CAPSULE REGULATION AND VIRULENCE IN CRYPTOCOCCUS NEOFORMANS

 描述(申请人提供)：新生隐球菌引起脑膜脑炎，每年导致超过625,000人死亡。这项研究的目标是从机制上理解这种微生物进入人体后所经历的环境变化如何刺激毒力因子的表达，从而促进真菌疾病的发生。在我们对新生葡萄球菌如何感觉到它在宿主中并协调导致毒力因子表达的转录反应的理解上存在重大差距。在这一系列事件的顶端，我们对宿主环境的个体特征如何刺激转录因子(TF)活性变化的了解是零散的。在中间水平上，我们可能还不知道隐球菌对环境变化的所有反应，也不知道诱导过程中TF活性是如何变化的。在转录因子的下游，我们不知道它们对基因表达的哪些影响对于毒力因子的发展是至关重要的。在这一新的应用中，我们建议使用分子和计算生物学的强大组合来填补这些空白，重点放在主要的隐球菌毒力因子，其多糖胶囊。在目标1中，我们将利用最新的技术进步来检测所有非必需转录因子缺失的菌株的基因表达(通过RNA-Seq)和衣壳表型(使用一种新的自动化检测和ELISA)。将这个数据集合并到我们最初的TF网络图中将大大增强它，为我们后续的研究提供信息。在目标2中，我们建议收集在被膜诱导过程中的多个时间点上所选择的TF的基因表达数据。这将允许我们通过计算将网络建模为处理来自环境的信息并协调复杂转录反应的动态电路。在目标3中，我们建议确定转导环境中每个胶囊诱导刺激的转运蛋白活性的关键变化。我们将通过计算估计这些活性变化，然后通过基因工程测试选定的TF活性变化是否足以替代给定的环境刺激。在目标4中，我们将沿着途径的下游移动，并通过计算产生假设，即编码非调控蛋白的基因的转录变化对于胶囊生长是必要的。然后，我们将通过从基因上消除相应的转录变化来检验每个假设。这一范围的研究将通过两个拥有互补技能的实验室的协同努力而得以实现。这项工作将建立在我们高生产率的第一次赠款期间提供的基础上，以阐明转录调控的基本机制，填补我们对隐球菌发病机制的理解空白，并提供资源，有力地促进从真菌发病机制到计算基因组学等领域的未来研究进展。

 DESCRIPTION (provided by applicant): The fungus Cryptococcus neoformans causes meningoencephalitis that kills more than 625,000 people each year. The goal of this research is to produce a mechanistic understanding of how the environmental changes experienced by this microbe upon entry into the human host stimulate the expression of virulence factors that advance fungal disease. There are major gaps in our understanding of how C. neoformans senses that it is in a host and coordinates a transcriptional response that leads to virulence factor expression. At the top of this chain of events, our knowledge of how individual characteristics of the host environment stimulate changes in transcription factor (TF) activity is patchy. At an intermediate level, we probably do not yet know all the TFs involved in the cryptococcal response to environmental change, nor how TF activities change during induction. Downstream of the TFs, we do not know which of their effects on gene expression are critical for the development of virulence factors. In this renewal application, we propose to use a powerful combination of molecular and computational biology to fill these gaps, focusing on the major cryptococcal virulence factor, its polysaccharide capsule. In Aim 1 we will exploit recent technical advances to assay strains deleted for all non-essential TFs for gene expression (by RNA-Seq) and capsule phenotypes (using a new automated assay and ELISAs). Incorporating this data set into our initial map of the TF network will greatly enhance it, informing our subsequent studies. In Aim 2 we propose to collect gene expression data on selected TFs at multiple time points during capsule induction. This will allow us to computationally model the network as a dynamic circuit that processes information from the environment and coordinates a complex transcriptional response. In Aim 3 we propose to identify the key changes in TF activity that transduce each capsule-inducing stimulus from the environment. We will computationally estimate these activity changes and then test, by genetic engineering, whether selected changes in TF activity are sufficient to substitute for a given environmental stimulus. In Aim 4, we will move downstream in the pathway and computationally generate hypotheses about which transcriptional changes in genes encoding non-regulatory proteins are necessary for capsule growth. We will then test each hypothesis by genetically eliminating the corresponding transcriptional change. This range of studies will be enabled by the synergistic efforts of two labs with complementary skill sets. This work will build on the foundation provided by our highly productive first grant period to elucidate fundamental mechanisms of transcriptional regulation, fill gaps in our understanding of the mechanisms of cryptococcal pathogenesis, and provide resources that will powerfully enable future research progress in areas ranging from fungal pathogenesis to computational genomics.