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Infect Immun. Oct 2003; 71(10): 5940–5950.
PMCID: PMC201066

An Attenuated Strain of the Facultative Intracellular Bacterium Francisella tularensis Can Escape the Phagosome of Monocytic Cells

Abstract

The facultative intracellular bacterium Francisella tularensis is a highly virulent and contagious organism, and little is known about its intracellular survival mechanisms. We studied the intracellular localization of the attenuated human vaccine strain, F. tularensis LVS, in adherent mouse peritoneal cells, in mouse macrophage-like cell line J774A.1, and in human macrophage cell line THP-1. Confocal microscopy of infected J774A.1 cells indicated that during the first hour of infection the bacteria colocalized with the late endosomal-lysosomal glycoprotein LAMP-1, but within 3 h this colocalization decreased significantly from approximately 60% to 30%. Transmission electron microscopy revealed that >90% of bacteria were not enclosed by a phagosomal membrane after 2 h of infection, and some bacteria were in vacuoles that were only partially surrounded by a limiting membrane. Similar findings were obtained with all three host cell types. Immunoelectron microscopy performed with an F. tularensis LVS-specific polyclonal rabbit antiserum showed that the antiserum stained a thick, evenly distributed capsule-like material in bacteria grown in broth. In contrast, intracellular F. tularensis LVS cells were only marginally stained with this antiserum. Instead, most of the immunoreactive material was diffusely localized in the phagosomes or was associated with the phagosomal membrane. Our findings indicate that F. tularensis LVS is able to escape from the phagosomes of macrophages via a mechanism that may involve degradation of the phagosomal membrane.

The facultative intracellular bacterium Francisella tularensis is a highly virulent microorganism capable of infecting many mammalian species (20). In nature, hares, rabbits, and rodents appear to be highly susceptible to the resulting disease, tularemia, and often the outcome is fatal in these animals. It is assumed that vectors such as ticks and mosquitoes feed on the infected animals, sustaining the life cycle of F. tularensis and spreading the disease to humans (8). The clinical presentations of tularemia are related to the virulence of the infecting bacterium. There are two clinically important subspecies of F. tularensis, F. tularensis subsp. tularensis and F. tularensis subsp. holarctica (41). Strains of the former subspecies cause a rapidly progressive disease that may be fatal even with a proper antibiotic regimen. In contrast, strains of F. tularensis subsp. holarctica cause a nonfatal infection that presents with flu-like symptoms and prominent lymph node enlargement (8).

The mouse, a natural host of F. tularensis, serves as an excellent experimental model for tularemia (17). Regardless of the port of entry, the bacterium eventually spreads to the liver and spleen, and if the infection is not controlled in these organs, a fatal outcome results. In this experimental model, the human live vaccine strain, F. tularensis LVS, is often used (10). Although attenuated for humans, this strain shows relatively high virulence in the mouse (17). The various phases and protective host defense mechanisms of the experimental infection have been well described (5, 11, 42, 46), but information regarding the intracellular life cycle is scarce. F. tularensis is capable of infecting many types of eukaryotic cells, and the primary target cells are tissue macrophages (43).

Electron microscopic studies have indicated that F. tularensis resides in a vacuolar compartment in phagocytic cells (4), and it has been suggested that the bacterium requires an acidified vacuole for intracellular growth since iron uptake is successful only under acidic conditions (15). However, other workers have indicated that after in vivo infection F. tularensis may escape the phagosome and reside in the cytoplasm of the host cells (24). In view of these contradictory findings and as part of ongoing work aimed at characterizing the effects of F. tularensis infections on mouse and human monocytic cells, we wanted to obtain a better understanding of the various phases of intracellular infection. Using transmission electron microscopy and confocal microscopy, we found that the majority of F. tularensis LVS cells, after residing in a vacuolar compartment expressing the late endosomal-lysosomal marker LAMP-1, escaped from the phagosome within a few hours after ingestion.

MATERIALS AND METHODS

Bacteria and mammalian cells.

