Genetic analysis of Cryptococcus neoformans virulence

描述：真菌生物学中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(S.cerevisiae、S.pombe、N.crassa、A.Cotsypii)的基因组已经完成，还有许多其他模式真菌正在进行中。人类真菌病原体新生隐球菌的基因组计划已经提供了D血清型菌株(JEC20)的完整基因组，为相关血清型产生了10到12个组合 D株B3501a和致病A型临床分离株H99，并对一株不同的B型菌株(WM276)进行6.5倍覆盖。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识别编码感染所必需的毒力属性的基因。新生葡萄球菌是一种优秀的模式病原菌。这种生物体是单倍体，所以隐性突变可以在诱变后直接分离出来。这种有机体有明确的性周期，便于进行基因分析。基因可以通过转化和同源重组来破坏，并且已经建立了健壮的动物模型。这些进展使得满足Falkow对这种真菌病原体毒力的分子假设成为可能。虽然基因可以被同源重组打乱，但靶向需要很长的同源区域(约1000bp)，效率并不是最优的。随机插入突变为识别感兴趣基因提供了一种强大的互补方法。我们使用显性遗传标记和农杆菌作为基因传递载体，优化了插入突变，开发了进行遗传杂交和建立连锁的同源菌株，并实施了鉴定突变基因的方法。在这里，我们将使用签名标记突变来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产生突变体库，以插入标记的显性标记来饱和基因组。在目标2中，我们将进行体外筛选，以鉴定毒力因子妥协的突变体，并结合异源宿主和培养的巨噬细胞的筛选来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，以从联合感染中识别毒力或组织特异性感染模式改变的突变。这些研究将能够在全基因组范围内定义导致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.