Outer membrane tube formation by Francisella novicida involves extensive envelope modifications and is linked with...

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.

Francisella tularensis is an intracellular bacterial pathogen that causes the zoonotic disease tularemia. Following uptake by host cells, the bacteria rapidly escape the phagosome and replicate intracellularly. In previous studies, we found that Francisella produces tubular extensions of its cell surface in response to specific cues and during macrophage infection. In the present study, we used cryogenic electron tomography to examine tube formation by the model Francisella sp., F. novicida. This analysis revealed that tube formation involves extensive bacterial envelope alterations and a dynamic cytoplasmic organelle. Furthermore, tubes produced by bacteria within infected macrophages were associated with the breakdown of the phagosomal membrane. In addition, we found that the Francisella type VI secretion system, which is essential for phagosomal escape, co-localized with the bacterial tubes. These findings reveal the cellular transformations that occur during membrane tubulation by Francisella and suggest a role for the tubes in phagosomal escape.