The live vaccine strain, F. tularensis LVS, was supplied by the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Md. It was grown on modified Thayer-Martin agar at 37°C to the logarithmic phase and was suspended in phosphate-buffered saline (PBS) before addition to cell cultures. A strain of Salmonella enterica serovar Typhimurium expressing green fluorescent protein (GFP) was a kind gift from Eduard Galyov, Institute for Animal Health, Compton, Berkshire, United Kingdom.

F. tularensis LVS expressing GFP was obtained by cloning the promoter for GroEL from F. tularensis LVS (12) into the SmaI/PstI sites upstream of the Cm gene in plasmid pKK214 (29). The M-2 mutant of GFP, which exhibits increased fluorescence compared to the fluorescence of the wild-type GFP, was a kind gift from Brendan Cormack (6). The M-2 gene was cloned into the PstI/EcoRI sites of the pKK214Cm plasmid and thus was under control of the GroEL promoter. The resulting plasmid was introduced into F. tularensis LVS by cryotransformation (34). When the GFP-expressing strain (F. tularensis LVS-GFP) was injected into mice and bacteria were recovered after 5 days of infection from the spleens, >98% of the isolates expressed GFP. The replication of this strain in macrophage cell line J774A.1 and in livers and spleens of infected mice was identical to that of the wild-type strain. F. tularensis LVS-GFP was maintained on modified Thayer-Martin agar containing 5 μg of tetracycline per ml.

Murine macrophage-like cell line J774A.1 and human monocytic cell line THP-1 were both obtained from the American Type Culture Collection, Manassas, Va. Peritoneal exudate cells (PEC) were obtained from BALB/cJ mice 3 days after intraperitoneal injection of 2 ml of 3% thioglycolate. They were grown in Dulbecco's modified Eagle medium (DMEM) (GIBCO BRL, Grand Island, N.Y.) with 10% (vol/vol) fetal calf serum. The THP-1 cell cultures were treated with of 100 ng of phorbol myristate acetate per ml 4 h prior to infection with F. tularensis to make the cells adherent.

F. tularensis LVS-specific antiserum.

An F. tularensis LVS-specific whole-cell antiserum (anti-FT) was obtained by suspending heat-killed F. tularensis LVS cells in complete Freund's adjuvant at a concentration of 109 bacteria/ml. The suspension was used for subcutaneous immunization of rabbits according to a previously used schedule (40).

In vitro infection of mammalian cells and assay of intracellular bacterial multiplication.

One day before the start of an experiment, J774A.1 cells or THP-1 cells were detached, resuspended in culture medium, and added to wells of a 24-well tissue culture plate (for enumeration of bacteria) or placed in a 6-cm-diameter tissue culture dish (for electron microscopy). PEC were washed with DMEM and suspended in the medium used for J774A.1 cells. After incubation for 2 h at 37°C, nonadherent cells were removed. After incubation overnight at 37°C, the wells were washed and reconstituted with fresh culture medium. To each well, a suspension of bacteria was added, and bacterial uptake was allowed to occur for 1 h at 37°C. After bacterial uptake, the monolayers were washed twice and incubated for different periods of time in culture medium with 2 μg of gentamicin per ml.

To determine an appropriate multiplicity of infection (MOI) (i.e., ratio of bacterial cells to J774A.1 cells), we used an immunofluorescence technique developed to differentiate intracellular bacteria from extracellular bacteria (38). A mouse monoclonal antibody specific for the F. tularensis lipopolysaccharide was used, and 500 cells were counted. At an MOI of 200, a mean of 92% (range, 86 to 97%; 10 experiments) of the J774A.1 cells were found to contain intracellular bacteria. This MOI was generally used in this study. Using the same technique, we determined that, on average, an MOI of 200 resulted in 1.7 intracellular bacteria per cell.

To determine the numbers of intracellular bacteria, cells were washed once and lysed with 0.2 ml of 0.1% sodium deoxycholate. After addition of 1.8 ml of PBS, 100-μl portions of each lysate, serially diluted in PBS, were plated on modified Thayer-Martin agar for determination of viable counts.

