Francisella tularensis subsp. tularensis strain:genetically engineered Genome sequencing. Francisella tularensis subsp. tularensis strain:genetically engineered

Purine biosynthetic mutants of intracellular bacterial pathogens exhibit significant attenuation in mammalian cells and mice. In this study, we found that growth of F. tularensis with knockout of purMCD was rescued by the cell permeable ribonucleoside, AICAR, which activates nutrient and energy sensing kinase, adenosine monophosphate protein kinase (AMPK). AICAR rescued F. tularensiswith knockout of purMCD intracellular growth in cultured eukaryotic cells but not free-living replication in chemically defined media. Analysis by high performance liquid chromatography (HPLC) revealed that addition of exogenous AICAR during infection of cultured cells led to accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) which activates AMPK. In contrast, AICAR did not rescue knockout purMCD mutant growth in AMPK knockout cells, demonstrating that AICAR rescue was dependent on AMPK. Lastly, we aimed to determine if AICAR mediated AMPK activation could rescue the knockout purMCD mutant in a mouse model but did not observe significant differences in growth compared to controls, which was likely due to low bioavailability or activity of AICAR in lung tissues as phosphorylated AMPK was not detected in lung tissue of AICAR treated mice. Overall, our studies demonstrate an interplay between bacterial biosynthetic purine mutants and AMPK activation which further demonstrates the importance of host metabolism in F. tularensis intracellular growth.