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 subseq...