Immunofluorescence confocal microscopy.

To identify the intracellular localization of bacteria and LAMP-1, glass coverslips were placed in 24-well tissue culture plates, and to each well 2 × 105 J774A.1 cells were added in 1.0 ml of DMEM. After overnight incubation, the cells were washed and infected with F. tularensis LVS-GFP as described above. S. enterica serovar Typhimurium expressing GFP was added to the J774A.1 cells at an MOI of 50. The plates were incubated in 5% CO2 at 37°C for 1 h, washed once, and then incubated for different periods of time. The medium was removed from the wells, and the coverslips were washed once in PBS. The cells were fixed with 4% formaldehyde for 10 min, washed three times in PBS, and permeabilized by incubation in PBS with 0.1% saponin. Then they were washed three times for 5 min with 2% bovine serum albumin in PBS (blocking buffer) at room temperature (RT). For staining, rat anti-mouse LAMP-1 antibody (1D4B; BD Pharmingen, Heidelberg, Germany) diluted 1:100 in 2% bovine serum albumin (BSA)-PBS supplemented with 0.1% saponin was used as a primary antibody, and incubation was carried out for 1 h. As a control, BSA-PBS supplemented with 0.1% saponin was added instead of the antibody. The coverslips were then washed and incubated with normal goat serum in PBS for 20 min and washed again, and a secondary goat anti-rat Cy3 antibody diluted 1:500 in the same buffer supplemented with 0.1% saponin was added and incubated for 30 min. The coverslips were washed and mounted. Samples were viewed with a Leica SP2 laser confocal microscope. Percentages of colocalization were calculated for an average of 100 cross-sectional images per time point.

Electron microscopy.

To evaluate the intracellular localization of F. tularensis LVS or S. enterica serovar Typhimurium by transmission electron microscopy, cells growing as monolayers in cell culture dishes with a diameter of 60 mm were infected by using the protocol described above. At different times, the cells were fixed in situ with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at RT for 2 h. After three washes with the same buffer, the cells were scraped off the dishes, suspended in PBS, transferred to tubes, and centrifuged. The pellets were fixed for 1 h with ferrocyanide-reduced osmium tetroxide in the dark for high contrast of cell membranes as described previously (22). Fifty microliters of 2% agarose was added to each of the pellets at 40°C and allowed to solidify at 4°C for 10 min. The agarose-embedded pellets were then cut into 1-mm3 pieces, which were stained en bloc with 1% uranyl acetate in 70% methanol overnight. After the pieces were washed in 70% methanol, they were dehydrated in a graded series of acetone solutions. Finally, the pieces were infiltrated and embedded under a weak vacuum in a mixture of Epon and Araldite (Fluka, Buchs, Switzerland). Ultrathin sections were cut, contrasted with uranyl acetate and lead citrate, and examined with a Zeiss EM 900 electron microscope. To visually estimate the content and behavior of the bacteria in the infected cells, short ribbons of ultrathin sections (thickness, 85 to 90 nm) were collected on Formvar-carbon-coated single-hole grids. The diameter of the holes (1 mm) allowed us to analyze a series of six or seven consecutive sections of one cell, representing a total thickness of 500 to 600 nm. Since F. tularensis bacterial cells are approximately 500 by 1,000 nm, at most seven ultrathin sections should have provided images covering a whole bacterium. As a rule, we analyzed 10 grids (around 10 μm), which were assumed to contain one-half of the eukaryotic cell volume. Ultrathin sections were cut, contrasted with uranyl acetate and lead citrate, and examined with a Zeiss EM 900 electron microscope. To determine the intracellular localization of F. tularensis LVS, 400 sections containing readily identifiable bacteria were counted.

Immunoelectron microscopy.

