Journal: Infection and Immunity

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/iai.72.6.3204-3217.2004

Figure Lengend Snippet: FIG. 2. Virulent RCI of F. tularensis colocalizes extensively with LAMP CD63 at 4 h but very little at 16 h after uptake by human THP-1 cells. THP-1 cells were infected with the RCI of F. tularensis, fixed 4 or 16 h after infection, stained for CD63 (Oregon Green), F. tularensis (Texas Red), and DNA (DAPI), and imaged by laser scanning confocal and two-photon fluorescence microscopy. After 4 h (top row), the majority of F. tularensis bacteria resided in compartments with intense staining around the rim for CD63 (arrows). After 16 h (bottom row), the bacteria had multiplied extensively and no longer colocalized with CD63. This experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Infection, Staining, Microscopy, Bacteria

Journal: Infection and Immunity

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/iai.72.6.3204-3217.2004

Figure Lengend Snippet: FIG. 4. Immunoelectron microscopy demonstrates that CD63, but not cathepsin D, is present on F. tularensis phagosomes, whereas both markers are present on latex bead phagosomes in human MDM 4 h after infection. Human monocytes were isolated from peripheral blood, differentiated for 5 days in Teflon wells, plated on tissue culture plastic, incubated with the F. tularensis RCI and latex beads as described in the text, and fixed and prepared for cryoimmunoelectron microscopy. (A) Sections were stained with immunogold for CD63 (5-nm-diameter immunogold; arrowheads) and F. tularensis (15-nm-diameter immunogold; arrows). Both the F. tularensis phagosome and the latex bead phagosome stained positively for CD63. (B) Sections were stained for cathepsin D (5-nm-diameter immunogold particles; arrowheads) and F. tularensis (15-nm-diameter immunogold particles; arrows). Whereas the latex bead phagosomes had abundant staining for cathepsin D, the bacterial phagosomes had no staining for cathepsin D. Some F. tularensis antigen (15-nm-diameter gold) was also present in compartments outside of the phagosomes (larger, open arrows).

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Immuno-Electron Microscopy, Infection, Isolation, Incubation, Microscopy, Staining

Journal: Infection and Immunity

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/iai.72.6.3204-3217.2004

Figure Lengend Snippet: FIG. 5. Quantitation of immunogold staining of F. tularensis RCI phagosomes 4 h after infection of human MDM. Histograms demon- strate the distribution of immunogold staining for CD63 (top) and cathepsin D (bottom) in phagosomes fixed 4 h after coincubation with the F. tularensis RCI and latex beads. Whereas the majority of both F. tularensis phagosomes and latex bead phagosomes acquired abundant staining for CD63, only latex bead phagosomes showed abundant staining for cathepsin D. Control sections incubated with iso- typic control mouse myeloma Igs had 0.25 gold particles per m of membrane. The experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Quantitation Assay, Staining, Infection, Control, Incubation, Membrane

Journal: Infection and Immunity

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/iai.72.6.3204-3217.2004

Figure Lengend Snippet: FIG. 6. Killed, but not live, F. tularensis RCI bacteria enter acidified compartments that stain positive for DAMP in human MDM. Human MDM were fixed 3 h after infection with formalin-killed (A) or live (B) F. tularensis RCI, and acidified compartments were identified by DAMP immunogold staining (15-nm-diameter gold particles; arrows) and immunoelectron microscopy as described in the text. F. tularensis antigen was identified by immunostaining with 5-nm-diameter gold particles (arrowheads). Abundant staining for DAMP was associated with killed F. tularensis (A) but not live F. tularensis (B). This experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Bacteria, Staining, Infection, Immuno-Electron Microscopy, Immunostaining

Journal: Infection and Immunity

Article Title: Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

doi: 10.1128/iai.72.6.3204-3217.2004

Figure Lengend Snippet: FIG. 8. F. tularensis LVS and RCI are within vacuoles with clearly discernible membranes at early times after infection, but not at late times after infection. THP-1 cells were incubated for 90 min with the F. tularensis LVS (A to D) or RCI (E to H) and fixed immediately (A and E) or after an additional 3 h (B and F), 6 h (C and G), or 14 h (D and H). Monolayers were fixed with osmium tetroxide and glutaraldehyde, stained with uranyl acetate, embedded in Epon resin, thin sectioned, enhanced for contrast with uranyl acetate and lead citrate, and viewed by electron microscopy. Host cell membranes were well preserved in all sections. Bacteria are indicated by asterisks. Immediately after infection, the majority of F. tularensis LVS (A) and RCI (E) bacteria resided in compartments with easily discernible phagosomal membranes. Many bacterial phagosomal membranes had a thick fibrillar coat radiating approximately 30 nm from the cytoplasmic aspect of the membrane (A, B, E, and F; solid arrow- heads). In other cases, the phagosomal membranes lacked these coats (e.g., white arrowheads in panel G). The coated membranes appeared to form buds (B and F [lower insert], open arrowheads), to pinch off and form vesicles (B and C, solid arrows), or to fragment (G, open arrows). By 14 h after infection, the majority of LVS (D) and RCI (H) bacteria (asterisks) lacked any identifiable phagosomal membranes. In all cases, the bacteria were separated from the host cell cytoplasm by electron lucent zones. Bars, 0.5 m. This experiment was performed twice, with similar results.

Article Snippet: Mouse monoclonal antibodies to the following antigens were purchased from the indicated sources: F. tularensis lipopolysaccharide, Serotec; transferrin receptor and CD63 (lysosomal integral membrane protein), AMAC; human LAMP-1 (clone H4A3) and LAMP-2 (clone H4B4), University of Iowa Hybridoma Bank; early endosomal antigen 1 (EEA1), Transduction Laboratories; and cathepsin D, CIBA-Corning Diagnostics.

Techniques: Infection, Incubation, Staining, Electron Microscopy, Bacteria, Membrane