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，一些毒力性状只在寄主中诱导，因此这些毒力性状的鉴定可能需要在体内进行检测。这项建议的主要目标是开发和实施基于秀丽线虫的全动物全基因组检测方法，以研究真菌的毒力。线虫模型为蠕虫的存活/死亡提供了一个明确的分析终点，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进行自动读数。这项建议中详细介绍的研究有望开发基于线虫真菌致病系统的高通量致病分析，并实施这些分析来鉴定毒力性状。这些研究将扩展线虫真菌致病机制的模型，并识别与致病相关的基本基因，而不依赖于所使用的模型系统。这一建议有两个目的：1.建立大量随机的新生隐球菌突变株，并实施线虫试验以鉴定与毒力相关的基因；2.实施全动物线虫试验以鉴定与白色念珠菌和光滑念珠菌感染相关的毒力性状。基于我们之前的秀丽隐杆菌库分析，包括使用与Aim 1中建议的文库类似的新生隐球菌库的分析，以及我们对念珠菌文库的研究，我们预计总共鉴定出800-1000个低毒突变株。根据我们在这个模型上的经验，我们预计我们将能够识别这些突变中的大多数被破坏的基因，并且这些基因中的大多数将参与哺乳动物的感染。重要的是，毒力基因将得到确认、研究和优先排序，突变株的所有表型数据将通过公共数据库访问，所有菌株都将公开可用。我们预计，这些收集将成为研究真菌发病机制的工作人员的重要资源。线虫是研究真菌致病机理的一种令人兴奋的、伦理上有利的、快速的和廉价的替代宿主。秀丽线虫的检测将哺乳动物的痛苦降至最低，并使在病原体和宿主都可以通过基因操作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大我们对真菌致病的进化保守元件的理解，并为研究真菌的毒力提供一种新的和令人兴奋的方法。此外，这些研究将成为未来研究其他病原体的范例，利用相对简单的模型宿主，可以在全基因组范围内研究体内的感染过程。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。这项建议的主要目标是发展高通量、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。这个模型可以最大限度地减少哺乳动物的痛苦，并有助于识别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.