Outer membrane tube formation by Francisella novicida involves extensive envelope modifications and is linked with type VI secretion and alterations to the host phagosomal membrane

Francisella tularensis is a gram-negative, intracellular pathogen that causes the zoonotic disease tularemia. Due to its ease of dissemination and high lethality, F. tularensis is classified as a tier 1 select agent with potential for misuse as a bioweapon. The mechanisms by which Francisella replicates intracellularly and interacts with the host during infection are not well understood. Francisella produces spherical outer membrane vesicles (OMVs) and novel tubular extensions of its cell surface that are also released extracellularly. These OMV and outer membrane tubes (OMTs) contain Francisella virulence factors and are produced in response to amino acid starvation and during infection of macrophages. To investigate how the OMTs are formed, we used cryogenic electron tomography to examine the model Francisella spp., Francisella novicida, during in vitro culture and within the macrophage phagosome. OMT formation involved progressive alterations of the bacterial envelope, resulting in extensions of both the inner and outer membranes. A dynamic cytoplasmic structure was present at the base of the OMT that extended into the tubes during elongation, together with cytoplasmic material. OMT produced within the macrophage phagosome was associated with changes to the phagosomal membrane, suggesting a role in phagosomal escape. Consistent with this, using confocal microscopy, we observed co-localization of the Francisella type VI secretion system with the OMT, both within bacteria and in released tubular vesicles. These findings reveal the cellular transformations that occur during membrane tubulation by Francisella and provide insights into the function of membrane-derived structures during host-pathogen interactions.