Regulation of Virulence of Cryptococcus neoformans

描述(申请人提供)：我们对允许真菌病原体成功解释和响应宿主环境的基因网络的了解是初级的，落后于管理细菌和病毒病原体毒力的调控电路的更复杂的知识。我们试图利用我们在人类真菌病原体新生隐球菌中开发的工具来解决这个问题。新生葡萄球菌是一种机会主义的酵母病原体，每年导致100多万人感染和60万人死亡。在已发表的研究中，我们确定了几种转录调控因子，它们控制着受感染小鼠的病原体适应性。其中最关键的是Gat201 GATA家族蛋白，它控制毒力、包膜形成和抑制吞噬的能力。我们最近确定，Gat201控制着~16%的新生葡萄球菌基因组对环境信号的反应。为了确定其直接靶点，我们建立了针对新生葡萄球菌的染色质免疫沉淀微阵列(CHIP-CHIP)方法。在Gat201直接结合和调控的基因中，有7个转录因子，这表明存在转录网络。其中两个调节子(Liv3和Cir1)与致病性有关，我们最近证明另一个调节子Gat204控制小鼠感染期间的适合性和体外吞噬的抑制。这些数据表明，新生假单胞菌实施了一个至少涉及四个调节器的网络来控制毒力。我们试图了解这个网络是如何运作的。鉴定该网络的靶标还为识别新的毒力机制提供了机会。我们寻求确定该网络必须激活以促进疾病的机制。因此，我们的目标如下：(1)确定Gat201毒力网络的结构和功能特性。我们将定义每个调控因子的直接和间接转录靶标。对网络架构的分析预计将产生可测试的预测，而这些预测无法从单个监管机构的研究中推断出来。同时，我们将确定这些转录调控因子在感染中是否具有相似或不同的作用。通过描述控制毒力的关键转录因子的功能以及它们之间的相互关系和靶基因，这些研究有望揭示调控因子如何协作控制病原菌的毒力特性。(2)利用调控网络识别新的非包膜非依赖毒力机制。有趣的是，Gat201激活了参与甲壳素合成的三种酶的编码基因的表达。因此，我们将检验这样的假设，即Gat201激活甲壳素和/或甲壳素衍生的多糖的产生，而这些是新生葡萄球菌抑制巨噬细胞吞噬从而提高毒力所必需的。如果我们反驳这一假设，我们将采取另一种方法，在监管网络的目标中测试基因敲除。这些研究有望揭示新的机制，其被网络激活是病原体成功所必需的。

DESCRIPTION (provided by applicant): Our understanding of gene networks that allow fungal pathogens to successfully interpret and respond to the host environment is rudimentary, and lags behind the more sophisticated knowledge of regulatory circuits that govern virulence of bacterial and viral pathogens. We seek to exploit tools we have developed in the human fungal pathogen Cryptococcus neoformans to approach this question. C. neoformans is an opportunistic yeast pathogen responsible for over 1 million infections and 600,000 deaths annually. In published studies, we identified several transcriptional regulators that control pathogen fitness in infected mice. Pivotal among these is the Gat201 GATA family protein that controls virulence, capsule formation, and the ability to inhibit phagocytosis. We recently determined that Gat201 controls the response of ~16% of the C. neoformans genome to environmental signals. To identify its direct targets we have developed chromatin immunoprecipitation-microarray (ChIP-chip) methods for C. neoformans. Among the genes that are directly bound and regulated by Gat201 are seven transcription factors, suggesting the existence of a transcriptional network. Two of these regulators (Liv3 and Cir1) have been implicated in pathogenicity and we have recently demonstrated that another, Gat204, controls fitness during infection of mice and the inhibition of phagocytosis in vitro. These data suggest that C. neoformans implements a network involving at least four regulators to control virulence. We seek to understand how this network functions. Characterizing targets of this network additionally provides an opportunity to identify novel virulence mechanisms. We seek to identify the mechanisms the network must activate to promote disease. Thus, our aims are as follows: (1) Determine the architecture and functional properties of the Gat201 virulence network. We will define the direct and indirect transcriptional targets of each regulator. Analysis of the network architecture is expected to yield testable predictions that could not be inferred from the study of individual regulators. In parallel, we will determine whether these transcriptional regulators have similar or distinct roles in infection. By delineating the functions of the key transcription factors that control virulence and their relationships to each other and target genes, these studies are anticipated to reveal how regulatory factors collaborate to control the virulence properties of the pathogen. (2) Exploit the regulatory network to identify novel capsule-independent virulence mechanisms. Intriguingly, Gat201 activates the expression of genes coding for three enzymes involved in chitin synthesis. Thus, we will test the hypothesis that Gat201 activates the production of chitin and/or chitin-derived polysaccharides and that these are necessary for C. neoformans to inhibit phagocytosis by macrophages thereby promoting virulence. Should we disprove this hypothesis, an alternative approach will be taken in which we test knockouts in targets of the regulatory network. These studies are anticipated to reveal novel mechanisms whose activation by the network is necessary for pathogen success.