Global Gene Expression Responses of Francisella tularensis to intracellular Infection of Human Alveolar Macrophages

土拉热弗朗西丝菌是引起土拉菌病的一种高致病力的细胞内病原体，属于1级选择 作为空气传播的生物武器，有可能导致严重疾病。对F.图拉纳西斯群岛 与其逃避宿主识别，抑制免疫反应和在体内复制的能力有关。 宿主细胞因为需要专门的设施来处理完全有毒的F。土拉热病， 该领域的研究已经用密切相关但毒性较小的弗朗西斯菌株进行，包括 新杀弗朗西丝氏菌，对小鼠有高度毒性，但对人无毒性。肺炎是最致命的一种 肺中弗朗西斯菌属的靶细胞是肺泡巨噬细胞。F. 还没有研究人AM的土拉热。本项目的总体目标是确定关键机制 潜在的人类AM的寄生，使用比较人类AM与 弗朗西丝氏菌属的完全毒力和减毒菌株。 具体目标1：剖面F。原代人肺泡内土拉热整体基因表达反应 巨噬细胞（AM）在细菌细胞内位置的不同阶段。确定弗朗西斯菌基因的变化 在i）细菌培养物和ii）原代人AM中，在对应于不同阶段的时间点， 细菌细胞内循环（吞噬体/胞质/复制）。AM会感染高毒力的 F.土拉热亚种土拉热杆菌（Ft），缺乏VI型分泌的Ft缺失突变体，或减毒土拉热杆菌 嗜杀亚种（Fn），以确定细菌对宿主环境的反应是唯一的毒性感染。 具体目标2：识别F。土拉热菌基因反应靶向宿主免疫途径， 与防御性蛋白质生产的相关性。Ft或Fn感染AM的天然免疫应答 将通过测量细胞因子和趋化因子来评估。将采用多种统计数据整合方法， 用于模拟宿主炎症与细菌基因反应和细菌生长之间的关系 在目标1中测量的动力学。这将鉴定对毒性Ft独特的细菌应答，并且与 在细菌细胞内生命周期的不同阶段抑制AM先天免疫应答。 具体目标3：F型。原代人AM中土拉菌代谢基因表达的变化。使用 F.土拉热亚种全北极星疫苗株（LVS）包括基因组 从Ft和Fn的注释，并使用这个模型来计算整个代谢网络的途径使用。基因 Aim 1中生成的表达数据将与此更新的模型进行映射，以探索途径的变化 在细胞内生命周期的不同阶段用于高毒力和减毒弗朗西斯菌菌株。 这些研究将对F.土拉热与人类AM的结合， 随后的工作旨在进一步了解的发病机制， 肺炎土拉菌病，为治疗干预的新策略的发展提供信息。

Francisella tularensis, the cause of tularemia, is a highly virulent intracellular pathogen and a Tier 1 select agent with the potential to cause severe disease as an airborne bioweapon. The virulence of F. tularensis is linked to its capacity to evade recognition by the host, suppress immune responses, and replicate inside of host cells. Because specialized facilities are required for work with fully virulent F. tularensis, much of the research in this field has been done with closely related but less virulent strains of Francisella, including Francisella novicida, which is highly virulent for mice but not for humans. Pneumonia is the most lethal form of tularemia, and the target cell of Francisella in the lungs is the alveolar macrophage. The interaction of F. tularensis with human AM has not been studied. The overall goal of this project is to identify key mechanisms underlying the parasitism of human AM, using an approach that compares the interactions of human AM with fully virulent and attenuated strains of Francisella. Specific Aim 1: Profile F. tularensis global gene expression responses within primary human alveolar macrophages (AM) at distinct phases of bacterial intracellular location. Identify changes in Francisella gene expression in i) bacterial cultures and ii) primary human AMs at time-points corresponding to distinct phases of the bacterial intracellular cycle (phagosomal/cytosolic/replicative). AMs will be infected with either highly virulent F. tularensis subsp. tularensis (Ft), a deletion mutant of Ft lacking type VI secretion, or attenuated F.tularensis subsp novicida (Fn) to identify bacterial responses to the host environment that are unique to virulent infection. Specific Aim 2: Identify F. tularensis gene responses that target host immune pathways through correlation with defensive protein production. Innate immune responses in AMs infected with either Ft or Fn will be assessed by measuring cytokine and chemokine. Multiple statistical data integration approaches will be used to model the relationship between host inflammation with bacterial gene responses and bacterial growth kinetics measured in Aim 1. This will identify bacterial responses unique to virulent Ft and that correlate with suppression of AM innate immune responses at distinct stages of the bacteria's intracellular lifecycle. Specific Aim 3: Model F. tularensis metabolic gene expression changes within primary human AM. Use a metabolic network reconstruction of F. tularensis subsp. holarctica vaccine strain (LVS) to include genome annotations from Ft and Fn and use this model to compute pathway usage across the metabolic network. Gene expression data generated in Aim 1 will be mapped against this updated model to explore shifts in pathway usage in highly virulent and attenuated Francisella strains at distinct stages of the intracellular lifecycle. These studies will yield novel insights into the interaction of F. tularensis with human AM that will guide subsequent work directed at further understanding of the mechanisms underlying the pathogenesis of pneumonic tularemia, informing the development of novel strategies for therapeutic intervention.