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Infect Immun. Dec 2008; 76(12): 5488–5499.
Published online Oct 13, 2008. doi:  10.1128/IAI.00682-08
PMCID: PMC2583578

The Early Phagosomal Stage of Francisella tularensis Determines Optimal Phagosomal Escape and Francisella Pathogenicity Island Protein Expression[down-pointing small open triangle]

Abstract

Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.

The gram-negative bacterium Francisella tularensis is the etiological agent of tularemia, a widespread zoonosis that accidentally affects humans. Human tularemia is a fulminating disease that can be caused by exposure to as few as 10 bacteria, the pneumonic form of which can lead to up to 25% mortality if untreated (23). Four subspecies of F. tularensis, F. tularensis subsp. tularensis (type A), F. tularensis subsp. holarctica (type B), “F. tularensis subsp. novicida,” and F. tularensis subsp. mediasiatica, are recognized, among which strains from F. tularensis subsp. tularensis and subsp. holarctica can cause tularemia in humans (9) while F. tularensis subsp. novicida strains are virulent in rodents (9). As a facultative intracellular pathogen, F. tularensis is capable of infecting and proliferating in a variety of host cell types, including hepatocytes, endothelial cells, fibroblasts, and mononuclear phagocytes (9). Macrophages are believed to be an important target for infection in vivo, and the pathogenesis of F. tularensis depends on the bacterium's ability to survive and replicate within these host cells (9). As such, the life cycle of F. tularensis inside macrophages has been the subject of intensive research. Our current understanding of the intracellular cycle of F. tularensis stems from various in vitro models of F. tularensis infection of murine and human macrophages or macrophage-like cell lines (3, 6, 11, 28). Following phagocytic uptake, the bacteria initially reside in a phagosome before escaping into the cytoplasm via degradation of the phagosomal membrane (3, 6, 11, 28). Phagosomal escape is followed by extensive cytosolic replication and eventual programmed cell death of the macrophage (13, 15), which is accompanied by bacterial egress. Additionally, we have recently demonstrated that cytosolic bacteria can reenter the endocytic compartment and reside in large autophagic vacuoles following cytoplasmic replication (3), although the function of these organelles remains to be elucidated.

Depending on the Francisella strains and macrophage models used, phagosomal escape has been shown to occur within 1 to >4 h postentry (3, 6, 11, 28), and it remains unclear why such different kinetics have been observed. In murine primary macrophages infected with the nonopsonized strain LVS, cytosolic bacteria are detectable as early as 20 min postinfection (p.i.) (3), indicating that phagosomal escape is a rapidly induced and efficient process. Phagosomal escape of LVS in murine macrophages is nonetheless preceded by phagosomal maturation events resembling a normal maturation process (3), including interactions with early and late endosomal compartments, which have also been observed in human macrophages (6). These maturation events are expected to include acidification of the Francisella-containing phagosome (FCP), a process driven by the activity of the vacuolar ATPase, which is both required for and a consequence of phagosomal maturation (14). However, FCPs in human macrophages apparently resist acidification and do not acquire the lysosomal protease cathepsin D (6), suggesting that Francisella is capable of impairing proper phagosomal maturation. These results are inconsistent with a previous report in which phagosomal acidification was shown to be required for intracellular growth of LVS (10). Altogether, the extent of FCP maturation prior to phagosomal escape remains unclear and deserves further investigation.

A ~30-kb locus within the F. tularensis genome, organized as the Francisella pathogenicity island (FPI) (22), potentially encodes a secretion system (7) similar to that of the recently identified gene clusters encoding IcmF homology-associated proteins or type VI secretion systems (21, 25). Functions encoded by the FPI have been associated with phagosomal escape and intracellular growth, since disruptions or deletions of FPI-encoded genes, such as iglA and iglB (12), iglC (11, 16, 29), and pdpA (22), result in a defect in the intramacrophage growth of F. tularensis. Furthermore, iglC mutants in F. tularensis subsp. novicida and F. tularensis subsp. holarctica LVS strains are reportedly defective in phagosomal escape (17, 29), suggesting that IglC-dependent FPI-encoded functions are involved in the early stages of Francisella intracellular trafficking.

