engleski

Acquisition of the vATPase proton pump and phagosome acidification are essential for escape of Francisella tularensis into the macrophage cytosol

The F. tularensis-containing phagosome (FCP) matures to a late endosome-like phagosome prior to bacterial escape into the cytosol of macrophages, where bacterial proliferation occurs. Our data show that within the first 15 min post-infection of primary human monocyte-derived macrophages (hMDMs) ; approximately 90% of the FCPs acquire the proton vATPase pump and the lysomotropic dye, LysoTracker, which concentrates in acidic compartments, similar to phagosomes harboring the Listeria monocytogenes control. Acquisition of the proton vATPase pump and the lysomotropic dye is gradually lost by 30-60 min post-infection, which coincides with bacterial escape into the cytosol of hMDMs. Co-localization of phagosomes harboring the iglD mutant with the vATPase and the LysoTracker was also transient and the loss of co-localization was faster than the wild type strain, which is consistent with faster escape of the iglD mutant into the macrophage cytosol. In contrast, co-localization of both makers with phagosomes harboring the iglC mutant was persistent, which is consistent with fusion to the lysosomes and failure of the iglC mutant to escape into the macrophage cytosol. We have utilized a fluorescence microscopy-based phagosome integrity assay for differential labeling of vacuolar vs. cytosolic bacteria using anti-bacterial antibodies loaded into the cytosol of live hMDMs. We show that specific inhibition of the proton vATPase pump by Bafilomycin A1 (BFA) blocks escape of the wild type strain of F. tularensis and the iglD mutant into the cytosol of hMDMs, similar to L. monocytogenes. The effect of BFA on blocking bacterial escape into the cytosol is completely reversible as the bacteria escape after removal of BFA. We also show that the limited fusion of the FCP to lysosomes is not due to failure to recruit the late endosomal fusion regulator, Rab7. Therefore, within few minutes of its biogenesis, the FCP transiently acquires the proton vATPase pump to acidify the phagosome, and this transient acidification is essential for subsequent bacterial escape into the macrophage cytosol.

Francisella; escape; acidification

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