The proton vATPase pump is essential for escape of Francisella tularensis from the phagosome into the cytosol of human macrophages.

Francisella tularensis is a facultative intracellular bacterium that causes tularemia in many mammalian species including humans. Due to its high infectivity and morbidity and mortality, F. tularensis has been classified as Category A bioterrorism agent.The F. tularensis-containing phagosome (FCP) matures to a late endosome-like phagosome prior to bacterial escape into the cytosol of human macrophages by 2-4h, as determined by ultrastructural examination of integrity of the phagosomal membrane. Whether the FCP acquires the vATPase proton pump is not known. To determine more accurately the kinetics of disruption of the FCP in human macrophages, we have utilized a fluorescence microscopy-based phagosome integrity assay. Our strategy is based on differential labeling of vacuolar vs. cytosolic bacteria using anti-bacterial antibodies loaded into the cytosol of live primary human monocyte-derived macrophages (hMDMs) using the glass beads “ loading” technique. We show that majority of the FCPs within hMDMs become disrupted within the first 15-60 min post-infection. Our data show that within the first 15 min post-infection, the FCPs acquire the proton vATPase pump, which is gradually lost by 30-60 min post-infection, consistent with bacterial escape into the cytosol of hMDMs. Importantly, using the phagosome integrity assay, we show that specific inhibition of the proton vATPase pump blocks bacterial escape into the cytosol of hMDMs. Therefore, the proton vATPase pump is essential for disruption of the FCP and subsequent bacterial escape into the cytosol within 15-60 min of phagosome biogenesis within hMDMs.