Genetic analysis of Cryptococcus neoformans virulence

新型隐球菌毒力的遗传分析

基本信息

批准号：
7571591
负责人：
JOSEPH HEITMAN
金额：
$ 41.57万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-06-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7571591
关键词：
Acquired Immunodeficiency Syndrome Affect Agrobacterium Alleles Anabolism Animal Model Animals Attention Biolistics Biological Process Biology Calcineurin Calmodulin Carbon Dioxide Cell Line Cells Chemotherapy-Oncologic Procedure Clinical Communities Complex Congenic Strain Coupled Cryptococcus Cryptococcus neoformans Defect Development Disease Progression Doctor of Philosophy Elements Fission Yeast Fostering Fruit Fungi Model Gene Delivery Genes Genetic Crosses Genetic Markers Genome Genomics Goals Growth Haploidy High temperature of physical object Human Human Genome Project Immunocompromised Host In Vitro Individual Infection Insertional Mutagenesis Inverse Polymerase Chain Reaction Iron Learning Libraries Life Cycle Stages Link Lung Mediating Melanins Modeling Molecular Molecular Analysis Morbidity - disease rate Mus Mutagenesis Mutate Mutation Neuraxis Nitric Oxide Nourseothricin Organ Transplantation Organism Partner in relationship Pathogenesis Pathway interactions Patients Pattern Phenotype Physiological Plasmids Process Production Research Personnel Resistance Resources Saccharomyces cerevisiae Series Serotyping Signal Transduction Site Specificity Spleen Staging Tissues Tropism Virulence Virulence Factors Yeasts base capsule congenic cost design genetic analysis genome sequencing genome-wide homologous recombination in vivo interest macrophage mortality mutant novel novel therapeutic intervention pathogen programs

项目摘要

DESCRIPTION: One of the most exciting advances in fungal biology is the application of genomics approaches. The genomes of four model fungi (S. cerevisiae, S. pombe, N. crassa, A. gossypii) are complete, and many others are in progress. The genome project for the human fungal pathogen Cryptococcus neoformans has provided the complete genome for the serotype D strain (JEC20), generated 10 to 12X assemblies for the related serotype D strain B3501A and the pathogenic serotype A clinical isolate H99, and 6.5X coverage for a divergent serotype B strain (WM276). Our challenge is to capitalize upon these genomic resources to elucidate the molecular basis of virulence, and to devise novel therapies. We propose to broadly apply Insertional mutagenesis to identify genes encoding virulence attributes necessary for infection. C. neoformans is an outstanding model pathogen. The organism is haploid, so recessive mutations can be directly isolated following mutagenesis. The organism has a defined sexual cycle, facilitating genetic analysis. Genes can be disrupted by transformation and homologous recombination, and robust animal models have been developed. These advances make it possible to satisfy Falkow's molecular postulates of virulence for this fungal pathogen. While genes can be disrupted by homologous recombination, targeting requires long regions of homology (about 1000 bp) and efficiency is not optimal. Random insertional mutagenesis provides a powerful complementary approach to identify genes of interest. We have optimized insertional mutagenesis using a dominant genetic marker and agrobacterium as the gene delivery vehicle, developed congenic strains to conduct genetic crosses and establish linkage, and implemented approaches to identify the mutated genes. Here, we will employ signature tagged mutagenesis to conduct a broad scale analysis of the molecular determinants of development and virulence. In aim 1, we will generate banks of mutants using agrobacterium-mediated gene delivery to insert tagged dominant markers to saturate the genome. In aim 2, we will conduct in vitro screens to identify mutants compromised for virulence factors, combined with screens in heterologous hosts and cultured macrophages to identify candidate virulence mutants. Finally, in aim 3, we will conduct studies in murine models to identify mutants from pooled infections that are altered in virulence or tissue-specific patterns of infection. These studies will enable a genome-wide definition of the gene set contributing to virulence of this common human fungal pathogen.

描述：真菌生物学最令人兴奋的进步之一是基因组学方法的应用。四种模型真菌的基因组（S. cerevisiae，S。Pombe，N。Crassa，A。Gossypii）是完整的，并且正在进行许多其他。人类真菌病原体加密球菌的基因组项目为Der型D菌株（JEC20）提供了完整的基因组，生成了相关血清型的10至12倍组件 D菌株B3501a和致病性血清型A临床分离株H99和6.5倍的覆盖范围，用于发散血清型B菌株（WM276）。我们的挑战是利用这些基因组资源来阐明毒力的分子基础，并设计新的疗法。我们建议广泛应用插入诱变，以识别编码感染所需的毒力属性的基因。 C. Neoformans是一种出色的模型病原体。生物体是单倍体，因此可以在诱变后直接分离隐性突变。该生物具有定义的性周期，促进了遗传分析。转化和同源重组可能会破坏基因，并且已经开发出健壮的动物模型。这些进步使得能够满足Falkow的这种真菌病原体的毒力分子假设。尽管基因可以通过同源重组而破坏基因，但靶向需要长的同源区域（约1000 bp），并且效率不是最佳的。随机插入诱变提供了一种强大的互补方法来识别感兴趣的基因。我们已经使用主要的遗传标记和农杆菌作为基因递送车进行了优化的插入诱变，开发了先天性菌株来进行遗传杂交并建立链接，并实施了鉴定突变基因的方法。在这里，我们将采用签名标记的诱变来对发育和毒力的分子决定因素进行广泛的分析。在AIM 1中，我们将使用农杆菌介导的基因输送来生成突变体的库，以插入带有标记的主要标记以使基因组饱和。在AIM 2中，我们将进行体外筛查，以鉴定因毒力因子而折衷的突变体，并结合异源宿主和培养的巨噬细胞中的筛查，以鉴定候选毒力突变体。最后，在AIM 3中，我们将在鼠模型中进行研究，以鉴定出在毒力或组织特异性感染模式中改变的聚集感染的突变体。这些研究将使全基因组的定义对基因集有助于这种常见的人类真菌病原体的毒力。