Acquisition of the vacuolar ATPase proton pump and phagosome acidification are essential for escape of Francisella tularensis into the macrophage cytosol.
Acquisition of the vacuolar ATPase proton pump and phagosome acidification are essential for escape of Francisella tularensis into the macrophage cytosol.
复制标题
获得液泡 ATP 酶质子泵和吞噬体酸化对于土拉弗朗西斯菌逃入巨噬细胞胞质至关重要。
DOI：
10.1128/iai.00185-08
复制
发表时间：
2008
影响因子：
3.1
通讯作者：
AbuKwaik,Yousef
中科院分区：
文献类型：
--
作者：
Santic,Marina;Asare,Rexford;Skrobonja,Ivana;Jones,Snake;AbuKwaik,Yousef
TheFrancisella tularensis-containing phagosome (FCP) matures to a late-endosome-like phagosome prior to bacterial escape into the cytosols of macrophages, where bacterial proliferation occurs. Our data show that within the first 15 min after infection of primary human monocyte-derived macrophages (hMDMs), ∼90% of the FCPs acquire the proton vacuolar ATPase (vATPase) pump and the lysomotropic dye LysoTracker, which concentrates in acidic compartments, similar to phagosomes harboring theListeria monocytogenescontrol. The acquired proton vATPase pump and lysomotropic dye are gradually lost by 30 to 60 min postinfection, which coincides with bacterial escape into the cytosols of hMDMs. Colocalization of phagosomes harboring theiglDmutant with the vATPase pump and the LysoTracker dye was also transient, and the loss of colocalization was faster than that observed for the wild-type strain, which is consistent with the faster escape of theiglDmutant into the macrophage cytosol. In contrast, colocalization of both makers with phagosomes harboring theiglCmutant was persistent, which is consistent with fusion to the lysosomes and failure of theiglCmutant to escape into the macrophage cytosol. We have utilized a fluorescence microscopy-based phagosome integrity assay for differential labeling of vacuolar versus cytosolic bacteria, using antibacterial antibodies loaded into the cytosols of live hMDMs. We show that specific inhibition of the proton vATPase pump by bafilomycin A1 (BFA) blocks rapid bacterial escape into the cytosols of hMDMs, but 30% to 50% of the bacteria escape into the cytosol by 6 to 12 h after BFA treatment. The effect of BFA on the blocking of 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.
