Collections > Electronic Theses and Dissertations > Identifying Host and Bacterial Factors Required for Francisella tularensis Intracellular Growth

Francisella tularensis is a small Gram negative coccobacillus that is the causative agent of the disease tularemia. A hallmark of F. tularensis pathogenesis is its extraordinary capacity to rapidly grow to high densities within the cytosol of host cells. To extensively replicate in the hostile environment of the host, the bacterium must expertly adapt to the cytosolic environment, evade destruction by immune defenses and acquire significant nutrients. However, few bacterial virulence factors have been identified and the mechanisms by which F. tularensis obtains nutrients and adapts to the cytosolic environment are poorly understood. Here we performed a large scale transposon mutagenesis screen to identify novel bacterial factors required for intracellular growth. From the screen we identified a gene of unknown function, FTT_0924, which we demonstrate is required for intracellular growth and virulence. We show FTT_0924 is required for resisting osmotic stress during bacterial replication indicating FTT_0924 is required for maintaining cell wall integrity. Together these data suggest F. tularensis requires FTT_0924 for strict control of cell wall dynamics to adapt to the cytosolic environment. To replicate to high densities within host cells, F. tularensis must efficiently acquire significant nutrient sources, yet the major sources of host derived carbon, as well as the strategies employed by the bacterium to acquire host carbon are unknown. Here we took a genetic approach to identify bacterial carbon metabolic pathways and define the major sources of host derived carbon that fuel bacterial replication. We show gluconeogenesis is essential for intracellular and in vivo growth and suggest glycerol 3-phosphate and amino acids are the primary carbon sources acquired by F. tularensis from host cells. We then investigated from where essential host derived nutrients are derived and identify host cell autophagy is required to provide F. tularensis with nutrients for bacterial replication. Specifically, we found F. tularensis infection induces flux through an ATG5-independent autophagy pathway to provide amino acids and bulk carbon to the bacterium. Overall, these studies provide significant steps in understanding how F. tularensis, and potentially other intracellular pathogens, adapt to the cytosolic environment and acquire essential nutrients for bacterial proliferation.