To localize ultrastructurally material from F. tularensis LVS that was reactive with the specific antiserum, the cultured bacteria were washed, centrifuged, resuspended in 4% paraformaldehyde in 0.1 M cacodylate buffer, and fixed for 30 min at RT. After the cells were washed with 0.1 M glycine to quench unreacted aldehyde groups, they were permeabilized with 0.05% saponin in PBS for 30 min. Then the cells were incubated with anti-FT antibody diluted in PBS containing 0.5% BSA, 0.1% normal donkey serum, and 0.05% saponin for 2 h at RT. After washing, the cells were incubated for 2 h at RT with peroxidase-conjugated F(ab′)2 fragments of donkey anti-rabbit immunoglobulin G (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) in PBS containing 0.5% BSA and 0.05% saponin. After washing in PBS, the peroxidase activity was revealed with a substrate containing 0.05% 3,3′-diaminobenzidine tetrahydrochloride and 0.03% H2O2 in 0.05% Tris HCl buffer (pH 7.6). The cells were then fixed in 1.33% OsO4 for 30 min and consecutively embedded in agarose and epoxy resins as described above. Ultrathin sections were examined without any additional counterstaining. The specificity of the staining was confirmed by replacing the primary polyclonal antibodies with normal rabbit serum.

Additionally, we performed pre-embedding immunoelectron microscopy to evaluate expression of the anti-FT-reactive material in F. tularensis LVS-infected macrophages. J774A.1 macrophages growing in cell culture dishes were infected for 2 h, fixed in situ in 4% paraformaldehyde in 0.1 M cacodylate buffer for 15 min, washed in the same buffer, scraped off the dishes, transferred to tubes, and centrifuged. After the cells were washed with 0.1 M glycine, they were treated with anti-FT serum diluted in 0.02 M PBS containing 0.5% BSA and 0.05% saponin for 3 h at RT, washed in PBS, and subsequently incubated with peroxidase-conjugated donkey anti-rabbit immunoglobulin G F(ab′)2 for 3 h at RT. The peroxidase activity was detected with diaminobenzidine-H2O2, and the cells were fixed in 1.33% of osmium tetroxide for 30 min, dehydrated in acetone, and embedded in a mixture of Epon and Araldite. Ultrathin sections were examined without additional staining. Cells incubated with normal rabbit serum instead of anti-FT serum served as a negative control. To quantify the localization of the F. tularensis-reactive material, 400 sections containing readily identifiable bacteria were counted.

RESULTS

Growth of F. tularensis LVS in monocytic cells.

The numbers of F. tularensis LVS cells in murine macrophage-like cell line J774A.1, adherent mouse PEC, and human macrophage cell line THP-1 were monitored over a 24-h period (Table (Table1).1). All types of cells supported growth of F. tularensis LVS, and the number of bacterial cells increased approximately 1.5 log10 in peritoneal cells, 2.0 log10 in THP-1 cells, and 2.5 log10 in J774A.1 cells.

TABLE 1.
Growth of F. tularensis LVS in THP-1 cells, PEC, and J774A.1 cells

Localization of F. tularensis LVS and the late endosomal-lysosomal marker LAMP-1 in J774A.1 cells.

Fusion of lysosomes to the phagosome is the primary bactericidal mechanism of macrophages (2). To determine whether vacuoles containing F. tularensis underwent fusion, we examined whether the bacteria colocalized with the late endosomal-lysosomal marker LAMP-1 using confocal microscopy. A GFP-expressing variant of F. tularensis LVS was used to localize the bacteria, and a GFP-expressing isolate of S. enterica serovar Typhimurium served as a control. At 1 h, approximately 60% of the F. tularensis LVS bacteria colocalized with LAMP-1, but the percentage decreased to 30% at 3 h (Fig. (Fig.1).1). In contrast, >60% of the S. enterica serovar Typhimurium bacteria showed colocalization at 3 h (Fig. (Fig.1),1), a result that is in agreement with the results of previous studies of Salmonella (37). A representative confocal microscopic image is shown in Fig. Fig.2.2.

