Evaluating the Progression of Pneumonic Plague

肺鼠疫是由吸入鼠疫耶尔森氏菌引起的。由于其致命性、疾病快速发展以及能够通过气雾化传播，鼠疫杆菌被美国疾病控制中心归类为最有可能在生物恐怖袭击中使用的六种A类毒剂之一。尽管它具有致命性和作为人类病原体的历史，但对肺鼠疫的发病机制知之甚少，因为大多数工作都集中在腺感染上，并且使用的毒力或无毒菌株较少。在小鼠感染模型中，肺炎鼠疫表现为两个不同的阶段。在疾病的头36小时内，在没有宿主炎症或疾病病理的情况下，细菌在肺部复制成巨大的负担。36-48小时后，开始进入强烈的炎症阶段，导致严重的支气管肺炎。为了生存，鼠疫杆菌必须适应哺乳动物肺中这两种截然不同的环境，最初逃避/抑制免疫，随后抵御宿主的炎症攻击。这项应用的最终目的是通过鉴定细菌毒力因子和靶向宿主细胞来更全面地了解肺炎鼠疫的进展。表达绿色荧光蛋白(GFP)的鼠疫杆菌将被用来经鼻感染小鼠，在整个感染过程中的不同时间，将采集肺部并通过流式细胞仪进行分析，以确定鼠疫菌所针对的细胞种群，以及这些种群如何随着时间的变化而变化。为了确定与疾病进展有关的细菌因素，将使用实时定量聚合酶链式反应来评估先前显示在感染后48小时受调控的288个鼠疫耶尔森氏菌基因的转录。相关基因的转录将在感染期间的多个时间点进行评估。受高度调控或在感染阶段表现出差异调控的基因将被删除，突变菌株将在小鼠感染模型中进行评估，以确定它们在肺炎鼠疫中的作用。

Pneumonic plague results from the inhalation of the bacterium Yersinia pestis. Due to its lethality, rapid disease progression, and ability to be disseminated via aerosolization, Y. pestis is categorized by the U.S. Centers for Disease Control as one of the six Category A agents most likely to be used during a biological terrorist attack. Despite its lethality and history as a human pathogen, very little is known regarding pneumonic plague pathogenesis, as a majority of work has focused on bubonic infection and with less virulent or avirulent strains. In a mouse infection model pneumonic plague was shown to progress in two distinct phases. During the first 36 hours of disease bacteria replicate to large burdens in the lung in the absence of host inflammation or disease pathology. After 36-48 hours, an intense inflammatory phase is initiated, resulting in severe bronchopneumonia. In order to survive, Y. pestis must adapt to these two very different environments in the mammalian lung, initially evading/suppressing immunity and subsequently withstanding the host inflammatory onslaught. The ultimate goal of this application is to obtain a more complete understanding of the progression of pneumonic plague by identifying bacterial virulence factors and target host cells. Y. pestis expressing green fluorescent protein (GFP) will be used to infect mice intranasally, and at different times throughout infection lungs will be harvested and analyzed by flow cytometry to identify cell populations targeted by Y. pestis, and how those populations change over time. To identify bacterial factors involved in disease progression, quantitative real-time PCR will be used to evaluate the transcription of 288 Y. pestis genes previously shown to be regulated at 48 hours post-infection. The transcription of genes of interest will be evaluated at multiple time points during infection. Genes that are highly regulated or demonstrate differential regulation between phases of infection will be deleted, and mutant strains evaluated in a mouse model of infection to determine their role in pneumonic plague.