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：
7371944
负责人：
JOSEPH HEITMAN
金额：
$ 41.4万
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/7371944
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 Condition 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 Other Therapy Partner in relationship Pathogenesis Pathway interactions Patients Pattern Phenotype Physiological Plasmids Polymerase Chain Reaction Process Production Research Personnel Resistance Resources Saccharomyces cerevisiae Series Serotyping Signal Transduction Site Specificity Spleen Staging Therapeutic Intervention Tissues Tropism Virulence Virulence Factors Yeasts base capsule congenic cost design genetic analysis genome sequencing homologous recombination in vivo interest macrophage mortality mutant novel novel therapeutics 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展之一是基因组学方法的应用。四种模式真菌（酿酒酵母、粟酒裂殖酵母、粗糙脉孢菌、棉曲霉）的基因组已完成，还有许多其他模式真菌的基因组正在研究中。人类真菌病原体新型隐球菌基因组计划提供了D血清型菌株（JEC20）的完整基因组，为相关血清型生成了10至12X的组装体 D 菌株 B3501A 和致病性血清型 A 临床分离株 H99，以及不同血清型 B 菌株 (WM276) 的 6.5 倍覆盖率。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来识别编码感染所需毒力属性的基因。新型隐球菌是一种杰出的模型病原体。该生物体是单倍体，因此可以在诱变后直接分离隐性突变。该生物体具有明确的性周期，有利于遗传分析。基因可以通过转化和同源重组来破坏，并且已经开发出稳健的动物模型。这些进展使得满足福尔科夫对这种真菌病原体毒力的分子假设成为可能。虽然基因可以通过同源重组来破坏，但靶向需要长同源区域（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补充方法来识别感兴趣的基因。我们使用显性遗传标记和农杆菌作为基因传递载体优化了插入诱变，开发了同源菌株来进行遗传杂交并建立连锁，并实施了识别突变基因的方法。在这里，我们将采用特征标记诱变对发育和毒力的分子决定因素进行广泛的分析。在目标 1 中，我们将使用农杆菌介导的基因传递来插入标记的显性标记以使基因组饱和，从而生成突变体库。在目标 2 中，我们将进行体外筛选，以确定毒力因子受损的突变体，并结合异源宿主和培养巨噬细胞中的筛选，以确定候选毒力突变体。最后，在目标 3 中，我们将在小鼠模型中进行研究，以识别来自混合感染的毒力或感染组织特异性模式发生改变的突变体。这些研究将对导致这种常见人类真菌病原体毒力的基因组进行全基因组定义。