FIG. 1.
Kinetics of colocalization of F. tularensis LVS-GFP or S. enterica GFP and the late endosomal-lysosomal glycoprotein LAMP-1. J774A.1 cells were infected with F. tularensis LVS or S. enterica at an MOI of 50 for 60 min, and extracellular bacteria were ...
FIG. 2.
Representative confocal microscopic images of localization of F. tularensis LVS-GFP or S. enterica GFP and the late endosomal-lysosomal marker LAMP-1 in J774A.1 cells. Macrophages were infected for 1 h with the bacteria and washed (time zero). Infected ...

Ultrastructural localization of F. tularensis LVS in mouse J774A.1 macrophage-like cells, PEC, and human THP-1 macrophages.

To determine if the reason for the relative lack of colocalization observed by confocal microscopy was escape of the bacteria or exclusion of LAMP-1, we determined the subcellular localization of the bacteria by electron microscopy. It has been demonstrated previously that infection with F. tularensis LVS has minimal cytopathogenic effects for 12 h, and therefore the cells were intact at the time used, 2 h (30).

At 2 h postinoculation, J774A.1 cells exhibited a morphology similar to that of the uninfected control cells. The majority of macrophages contained single internalized F. tularensis LVS cells in the peripheral parts of the cytoplasm, and we observed on average one or two bacteria per section (Fig. 3A to D). At high magnifications, some intracellular bacteria were clearly observed within small phagosomes (Fig. (Fig.3A),3A), while other bacteria were present in vacuoles that were only partially surrounded by the limiting membrane (Fig. (Fig.3B).3B). More than 90% of the bacteria were not enclosed by a phagosomal membrane (representative images are shown in Fig. 3C and D). These bacteria were separated from the cytoplasm only by a narrow electron-lucent space often containing vesicular, membranous, or amorphous bodies (Fig. 3C and D). Most of the intracellular bacteria exhibited an intact morphology. At 18 h, the majority of J774A.1 macrophages contained high numbers of intracellular bacteria. These bacteria were always free in the cytoplasm (Fig. (Fig.3E).3E). Although a proportion of the macrophages contained large numbers of bacteria in the cytoplasm (Fig. 3G to I), not all macrophages were apoptotic (Fig. (Fig.3H).3H). It has been shown previously that apoptosis occurs in F. tularensis LVS-infected J774 cells within 24 h (30). To better illustrate the integrity of the infected cells, lower-magnification images are shown in Fig. Fig.4.4. The subcellular localization of S. enterica serovar Typhimurium in J774 cells was assessed as a control. In this case, >90% of the bacteria were localized inside phagosomes (Fig. (Fig.5).5).

FIG.3.
Transmission electron micrographs of mouse J774A.1 macrophages infected with F. tularensis LVS at 2 h (A to D) and 18 h (E to H). (A) F. tularensis LVS localized in a phagosome. The arrowheads indicate a clearly visible phagosomal membrane. The arrow ...
FIG. 4.
Transmission electron micrographs of mouse J774A.1 macrophages infected with F. tularensis LVS at 2 h. The macrophages contain single bacteria in the cytoplasm (enclosed in boxes). The insets show higher magnifications of the bacteria. The bacteria are ...
FIG. 5.
Transmission electron micrographs of mouse J774A.1 macrophages infected with S. enterica serovar Typhimurium at 2 h. (A) S. enterica (arrow) localized in a phagosome. Magnification, ×16,000. (B) Arrowheads indicate an intact phagosomal membrane. ...

Altogether, these findings suggest that F. tularensis LVS had escaped phagosomes of J774A.1 macrophages by 2 h after infection. Therefore, we selected this time to determine whether escape also occurred after infection of other types of macrophages with F. tularensis LVS.