Cytosolic bacterial pathogens have evolved strategies to efficiently disrupt their initial phagosome and be released in the replication-permissive cytosol. Among them, Listeria monocytogenes uses listeriolysin O for phagosomal escape, a hemolysin whose optimal activity depends upon acidification of the initial phagosome (1, 24), indicating that cytosolic pathogens can take advantage of phagosomal maturation processes to efficiently reach their replication niche. As an early event in the intracellular trafficking of Francisella, phagosomal escape seems to be an essential step in the intracellular cycle of Francisella, since mutants impaired in their ability to disrupt phagosomal membranes are compromised for intracellular growth (17, 29). We have postulated that phagosomal escape is determined upon entry into host cells by the immediate environment encountered by phagocytosed bacteria, such as early intraphagosomal conditions. We have therefore examined whether trafficking events preceding phagosomal escape determine the efficiency of this process. Here we report that early FCPs containing the highly virulent F. tularensis subsp. tularensis strain Schu S4 interact with late endocytic compartments and become acidified and that these early maturation events are required for optimal phagosomal escape and intracellular proliferation. We further show that the early FCP provides signals conducive to the expression of FPI-encoded proteins independently of vacuole acidification, therefore allowing a rapid bacterial response to the intracellular environment.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

The prototypic type A virulent strain, F. tularensis subsp. tularensis Schu S4, was obtained from Anders Sjöstedt (University of Umea, Umea, Sweden). The F. tularensis subsp. novicida strain Utah112 (U112; ATCC 15482) was provided by Francis Nano (University of Victoria, Victoria, Canada). Green fluorescent protein (GFP)-expressing Schu S4 was constructed by introducing pFNLTP6ΩPgroE-gfp (18) into Schu S4 by electroporation, as described previously (18). F. tularensis subsp. novicida U112 ΔiglC::ermC (strain KKF24) has been described elsewhere (16). This mutant was complemented (strain KKF87) by introduction of the plasmid pKEK974 by electroporation (18). To construct pKEK974, both iglC and iglD were amplified by PCR using the primers iglC-down-NcoI (5′-CGCCGCCCATGGCAATGATTATGAGTGAGATGATAAC-3′) and iglD-up-EcoRI (5′-GCGAATTCTTAAGAAAAGGCTATAAAGAAATC-3′), and the resulting 1.8-kb fragment was digested with NcoI and EcoRI and cloned into the similarly digested pKEK894 (31) to generate pKEK974. This plasmid allows expression of both iglC and iglD under the control of the Francisella omp26 (FTN_1451) promoter region (31).

F. tularensis subsp. tularensis Schu S4 was grown on modified Mueller-Hinton (mMH) or cystine heart agar blood plates for 3 days at 37°C under 7% CO2. F. tularensis subsp. novicida strains were grown on tryptic soy agar supplemented with 0.1% l-cysteine (TSAC) for 1 day at 37°C. Immediately prior to infection of murine bone marrow-derived macrophages (BMMs), a few colonies from freshly streaked mMH, cystine heart agar blood (Schu S4), or TSAC (F. tularensis subsp. novicida) were resuspended in tryptic soy broth supplemented with 0.1% l-cysteine and the optical density at 600 nm was measured to estimate bacterial numbers. All manipulations of F. tularensis subsp. tularensis strain Schu S4 were performed in a biosafety level 3 facility according to standard operating procedures approved by the Rocky Mountain Laboratories Institutional Biosafety Committee.

Macrophage culture and infection.

Bone marrow cells were isolated from femurs of 6- to 10-week-old C57BL/6J female mice (Jackson Laboratories, Bar Harbor, ME) and differentiated into macrophages for 5 days at 37°C and 7% CO2 in 1 g/liter glucose Dulbecco's modified Eagle medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 10% L-cell-conditioned medium (8), and 2 mM l-glutamine in non-tissue-culture-treated petri dishes. After 5 days, loosely adherent BMMs were washed with phosphate-buffered saline (PBS), harvested by incubation in chilled cation-free PBS supplemented with 1-g/liter d-glucose on ice for 10 min, resuspended in complete medium, and replated in either 6- or 24-well cell culture-treated plates at a density of 1 × 106 or 1 × 105 macrophages/well, respectively. BMMs were further incubated at 37°C under a 7%-CO2 atmosphere for 48 h and replenished with complete medium 24 h before infection. For infections, bacterial suspensions were diluted in complete medium, and 1.5 ml or 0.5 ml was added to chilled BMMs at a theoretical multiplicity of infection (MOI) of 50 unless stated otherwise. Bacteria were centrifuged onto macrophages at 400 × g for 10 min at 4°C and infected BMMs incubated for 20 min at 37°C under a 7%-CO2 atmosphere, including an initial, rapid warmup in a 37°C water bath to synchronize bacterial uptake. Infected BMMs were then washed five times with DMEM to remove extracellular bacteria and incubated for 40 min in complete medium and then for an additional 60 min in complete medium containing 100 μg/ml gentamicin to kill extracellular bacteria. Thereafter, infected BMMs were incubated in gentamicin-free medium until processing. All infections with strains of F. tularensis subsp. novicida were performed in the absence of gentamicin. When required, BMMs were treated with 100 nM bafilomycin A1 (BAF) (AG Scientific) or 100 nM concanamycin A (ConA) (AG Scientific) or corresponding dilutions of dimethyl sulfoxide (DMSO) as a carrier control 1 h prior to infection, unless stated otherwise, and maintained throughout.

