title: Francisella tularensis phagosomal escape does not require acidification of the phagosome.
abstract: Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated 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.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, UCLA School of Medicine, CHS 37-121, 10833 LeConte Avenue, Los Angeles, CA 90095-1688, USA. dclemens@mednet.ucla.edu
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Clemens
authors.forename: Bai-Yu
authors.initials: BY
authors.lastname: Lee
authors.forename: Marcus A
authors.initials: MA
authors.lastname: Horwitz
mesh_terms: D000588:Amines / Q000494:pharmacology; D002478:Cells, Cultured; D004396:Coloring Agents / Q000494:pharmacology; D005604:Francisella tularensis / Q000502:physiology*; D006801:Humans; D008264:Macrophages / Q000737:chemistry* / Q000382:microbiology*; D010539:Permeability; D010588:Phagosomes / Q000737:chemistry* / Q000382:microbiology*; D013194:Staining and Labeling; D025262:Vacuolar Proton-Translocating ATPases / Q000032:analysis
chemical_list: D000588:Amines; D004396:Coloring Agents; C493330:Red DND-99; D025262:Vacuolar Proton-Translocating ATPases