Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages

Abstract

Francisella tularensis, the agent of tularemia, is an intracellular pathogen, but little is known about the compartment in which it resides in human macrophages. We have examined the interaction of a recent virulent clinical isolate of F. tularensis subsp. tularensis and the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy. We assessed the maturation of the F. tularensis phagosome by examining its acquisition of the lysosome-associated membrane glycoproteins (LAMPs) CD63 and LAMP1 and the acid hydrolase cathepsin D. Two to four hours after infection, vacuoles containing live F. tularensis cells acquired abundant staining for LAMPs but little or no staining for cathepsin D. However, after 4 h, the colocalization of LAMPs with live F. tularensis organisms declined dramatically. In contrast, vacuoles containing formalin-killed bacteria exhibited intense staining for all of these late endosomal/lysosomal markers at all time points examined (1 to 16 h). We examined the pH of the vacuoles 3 to 4 h after infection by quantitative immunogold staining and by fluorescence staining for lysosomotropic agents. Whereas phagosomes containing killed bacteria stained intensely for these agents, indicating a marked acidification of the phagosomes (pH 5.5), phagosomes containing live F. tularensis did not concentrate these markers and thus were not appreciably acidified (pH 6.7). An ultrastructural analysis of the F. tularensis compartment revealed that during the first 4 h after uptake, the majority of F. tularensis bacteria reside within phagosomes with identifiable membranes. The cytoplasmic side of the membranes of approximately 50% of these phagosomes was coated with densely staining fibrils of approximately 30 nm in length. In many cases, these coated phagosomal membranes appeared to bud, vesiculate, and fragment. By 8 h after infection, the majority of live F. tularensis bacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm. These results indicate that F. tularensis initially enters a nonacidified phagosome with LAMPs but without cathepsin D and that the phagosomal membrane subsequently becomes morphologically disrupted, allowing the bacteria to gain direct access to the macrophagic cytoplasm. The capacity of F. tularensis to alter the maturation of its phagosome and to enter the cytoplasm is likely an important element of its capacity to parasitize macrophages and has major implications for vaccine development.

FIG. 1.

Live and formalin-killed F. tularensis bacteria exhibit similar kinetics of colocalization with the early endosomal marker EEA1. (A) Preopsonized formalin-killed (a to c) or live F. tularensis RCI (d and e) was incubated with THP-1 cells at 0°C to allow adherence, but not uptake, washed, warmed to 37°C to allow for internalization, fixed after 10 to 60 min, and stained by immunofluorescence for EEA1 (green) (a and d) and for F. tularensis antigen (red) (b and e). Panels c and f show the merged green and red images. The panels show THP-1 cells fixed after 15 min. Arrows indicate bacteria that colocalized with EEA1. (B) Percentages of live (RCI or LVS) or formalin-killed (FK) F. tularensis RCI bacteria colocalizing with EEA1. The data represent the means ± standard errors. The experiment was performed three times, with similar results.

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.

FIG. 3.

Quantitation of immunofluorescence staining for CD63 in THP-1 cells infected with F. tularensis RCI or LVS. Monolayers of human THP-1 cells were incubated with latex beads, formalin-killed F. tularensis, or live F. tularensis LVS or RCI as described in the text. Monolayers were washed, incubated for an additional 1 to 16 h, fixed, permeabilized, stained for CD63 and F. tularensis by using immunofluorescence reagents, and examined by confocal and two-photon laser scanning fluorescence microscopy, and the numbers of bacteria and latex beads that colocalized with CD63 were enumerated. Colocalization of the F. tularensis RCI and LVS with CD63 reached a maximum of 60 to 70% 2 to 4 h after infection. Thereafter, with continued intracellular multiplication, the colocalization of F. tularensis with CD63 steadily declined, falling to 15% by 16 h. Latex beads and formalin-killed F. tularensis colocalized extensively (70 to 95%) with CD63 at all time points. This experiment was performed twice, with similar results. Similar results were also obtained with LAMP1 and LAMP2 and when MDM were used.

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).

FIG. 5.

Quantitation of immunogold staining of F. tularensis RCI phagosomes 4 h after infection of human MDM. Histograms demonstrate 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 isotypic control mouse myeloma Igs had <0.25 gold particles per μm of membrane. The experiment was performed twice, with similar results.

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.

FIG. 7.

Calculation of phagosomal pH by quantitative immunogold staining. Human MDM were infected with the live or killed F. tularensis RCI as described for Fig. 6, and DAMP immunogold particles were enumerated in the bacterial compartments and in a reference compartment (the nucleus). The pH was calculated as described in Materials and Methods. Values shown are the means ± standard errors of at least 20 bacterial compartments from each of at least two electron microscopy grids. The experiment was performed twice, with similar results.

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 arrowheads). 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 μm. This experiment was performed twice, with similar results.

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 arrowheads). 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 μm. This experiment was performed twice, with similar results.

FIG. 9.

Quantitation of bacteria surrounded by phagosomal membranes. THP-1 cells were infected with the F. tularensis LVS or RCI and prepared for transmission electron microscopy as described in the text. Bacteria were scored as having a phagosomal membrane if at least 50% of their circumference was surrounded by a morphologically discrete membrane bilayer. Eighty to 90% of the bacteria had identifiable membrane bilayers when they were fixed immediately after a 90-min incubation with THP-1 cells (0 h); the percentage dropped to <20% by 14 h after infection. In the first 6 h after infection, 40 to 50% of the RCI phagosomes and 58 to 88% of the LVS phagosomes had densely staining fibrillar coats (data not shown). At 14 h, it was difficult to quantify the fraction of phagosomes with fibrillar coats because very few phagosomal membranes were visible. This experiment was performed twice, with similar results.