Acquisition of the Vacuolar ATPase Proton Pump and Phagosome Acidification Are Essential for Escape of Francisella tularensis into the Macrophage Cytosol

Abstract

The Francisella 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), ϳ90% of the FCPs acquire the proton vacuolar ATPase (vATPase) pump and the lysomotropic dye LysoTracker, which concentrates in acidic compartments, similar to phagosomes harboring the Listeria monocytogenes control. 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 the iglD mutant 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 the iglD mutant into the macrophage cytosol. In contrast, colocalization 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 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.

A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1

“…The phagosome subsequently gains late endosomal markers such as Rab7, CD63, LAMP-1 and LAMP-2 (Clemens et al, 2004;Santic et al, 2005a). This late endosome-like compartment acquires the proton vacuolar ATPase pump and becomes transiently acidified but does not associate with lysosomal markers, such as cathepsin D (Clemens et al, 2004;Santic et al, 2005aSantic et al, , 2008. Francisella is thus able to prevent phagosomal-lysosomal fusion, and within 4 h of uptake actively breaks down the phagosomal membrane in order to escape into the host-cell cytosol and replicate.…”

“…A number of genes, particularly within the FPI, have been shown necessary for phagosomal escape and intracellular replication; however, most of these studies have not investigated the nature of the gene products nor provided any evidence to support a mechanism of action of the gene products (Bonquist et al, 2008;de Bruin et al, 2007;Nano et al, 2004;Santic et al, 2005bSantic et al, , 2008. Studies of mutants of Francisella novicida that have knockouts of genes encoding phosphatases suggest that one or more of them play a role in phagosome membrane degradation.…”

Putting the Jigsaw Together - A Brief Insight Into the Tularemia

“…It is still under debate whether the FCP is acidified before disruption of the membrane by F. tularensis. There are experimental data demonstrating the progressive acidification of the vacuole by acquiring vacuolar ATPase before phagosomal disruption [35,36], as well as contradictory data that exclude FCP acidification [32,37,38]. Whether this discrepancy is due to different experimental conditions or different infectious agents is not yet clear.…”

Avirulent strains of Toxoplasma gondii infect macrophages by active invasion from the phagosome

“…Previous studies have described a similar process of entry into bone marrow-derived macrophages by using live video microscopy, although this phenomenon was seen only with a small portion of virulent type I strain RH parasites (13). Many intracellular pathogens (such as Listeria and Francisella) were reported able to escape from phagosome into the cytosol for multiplication, a process that depends on phagosome acidification and can be blocked by inhibition of vacuolar-ATPase (14,15). To determine whether PVM formation by phagocytosed PTG depends on phagosome maturation, RAW264.7 cells were pretreated with Bafilomycin A1 (Baf A1) followed by infection with parasites in the absence of the drug.…”