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小鼠模型系统来发现 并评估对疾病发生和进展至关重要的鼠疫耶尔森氏菌基因。我们将查明这些 候选者使用两种方法：转录分析以揭示在 肺鼠疫的各个阶段；和正向遗传学方法来筛选/选择鼠疫耶尔森氏菌基因 这对于肺部疾病的发展是必不可少的。 具体目标 1. 鼠疫耶尔森氏菌在肺鼠疫阶段的转录反应比较。 我们之前开发了全基因组微阵列来表征细菌转录组 肺鼠疫，但这种分析在技术上仅限于感染的晚期阶段。因此，我们将 使用定量 RT-PCR 来检查 Y. peso 基因的子集在整个时间过程中 疾病。这个大约 250 个基因的子集基于显示差异证据的基因 感染期间的表达，以及微阵列技术未充分探索的基因。 具体目标 2. 正向遗传学鉴定在肺炎发展中重要的细菌基因 瘟疫。转座子位点杂交 (TraSH) 是一种利用负选择来发现基因的策略 鉴定感染过程中必需的细菌基因。我们构建的微阵列将允许 我们利用基于 TraSH 的方法，使用阵列杂交来识别鼠疫耶尔森氏菌基因 在肺部感染的各个阶段重复三次。 具体目标 3. 分析候选毒力相关基因的重要性和作用。基因 在前两个目标中选择的菌株将通过创建确定的 Y 突变株进行进一步分析。 鼠疫。突变株和对照菌株将在肺鼠疫鼠模型中进行毒力测试， 监测肺部细菌增殖、扩散至脾脏以及组织病理学以评估 疾病表现或动力学的差异。突变菌株的特征将是 扩展到感染期间宿主转录反应的微阵列分析，合作完成 与弗吉尼亚·米勒博士。

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.