Determination of bacterial CFU.

The number of viable intracellular bacteria per well was determined in triplicate for each time point. Infected BMMs were washed three times with sterile PBS and then lysed with 1 ml of sterile deionized water for 3 min at room temperature, followed by repeated pipetting to complete lysis. Serial dilutions of the lysates were rapidly plated onto mMH or TSAC plates, and plates were incubated for either 3 days (Schu S4) or 1 day (U112) at 37°C under 7% CO2 before enumeration of CFU. To estimate the intracellular growth rate of bacteria within a particular time interval, CFU doubling times were established by dividing the time interval considered with the number of rounds of replication (n), which was calculated as follows: n = ln(CFU2/CFU1)/ln2, where CFU1 and CFU2 are the numbers of CFU obtained at the beginning and end of the time interval, respectively. Intracellular doubling times were calculated from three independent experiments and expressed as means ± standard deviations (SD).

Immunofluorescence microscopy.

Macrophages grown on 12-mm glass coverslips in 24-well plates were infected, washed three times with PBS, fixed with 3% paraformaldehyde, pH 7.4, at 37°C for 20 min, washed three times with PBS, and then incubated for 10 min in 50 mM NH4Cl in PBS in order to quench free aldehyde groups. Samples were blocked and permeabilized in blocking buffer (10% horse serum, 0.1% saponin in PBS) for 30 min at room temperature. Cells were labeled by incubating inverted coverslips on drops of primary antibodies diluted in blocking buffer for 45 min at room temperature. Primary antibodies used were mouse anti-F. tularensis lipopolysaccharide (LPS) (US Biological, Swampscott, MA), mouse anti-F. tularensis subsp. novicida (clone 8.2; Immuno-Precise Antibodies Ltd., Victoria, Canada), goat polyclonal anti-EEA-1 (N-19; Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-mouse LAMP-1 (clone 1D4B; developed by J. T. August and obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City). Bound antibodies were detected by incubation with 1:500 dilutions in blocking buffer of Alexa Fluor 488-donkey anti-mouse, Alexa Fluor 586-donkey anti-goat, and Alexa Fluor 568-donkey anti-rat antibodies for 45 min at room temperature. Cells were washed twice with 0.1% saponin in PBS, once in PBS, and once in H2O and then mounted in Mowiol 4-88 mounting medium (Calbiochem, Gibbstown, NJ). Samples were observed on a Nikon Eclipse E800 epifluorescence microscope equipped with a Plan Apo ×60/1.4 objective for quantitative analysis or a Carl Zeiss LSM 510 confocal laser scanning microscope for image acquisition. Confocal images of 1,024 by 1,024 pixels were acquired and assembled using Adobe Photoshop CS.

Phagosomal integrity assay.

To quantify escape of Francisella from its initial phagosome, phagosomal integrity assays were performed as described previously (3) with minor modifications. Briefly, infected BMMs were washed three times with KHM buffer (110 mM potassium acetate, 20 mM HEPES, 2 mM MgCl2, pH 7.3) and the plasma membrane selectively permeabilized by incubation with 50 μg/ml digitonin (Sigma) in KHM buffer for 1 min at room temperature. Cells were then washed immediately with KHM buffer and rabbit polyclonal anticalnexin (specific to the cytoplasm-facing C-terminal tail; Stressgen Biotechnologies), and Alexa Fluor 488-conjugated mouse monoclonal anti-F. tularensis LPS antibodies (US Biological) were delivered to the macrophage cytosol for 12 min at 37°C to label the endoplasmic reticulum of permeabilized cells and accessible intracellular bacteria, respectively. Alexa Fluor-conjugated mouse monoclonal anti-F. tularensis LPS antibodies were generated using Alexa Fluor monoclonal antibody labeling kits (Invitrogen) according to the manufacturer's instructions. BMMs were then washed with PBS, fixed with 3% paraformaldehyde, pH 7.4, at 37°C for 10 min, washed three times with PBS, and then incubated for 10 min in 50 mM NH4Cl in PBS in order to quench free aldehyde groups. All host cell membranes were then permeabilized in 10% horse serum-0.1% saponin in PBS for 30 min at room temperature. Bound anticalnexin antibodies were detected using cyanin 5-conjugated donkey anti-rabbit antibodies (Jackson ImmunoResearch Laboratories), and all intracellular bacteria were labeled using Alexa Fluor 568-conjugated anti-Francisella antibodies. To label F. tularensis subsp. novicida strains, a nonconjugated anti-F. tularensis subsp. novicida LPS antibody was used and detected using either Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies. This technique resulted in a differential staining of cytoplasmic bacteria (Alexa Fluor 488/568 dual fluorescence) and those enclosed within an intact phagosome (Alexa Fluor 568 single fluorescence). Cells were washed twice with 0.1% saponin in PBS, once in PBS, and once in H2O and then mounted in Mowiol 4-88 mounting medium (Calbiochem). Samples were observed using a Nikon Eclipse E800 epifluorescence microscope equipped with a Plan Apo ×60/1.4 objective for quantitative analysis or a Carl Zeiss LSM 510 confocal laser scanning microscope.

FCP acidification measurements.

BMMs were seeded 48 h before infection on WillCo-dish glass-bottom 35-mm dishes (WillCo Wells BV) at 5 × 104/dish. BMMs were left untreated or treated with 100 nM BAF or 100 nM ConA 1 h prior to infection. When required, GFP-expressing Schu S4 was killed by incubation for 10 min at room temperature in 1 ml of 3% paraformaldehyde in PBS, washed five times with PBS, and resuspended in complete DMEM. For infections, bacterial cultures were diluted in complete medium and added to chilled cells at a theoretical MOI of either 100 (live bacteria) or 1,000 (killed bacteria). Bacteria were centrifuged onto cells at 400 × g for 10 min at 4°C, and infected cells were incubated in a 37°C water bath for 3 min, followed by incubation at 37°C under a 7%-CO2 atmosphere. At 15 min p.i., the infected cells were washed five times with DMEM to remove extracellular bacteria and either processed for Lysotracker staining or incubated in complete medium, supplemented with BAF or ConA where required, at 37°C under a 7%-CO2 atmosphere for later time points. At each time point, the infected cells were incubated for 2 min at 37°C under a 7%-CO2 atmosphere with 0.5 μM Lysotracker Red DND-99 (Invitrogen) diluted in complete medium, washed seven times with CO2-independent medium (Invitrogen), and incubated with CO2-independent medium supplemented with 10% fetal bovine serum, 10% L-cell-conditioned medium, and 4 mM l-glutamine during live cell imaging. Quantification was performed immediately on a Carl Zeiss LSM 5 Live confocal live cell imaging microscope fitted with a Pecon heated stage insert and LCI Plan ×63/1.45 numerical aperture objective, using 488-nm and 532-nm solid-state lasers for sequential excitation. Colocalization of bacteria with a Lysotracker Red signal was scored for at least 60 bacteria per time point for each experiment. Values are the means and SD of three independent experiments. For image acquisition, the cells were washed seven times with PBS after Lysotracker staining and then fixed at room temperature for 3 min with 3% paraformaldehyde in PBS and imaged immediately. Images (1,024 by 1,024 pixels) were acquired using the Carl Zeiss LSM 5 Live 4.0 SP2 software program and assembled in Adobe Photoshop CS.

Transmission electron microscopy.

