的新生秀丽隐杆线虫实验，以及我们对念珠菌文库的研究，我们预计将鉴定出总共800- 1000个低毒突变体。根据我们对这个模型的经验，我们预计我们将能够在大多数这些突变体中识别出被破坏的基因，这些基因中的大多数将参与哺乳动物感染。重要的是，毒力基因将被确认、研究和优先排序，来自突变体的所有表型数据将通过公共数据库访问，所有菌株将公开可用。我们预计这些收集将成为研究真菌发病机制的重要资源。秀丽隐杆线虫是一种令人兴奋的、伦理上有利的、快速和廉价的替代宿主来研究真菌的发病机制。秀丽隐杆线虫试验可以最大限度地减少哺乳动物的痛苦，并使在病原体和宿主都可以基因操纵的系统中研究真菌发病机制成为可能。我们期望这个模型系统将扩展我们对真菌发病机制的进化保守元素的理解，并为研究真菌毒力提供一种新的令人兴奋的方法。此外，这些研究将作为未来研究其他病原体的范例，即利用相对简单的模型宿主在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。本提案的主要目的是开发高通量，基于全动物秀丽隐杆线虫的检测方法，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，并有助于确定真菌发病机制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.