Regulation of Virulence of Cryptococcus neoformans

描述（由申请人提供）：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理解是初步的，并且落后于控制细菌和病毒病原体毒力的调控回路的更复杂的知识。我们试图利用我们在人类真菌病原体新型隐球菌中开发的工具来解决这个问题。C.新型酵母菌是一种机会性酵母菌病原体，每年造成超过100万例感染和60万例死亡。在已发表的研究中，我们确定了几个转录调节因子，控制感染小鼠的病原体适应性。其中最重要的是Gat 201加塔家族蛋白，其控制毒力、荚膜形成和抑制吞噬作用的能力。我们最近确定Gat 201控制了约16%的C.环境信号的新形式。为了鉴定其直接靶点，我们开发了染色质免疫沉淀芯片（ChIP芯片）方法。新人类在Gat 201直接结合和调控的基因中，有7个转录因子，这表明存在一个转录网络。其中两种调节剂（Liv 3和Cir 1）与致病性有关，我们最近证明了另一种调节剂Gat 204在小鼠感染过程中控制适应性，并抑制体外吞噬作用。这些数据表明C.新形虫实施一个涉及至少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。表征该网络的靶标还提供了鉴定新毒力机制的机会。我们试图确定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Gat 201毒力网络的结构和功能特性。我们将定义每个调节因子的直接和间接转录靶点。网络架构的分析，预计将产生可测试的预测，不能推断出从个别监管机构的研究。同时，我们将确定这些转录调节因子是否在感染中具有相似或不同的作用。通过阐明控制毒力的关键转录因子的功能及其相互关系和靶基因，这些研究有望揭示调控因子如何协同控制病原体的毒力特性。(2)利用调控网络来识别新的不依赖于胶囊的毒力机制。有趣的是，Gat 201激活了编码三种参与几丁质合成的酶的基因的表达。因此，我们将测试Gat 201激活几丁质和/或几丁质衍生的多糖的产生并且这些是C.新形式的细菌抑制巨噬细胞的吞噬作用，从而促进毒力。如果我们反驳这一假设，我们将采取另一种方法，在该方法中，我们在调控网络的目标中测试敲除。这些研究预计将揭示新的机制，其激活的网络是必要的病原体的成功。

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.