Electron microscopic analysis of infected adherent mouse PEC and human THP-1 cells at 2 h postinoculation resulted in findings similar to those obtained with J774A.1 cells (Fig. (Fig.6).6). Some internalized bacteria were clearly present within phagocytic vacuoles which were completely (Fig. 6A and D) or partially (Fig. (Fig.6B)6B) surrounded by a phagosomal membrane. In a majority of the cases, however, bacterium-containing phagosomes had lost the limiting membrane and bacteria were observed directly in the cytoplasm (Fig. 6C, E, and F). The majority of bacteria were separated from the cytoplasmic matrix only by an electron-translucent space, which frequently included polymorphic bodies (Fig. (Fig.6F6F).

FIG. 6.
Transmission electron micrographs of adherent mouse PEC (A to C) and human THP-1 cells (D to F) infected with F. tularensis LVS for 2 h. (A) F. tularensis LVS localized in a phagosome. The arrowheads indicate a clearly visible phagosomal membrane. Magnification, ...

Localization of anti-FT-reactive material in extracellular F. tularensis LVS.

We performed immunoelectron microscopy analysis of extracellular F. tularensis LVS grown in broth to identify the localization of the antigens recognized by the anti-FT antiserum. As shown in Fig. Fig.7A,7A, the electron-dense peroxidase reaction product selectively stained a thick and evenly distributed capsule-like material along the bacterial envelope. The thickness of the labeled layer usually varied between 80 and 100 nm. The anti-FT-reactive material was very compact and consisted of fine granular masses masking the underlying cell structures. In control experiments, anti-FT serum was replaced by normal rabbit serum, and no labeling of bacteria was observed (Fig. (Fig.7B7B).

FIG. 7.
Immunoelectron microscopic localization of the anti-FT-reactive material in extracellular F. tularensis LVS. (A) The electron-dense peroxidase reaction product stains a thick surface layer (arrowheads) of extracellular F. tularensis LVS cells. (B) No ...

Localization of anti-FT-reactive material intracellularly.

To ascertain whether intracellular F. tularensis LVS cells were stained with the anti-FT-reactive material like the bacteria grown in broth, immunoelectron microscopy of J774A.1 macrophages infected with F. tularensis LVS for 2 h was performed. A variety of bacterium-containing phagosomes are shown in Fig. Fig.8.8. Most intracellular bacteria contained only a little of the material stained with anti-FT antiserum (Fig. 8A to D) or were devoid of the stained material (Fig. 8E and F). Instead, the electron-dense peroxidase reaction product was diffusely localized in the phagosomes (Fig. (Fig.8B)8B) or in the limiting phagosomal membrane (Fig. 8C and D). In some bacterium-containing phagosomes, large defects in the stained limiting membrane were clearly seen (Fig. (Fig.8E).8E). There were intracellular bacteria that were surrounded by positively stained vesicular and membranous structures (Fig. (Fig.8F)8F) resembling the bodies shown in Fig. 3C and D but no phagosomal membrane. Less than 1% of the intracellular bacteria were evenly stained with the peroxidase reaction product in the same way as the extracellular bacteria (Fig. (Fig.7A).7A). A minority of the bacteria, approximately 20%, exhibited localized staining, as shown in Fig. 8A to D. A majority of the intracellular bacteria, approximately 80%, exhibited no staining, as shown in Fig. 8E and F.

FIG. 8.
Immunoelectron microscopic localization of the anti-FT-reactive material in infected J774A.1 macrophages at 2 h. (A) F. tularensis LVS localized in the phagosome. The electron-dense reaction product partially stains the bacterial surface (arrows). (B) ...

