Controlling the progression of pneumonic plague

我们这项研究计划的总体目标是使用肺鼠疫的小鼠模型系统来发现 并评估Y。鼠疫基因对疾病的发展和进展至关重要。我们将精确定位这些 候选人使用两种方法：转录谱，以揭示基因的差异调节，在 肺鼠疫的各个阶段;和正向遗传学方法来筛选/选择Y。鼠疫基因 是肺部疾病发展不可缺少的。 具体目标1。比较Y.在肺鼠疫的各个阶段。 我们以前开发了一个全基因组微阵列，以表征细菌转录组， 肺鼠疫，但这种分析在技术上限于感染的晚期阶段。所以我们会 使用定量RT-PCR检测Y的一个子集。比索的基因在整个时间过程中， 疾病这个大约250个基因的子集是基于显示出差异表达证据的基因。 感染期间的表达，以及未被微阵列技术充分探索的基因。 具体目标2。正向遗传学用于识别在肺炎发展中重要的细菌基因 瘟疫转座子位点杂交（TraSH）是一种利用负选择的基因发现策略， 识别在感染过程中必不可少的细菌基因。我们构建的微阵列将允许 我们利用一种基于TraSH的方法，使用阵列杂交来鉴定Y。鼠疫基因 在肺部感染的不同阶段出现了三次 具体目标3。分析候选毒力相关基因的重要性和作用。的基因 在前两个目标中选择的菌株将通过创建确定的Y. 鼠疫将在肺鼠疫鼠模型中检测突变株和对照株的毒力， 监测肺中的细菌增殖、向脾的传播和组织病理学，以评估 疾病表现或动力学的差异。突变菌株的表征将是 扩展到在感染期间宿主转录反应的微阵列分析， 和弗吉尼亚米勒医生在一起

Our overall goal for this research plan is to use a mouse model system for pneumonic plague to discover and evaluate Y. pestis genes critical for the development and progression of disease. We will pinpoint these candidates using two methods: transcriptional profiling to reveal genes that are differentially regulated in the various stages of pneumonic plague; and forward genetics approaches to screen/select for Y. pestis genes that are indispensable for development of pulmonary disease. Specific Aim 1. Comparative transcriptional responses by Y. pestis during the stages of pneumonic plague. We previously developed a whole genome microarray to characterize the bacterial transcriptome during pneumonic plague, but this analysis was technically limited to a late stage of infection. Therefore, we will use quantitative RT-PCR to examine a subset of Y. peso's genes throughout the entire time course of disease. This subset of approximately 250 genes is based on genes that show evidence of differential expression during infection, as well as genes that were not sufficiently explored by microarray technology. Specific Aim 2. Forward genetics to identify bacterial genes important in the development of pneumonic plague. Transposon site hybridization (TraSH) is a gene discovery strategy using negative selection to dentify bacterial genes that are essential during infection. The microarrays we have constructed will allow us to take advantage of a a TraSH-based approach using array hybridizations to identify Y. pestis genes triplicated in various stages of the pulmonary infection. Specific Aim 3. Analyzing the importance and role of candidate virulence-associated genes. The genes selected in the first two Aims will be targeted for further analysis by creating defined mutant strains of Y. pestis. Mutant and control strains will be tested for virulence in the murine model of pneumonic plague, monitoring bacterial proliferation in the lung, dissemination to the spleen, and histopathology to evaluate differences in the manifestation or kinetics of disease. The characterization of mutant strains will be extended to a microarray analysis of host transcriptional responses during infection, done in collaboration with Dr. Virginia Miller.