Francisella tularensisPhagosomal Escape Does Not Require Acidification of the Phagosome

Abstract

Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosomeassociated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by "F. tularensis subsp. novicida," indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.

Disease and mortality among Yesso scallops Patinopecten yessoensis putatively caused by infection with Francisella halioticida

“…Although the ultrastructural appearance of the bacteria in this study (Fig. 3) is similar to some specimens of F. tularensis in host cells depicted by Clemens et al (2009) andLian (2016), they look different from the electron micrographs presented for F. halioticida by Brevik et al (2011). Also, the bacteria measurements re corded in this study via TEM fall within the size range of Francisella spp.…”

Subversion of Host Recognition and Defense Systems by Francisella spp

“…It is interesting to note that acidification is actually required for the rapid phagosomal escape of the intracellular pathogen Listeria monocytogenes (27). Studies with Francisella have shown a brief acidification of the FCP that is dependent on the route of uptake, although there has been debate about whether this step is required for rapid escape and about its effect on the outcome of infection, as detailed below (49,52,53,194).…”

“…Additionally, the authors found that the maturing FCP does not acquire high levels of the acid hydrolase cathepsin D, which is a cellular marker of acidification, or the endosomal-lysosomal markers CD63, LAMP1, and LAMP2, which are cellular markers of phagosomal maturation (52,53). In contrast to the previously mentioned work using unopsonized bacteria, BFA did not significantly delay infection kinetics in these experiments using serum-opsonized bacteria (53). These data demonstrate that acidification of the FCP is not required for escape and replication of unopsonized or serum-opsonized Francisella but is required for rapid escape of unopsonized Francisella.…”

“…Concurrently, it has been reported that uptake of serum-opsonized Francisella results in acidification of a modest 20% to 30% of phagosomes containing serum-opsonized LVS and that it is not required for escape or replication (53). Additionally, the authors found that the maturing FCP does not acquire high levels of the acid hydrolase cathepsin D, which is a cellular marker of acidification, or the endosomal-lysosomal markers CD63, LAMP1, and LAMP2, which are cellular markers of phagosomal maturation (52,53).…”

Working toward the Future: Insights intoFrancisella tularensisPathogenesis and Vaccine Development

“…It also remains controversial whether exposure to acidic pH is necessary and/or sufficient for F. tularensis egress from the phagosome. Studies conducted by Clemens et al using THP1 cells (a human macrophage-like cell line) or primary macrophages derived from peripheral blood monocytes have demonstrated that FCVs harboring LVS or type A F. tularensis become only minimally acidified (pH of 6.7) and acquire limited amounts of the proton vacuolar ATPase (34,35). Additionally, use of the proton pump inhibitor bafilomycin A prior to infection of these macrophage types with F. tularensis strains does not alter the efficiency of F. tularensis phagosomal escape (34).…”