Controlling the progression of pneumonic plague

我们这项研究计划的总体目标是使用肺炎鼠疫的小鼠模型系统来发现 并评估鼠疫耶尔森氏菌对疾病发展和进展至关重要的基因。我们会找出这些 候选人使用两种方法：转录图谱，以揭示差异调控的基因 肺鼠疫的不同阶段；以及筛选鼠疫耶尔森氏菌基因的正向遗传学方法 对于肺部疾病的发展是不可或缺的。 具体目的1.比较鼠疫耶尔森氏菌在肺鼠疫阶段的转录反应。 我们之前开发了一个全基因组微阵列来表征细菌转录组在 肺鼠疫，但从技术上讲，这一分析仅限于感染的晚期。因此，我们将 使用定量RT-PCR在整个时间过程中检测Y.Peso的基因子集 疾病。这个大约250个基因的子集是基于显示出差异的证据的基因 感染过程中的基因表达，以及微阵列技术没有充分探索的基因。 具体目标2.正向遗传学鉴定肺炎发病过程中重要的细菌基因 瘟疫。转座子位点杂交(Trash)是一种利用负选择来发现基因的策略 确定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 美国将利用一种基于垃圾的方法，使用阵列杂交来鉴定鼠疫杆菌基因 在肺部感染的不同阶段有三重感染。 具体目的3.分析候选毒力相关基因的重要性和作用。基因 在前两个AIMS中选择的将通过创建Y.突变株进行进一步分析。 鼠疫。突变株和对照株将在肺炎鼠疫小鼠模型中进行毒力测试， 监测肺部细菌增殖、扩散至脾和组织病理学，以评估 疾病表现或动力学上的差异。突变菌株的特征将是 扩展到与合作完成的感染期间宿主转录反应的微阵列分析 弗吉尼亚·米勒医生。

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.