DISCUSSION

F. tularensis is considered a facultative intracellular bacterium that survives in the phagosomes or phagolysosomes of mononuclear cells (43). In a previous study in which electron microscopy was used the workers questioned whether phagolysosome fusion occurs in F. tularensis-infected monocytic cells, however (4). Moreover, previous electron microscopy studies of infected animals demonstrated that bacteria may be localized in the cytoplasm of infected cells (24). Also, indirect evidence has suggested that F. tularensis may not reside in a typical phagolysosome. For example, F. tularensis LVS appears to be relatively susceptible to agents present in the phagolysosome, like hydrogen peroxide (13, 28, 33). It has been shown previously that unlike the proteins of pathogens that survive in the phagolysosomal compartment, few F. tularensis LVS proteins are differentially expressed during intracellular infection (18). Thus, the long-held belief that F. tularensis is capable of surviving in a phagolysosomal compartment has been directly or indirectly questioned. The present findings may provide an explanation for some of the previous findings. Although F. tularensis LVS initially is localized in a phagosome and subsequently colocalizes with the late endosomal-lysosomal marker LAMP-1, soon thereafter most bacteria do not colocalize. The electron microscopic findings strongly indicate that F. tularensis LVS escapes from the late endosome/phagosome within a few hours after uptake and appears to be free in the cytoplasm or in organelles with incomplete membranes.

In an attempt to understand the mechanisms of escape from the phagosome, we investigated the localization of material from F. tularensis LVS that was reactive with a polyclonal anti-F. tularensis LVS rabbit antiserum. In extracellular bacteria grown in broth, most of the material localized in association with the bacterial envelope, and one possibility is that the immunoreactive material comprises the bacterial capsule. This capsule has not been well characterized, and the nature of its components is unknown (19, 39). Alternatively, the immunoreactive material may be part of the bacterial envelope since it is known that gram-negative bacteria may release membrane vesicles (21). In contrast, little or no such material was found in association with the intracellular F. tularensis LVS cells. Although it is difficult on the basis of a static technique such as an electron microscopy assay to interpret images, we hypothesize that once bacteria are inside the late endosomes/phagosomes, certain components of the bacterial capsule are rapidly released. After this shedding, there appears to be a preferential association of the immunoreactive material and the vacular membrane. This event results in the degradation of the membrane, leading to the release of F. tularensis LVS into the cytoplasm. We believe that the results of confocal microscopy showing that 30% of the bacteria still colocalized with LAMP-1 at 3 h may be an indication that F. tularensis LVS harbored in vacuoles with incomplete membranes may still appear to colocalize with LAMP-1. In a previous study Anthony et al. noted that F. tularensis LVS-infected cells contained intra- and extraphagosomal vesicles, and it was suggested that these vesicles were of bacterial origin (4). Morphologically, these vesicles are similar to those which we identified in Fig. 3C and D, and they may contain the immunoreactive material which we observed.

Previous studies on the intracellular localization of F. tularensis LVS have indicated that the bacterium is localized in a phagosomal compartment. In one study Fortier et al. used an ex vivo model (15). After 3 days of in vivo infection, peritoneal cells were obtained, and the localization of F. tularensis LVS was determined by electron microscopy. It was found that the bacterium was confined to a membrane-enclosed vacuole. One explanation for the apparent discrepancy between our findings and those of Fortier et al. may be that a mixed population of peritoneal cells was used in the latter study. It is likely that after 3 days the infected macrophages had become activated by gamma interferon (IFN-γ) produced by T cells or NK cells. Control of F. tularensis by macrophages depends strictly on IFN-γ-induced activation of the cells (3, 31). In the Listeria monocytogenes model, IFN-γ activation prevents escape of the bacterium from the phagosomes and leads to subsequent killing of the organism (7, 35). By analogy, it is possible that escape of F. tularensis may be prevented if the infected macrophages are activated with IFN-γ and that this was case in the study of Fortier et al. In another study in which electron microscopy was used, Anthony et al. infected peritoneal and bone marrow cells from mice and rats (4). Also in this study, it was claimed that the F. tularensis LVS cells were localized in phagocytic vesicles, although it was not stated if this was true for all bacteria. The only notable difference between our study and this study is that F. tularensis LVS was added to the cell cultures by centrifugation. It is not known if the protocol used for cell infection affects the intracellular localization of F. tularensis.

