Link between intraphagosomal biotin and rapid phagosomal escape in Francisella

Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15-60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens.