AIDS Opportunistic Infections: Virulence of C. glabrata

描述（由申请人提供）：hiv阳性人群发生粘膜念珠菌病的风险增加，在艾滋病晚期，发生播散性念珠菌病的风险增加。与阴道和食道念珠菌病HIV阳性人群的情况相呼应，临床系列显示，现在弥散性念珠菌病中约有20%是由光滑念珠菌引起的。本续的目的是了解在播散性感染中重要的毒力基因的调控。C. glabrata在染色体的亚端粒区编码大量细胞壁蛋白，其中一些我们已经证明在毒力中是重要的。然而，由于从鸟枪序列中组装亚端粒区域的困难，对亚端粒区域的描述很差。我们建议克隆和测序单个亚端粒，以确定在那里编码的基因的完整补充；我们假设注释的亚端粒假基因实际上是功能性的，并有助于毒力。我们之前已经证明亚端粒区域受转录沉默的影响，我们已经从基因上定义了沉默所需的许多成分。我们建议生成关键沉默蛋白的功能标记版本，并使用ChIP来探测沉默染色质结构。沉默黏附素基因在响应环境信号时被转录抑制；我们将使用染色质免疫沉淀来识别染色质的结构变化，这些变化是对这些生理信号的反应。我们筛选了182株因非必需酿酒葡萄球菌转录因子的同源或旁同源而被删除的光斑葡萄球菌菌株。我们已经确定了两个重要的毒力调节机制。Mediator的Ssn2/Ssn3/Ssn8/Srb8模块的缺失导致毒力增加10倍，而缺失酿酒酵母中一个鲜为人知的应激基因调控因子Imp2′，则大大降低了对C. glabrata的毒力。我们将确定这两个调控子下游的调控基因。我们的初步证据表明，亚端粒基因受到基于染色质的沉默，是这两个调节因子的关键目标。我们假设它们通过改变RNA聚合酶II c端结构域（CTD）的磷酸化状态来影响感染特异性转录。这项拨款建立在我们以前的努力，这已经独特地专注于毒力策略的光棘球蚴。在白色念珠菌的研究中，对调控毒力相关性状的转录因子的分析已经取得了很高的成果，我们相信，我们的研究将共同关注亚端粒基因的转录调控，这将为研究光滑念珠菌的毒力策略提供新的思路。

DESCRIPTION (provided by applicant): The HIV-positive population is at increased risk for mucosal candidiasis and, in late stage AIDS, for disseminated candidiasis. Mirroring what is seen in the HIV positive population for vaginal and esophageal candidiasis, clinical series show that C. glabrata now accounts for about 20% of disseminated candidiasis. The objective of this continuation is to understand the regulation of virulence genes important in disseminated infections. C. glabrata encodes a large number of cell wall proteins in the subtelomeric regions of the chromosome, some of which we have demonstrated to be important in virulence. The subtelomeric regions are poorly described, however, due to difficulties in assembling subtelomeric regions from shotgun sequence. We propose to clone and sequence individual subtelomeres to identify the full complement of genes encoded there; we hypothesize that the annotated subtelomeric pseudogenes are in fact functional and contribute to virulence. We have previously shown that the subtelomeric regions are subject to transcriptional silencing and we have genetically defined many components required for silencing. We propose to generate functional tagged versions of the key silencing proteins and use these to probe the silent chromatin structure using ChIP. Silent adhesin genes are transcriptionally de-repressed in response to environmental signals; we will use chromatin immunoprecipitation to identify the structural changes in chromatin that occur in response to those physiological signals. We have screened a set of 182 C. glabrata strains deleted for orthologues or paralogues of non- essential S. cerevisiae transcription factors. We have identified two regulatory mechanisms important in virulence. Deletions of the Ssn2/Ssn3/Ssn8/Srb8 module of Mediator results in a 10-fold increase in virulence, and deletion of Imp2', a poorly understood regulator of stress genes in S. cerevisiae, profoundly decreases virulence in C. glabrata. We will identify the regulated genes downstream of these two regulators. Our preliminary evidence suggests that sub-telomeric genes, subject to chromatin based silencing, are key targets of both regulators. We hypothesize that they affect infection-specific transcription by altering the phosphorylation status of the RNA polymerase II C-terminal domain (CTD). This grant builds on our previous efforts, which have been uniquely focused on virulence strategies of C. glabrata. Analysis of transcription factors regulating virulence-associated traits has been highly productive in the study of C. albicans, and we believe that our proposed studies, jointly focused on transcriptional regulation of subtelomeric genes will shed new light on virulence strategies in C. glabrata. PUBLIC HEALTH RELEVANCE: The ability of Candida glabrata, an important yeast pathogen, to cause disease is not well understood. We are studying the regulation of virulence genes encoded in the subtelomeric regions of the genome. We have identified two key transcriptional regulators that dramatically alter virulence and are studying how they control the infectious process.