The demonstration by Fortier et al. that F. tularensis requires an acidified vacuole for intracellular growth (15) may appear to contradict the findings of the present study. However, similar findings have been reported for the intracellular pathogen Trypanosoma cruzi (32). Escape of this organism from the phagolysosome and multiplication in the cytoplasm have been well documented. Despite this survival strategy, multiplication of the pathogen is inhibited when phagosomal acidification is inhibited (32). It has been suggested that this is due to an inability to escape the phagolysosome. Thus, acidification may be a prerequisite not only for effective uptake of iron but also for expression of other virulence mechanisms, such as escape into the cytoplasm.

A notable finding of the present study was the relatively ineffective uptake of F. tularensis in the monocytic cells, as described previously by other workers (4, 16). This seems paradoxical considering that F. tularensis presumably requires an intracellular habitat for replication (15). Although a high MOI (200) was used, the microscopic analyses indicated that during the initial phase of the infection, on average only one or two intracellular bacteria per cell were observed regardless of the host cell type. Additional investigations are needed to determine which factors affect the uptake of F. tularensis.

F. tularensis LVS is attenuated for humans but exhibits high virulence for mice, and the histological findings for F. tularensis LVS infection in mice are similar to those for human infection caused by fully virulent F. tularensis strains. Thus, F. tularensis LVS appears to be a relevant model organism for studies of tularemia in vitro and in vivo. Nevertheless, an important future task is to determine if the intracellular survival mechanisms of fully virulent F. tularensis strains are similar to those of F. tularensis LVS.

Several other intracellular bacteria, including Listeria, Shigella, and Rickettsia, have been shown to escape the phagosome and multiply in the cytoplasm of infected cells (14, 35, 44). In most of these cases, specific mechanisms have been identified that lead to the disruption of the phagosomal membrane, thus allowing bacterial entry into the cytoplasm. In L. monocytogenes, the required proteins have been characterized and comprise the pore-forming lysteriolysin (LLO) and two phospholipases C, PlcA and PlcB. An ongoing sequencing analysis of the genome of the highly virulent isolate Schu S4 has not revealed the presence of any hemolysins or phospholipases (26, 36). Thus, it is likely that F. tularensis utilizes mechanisms distinct from those of other intracellular bacterial pathogens, like L. monocytogenes, to escape from the phagosome. Moreover, during L. monocytogenes infection, the recruitment of lysosomal proteins, such as LAMP-1, to the phagosome is blocked (1). This further emphasizes the difference in the survival strategies of L. monocytogenes and F. tularensis. The strategy of F. tularensis that has been described, escape from a compartment containing markers of a late endosome/phagosome, is unusual for intracellular pathogens. One other example is the finding that Bacillus anthracis can escape the phagolysosome (9). A recent report indicated, however, that B. anthracis is capable of expressing both hemolysins and phospholipases under anaerobic conditions (27) and thus has virulence mechanisms that are similar to those of L. monocytogenes and probably distinct from those of F. tularensis. A strategy that resembles the strategy of F. tularensis has been demonstrated for the fungus Cryptococcus neoformans, which after phagocytosis is able to disrupt the phagosomal membrane by release of polysaccharide-containing vesicles (45).

In conclusion, we demonstrated that F. tularensis LVS can survive in several types of human and murine macrophages in vitro and that the bacteria can escape from the phagosome into the cytoplasm. Although the exact mechanism of this escape is unknown, our findings support the hypothesis that this occurs as a result of bacterium-induced degradation of the phagosomal membrane. This conclusion is based on three major lines of evidence: (i) intracellular bacteria are intact and replicate; (ii) some bacterium-containing phagosomes have large defects in the limiting membrane; and (iii) there is no phagosomal membrane around the majority of internalized bacteria.

Acknowledgments

Grant support was obtained from the Defense Advanced Research Project Agency (DARPA), from the Swedish Medical Research Council, from the Medical Faculty, Umeå University, Umeå, Sweden, and from the Ministry of Education, Sport and Youth, Czech Republic (grant LN00A033).

We thank Roland Rosquist for help with confocal microscopy.

Notes

Editor: V. J. DiRita

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