Genetic analysis of Cryptococcus neoformans virulence

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。对四种模式真菌（S. cerevisiae，S. pombe，N. crassa、红腹锦鸡儿A. gossypii）已经完成，还有许多其他的正在进行中。人类真菌病原体新型隐球菌的基因组计划提供了血清型D菌株（JEC20）的完整基因组，产生了相关血清型的10至12倍组装体 D菌株B3501 A和致病性血清型A临床分离株H99，以及6.5倍覆盖率的不同血清型B菌株（WM 276）。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，并设计新的治疗方法。我们建议广泛应用插入突变来鉴定编码感染所必需的毒力属性的基因。C. neoformans是一种杰出的模式病原体。该生物体是单倍体，因此隐性突变可以在诱变后直接分离。生物体有一个明确的性周期，便于遗传分析。基因可以通过转化和同源重组来破坏，并且已经开发了稳健的动物模型。这些进展使得有可能满足Falkow的分子假设的毒力，这种真菌病原体。虽然基因可以通过同源重组破坏，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有力的互补方法来鉴定感兴趣的基因。我们已经优化了插入诱变，使用显性遗传标记和农杆菌作为基因传递载体，开发同源菌株进行遗传杂交和建立连锁，并实施方法来识别突变基因。在这里，我们将采用签名标记诱变进行大规模的发展和毒力的分子决定因素的分析。在目标1中，我们将使用农杆菌介导的基因传递插入标记的显性标记以使基因组饱和来产生突变体库。在目标2中，我们将进行体外筛选以鉴定毒力因子受损的突变体，结合异源宿主和培养的巨噬细胞中的筛选以鉴定候选毒力突变体。最后，在目标3中，我们将在小鼠模型中进行研究，以鉴定合并感染中毒力或组织特异性感染模式发生改变的突变体。这些研究将能够在全基因组范围内定义有助于这种常见人类真菌病原体毒力的基因集。

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.