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

引起图拉热症的图拉氏方济氏菌是一种高度毒力的细胞内病原体，是一种一级选择 有可能导致严重疾病的制剂，如空气中的生物武器。图拉氏丝孢子虫的毒力是 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。由于需要专门的设施来处理完全毒力的图拉氏菌，因此许多 这一领域的研究是用密切相关但毒性较小的弗朗西塞氏菌菌株进行的，包括 新城疫杆菌对小鼠有很强的毒力，但对人类没有毒力。肺炎是最致命的一种 图拉热病，而方济氏菌在肺部的靶细胞是肺泡巨噬细胞。F。 图拉尔牛与人AM的关系尚未见报道。该项目的总体目标是确定关键机制 人类AM寄生的基础，使用一种方法将人类AM与 完全毒力和减毒的弗朗西塞氏菌菌株。 特异靶1：人肺泡内图拉氏丝虫的全球基因表达反应 巨噬细胞(AM)在细菌细胞内定位的不同阶段。鉴定弗朗西斯氏菌基因的变化 在i)细菌培养和ii)原代人AM中的表达对应于不同的阶段 细菌的细胞内周期(吞噬/胞质/复制)。AM会感染任何一种高毒力 Tularensis亚种。土拉氏菌(Ft)，缺乏VI型分泌物的Ft的缺失突变体，或减毒的图拉氏菌 新冠亚种(subspnovicida，FN)识别细菌对强毒侵染所特有的寄主环境的反应。 特定目标2：通过以下途径识别针对宿主免疫途径的图拉氏杆菌基因应答 与防御蛋白产量的相关性。感染Ft和Fn的AM的先天免疫反应 将通过测量细胞因子和趋化因子进行评估。将采用多种统计数据集成方法 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。这将识别细菌对毒力Ft的独特反应，并与 在细菌细胞内生命周期的不同阶段抑制AM的先天免疫反应。 特定目标3：原代人AM中图拉氏丝虫代谢基因表达的变化使用 图拉曲霉亚种代谢网络的重建。冬虫夏草疫苗株(LVS)将包括基因组 来自Ft和Fn的注释，并使用此模型来计算代谢网络中的途径使用量。基因 在AIM 1中产生的表达数据将被映射到这个更新的模型中，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的弗朗西塞氏菌菌株。 这些研究将对图拉氏疟原虫与人AM的相互作用产生新的见解，这将指导 随后的工作旨在进一步了解其发病机制。 肺炎图拉热症，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.