Infected BMMs on 12-mm Aclar coverslips were fixed in 2.5% glutaraldehyde-4.0% paraformaldehyde-50 mM sucrose in 0.1 M sodium cacodylate buffer (pH 7.0) at 4°C for 24 h and briefly refixed with a Pelco 3451 laboratory microwave system (Ted Pella, Inc., Redding, CA) (MW) at 650 W (10 s on, 20 s off, and 10 s on) before further processing. Samples were rinsed with 0.1 M sodium cacodylate buffer for 40 s and then distilled H2O for 40 s and postfixed in 0.5% OsO4-0.8% K4Fe(CN)6 in 0.1 M sodium cacodylate buffer twice for 2 min each with MW (80 W under a 20-in. Hg vacuum). After three washes in distilled H2O, samples were treated with 1.0% tannic acid two times for 2 min each with MW at 80 W, rinsed again in distilled H2O, and stained en bloc in 0.1% aqueous uranyl acetate, pH 3.9, at 80 W two times for 2 min each. Samples were then dehydrated with ethanol for 45 s in MW without vacuum at 250 W and then with propylene oxide (PO) three times for 45 s and infiltrated with 3:1 PO:Spurrs and then 1:1 PO:Spurrs at 250 W for 6 min under vacuum. The final infiltration with 100% Spurrs was carried out for 12 min. The samples were left under vacuum for 30 min and then embedded in BEEM capsules. Following poststaining with uranyl acetate and lead citrate, thin sections were viewed in a Hitachi H7500 transmission electron microscope at 80 kV, fitted for image capture with a Hamamatsu charge-coupled-device camera, C4742-95, and Advantage HR/HR-B digital image software (AMT, Danvers, MA). Images were assembled in Adobe Photoshop CS.

Intracellular expression of FPI proteins.

To examine the expression of FPI proteins by intracellular bacteria, BMMs were infected with Francisella strains at a MOI of 200 as described above. At each time point, cells were washed three times with PBS and lysed in sterile distilled water to release intracellular bacteria. An aliquot was used to enumerate bacteria by CFU analysis, and the remaining volume was centrifuged at 16,100 × g for 5 min at 4°C to pellet bacteria. The pellet was lysed in sample buffer (0.035 M Tris-HCl-sodium dodecyl sulfate (SDS), pH 6.8, 30% glycerol, 10% SDS, 10% dithiothreitol, 0.012% bromophenol blue) and boiled for 5 min. For Western blot analysis, samples were normalized to CFU equivalents, resolved on 15% SDS-polyacrylamide gels, and transferred to Hybond-ECL nitrocellulose membranes (Amersham Biosciences) using a semidry apparatus. Membranes were blocked for 2 h at room temperature in 0.1% Tween 20-5% nonfat milk Tris-buffered saline and probed overnight at 4°C with either a mouse monoclonal anti-IglC antibody (1:250 dilution in blocking buffer; gift from Francis E. Nano) or a rabbit polyclonal anti-PdpC antibody (1:10,000 dilution in blocking buffer), which was raised against the synthetic peptide DDINVDRENRRELVAK, corresponding to residues 168 to 183 of PdpC, and affinity purified by New England Peptides, Inc. The mouse monoclonal anti-IglD antibody was a gift from Francis E. Nano. Membranes were then washed in 0.1% Tween 20-5% nonfat milk Tris-buffered saline and incubated with either horseradish peroxidase-linked anti-rabbit immunoglobulin G or anti-mouse immunoglobulin G (1:12,000 dilution in blocking buffer; Cell Signaling Technology) for 1 h at room temperature. Proteins were then detected using an ECL Advance Western blotting detection kit (Amersham Biosciences) and either autoradiography or densitometry on a Kodak Image Station 4000MM digital imaging system (Eastman Kodak, Rochester, NY) to quantify the relative amounts of proteins in samples.

RESULTS AND DISCUSSION

FCPs mature along the endocytic pathway prior to bacterial escape.

Our previous work has shown that early phagosomes containing the attenuated F. tularensis subsp. holarctica strain LVS sequentially interact with early and then late endocytic compartments in murine primary macrophages before phagosomal disruption (3). To examine early trafficking events of phagosomes containing the highly virulent strain Schu S4, we analyzed interactions of early FCPs with early and late endocytic compartments by confocal microscopy. At 5 min p.i., 33% ± 4.6% of FCPs were positive for the early endosomal antigen EEA-1 (Fig. 1A and B), indicating interactions with early endosomes. These interactions were transient, since FCP colocalization with EEA-1 rapidly decreased to 7.1% ± 1.0% by 20 min p.i. (Fig. 1A and B). Thereafter, FCPs progressively acquired the late endocytic lysosome-associated membrane protein 1 (LAMP-1), indicating interactions with late endosomes/lysosomes (Fig. 1C and D). LAMP-1 acquisition by FCPs peaked at 20 min p.i. (48 ± 5.3%; Fig. Fig.1C),1C), and then decreased to 11 ± 2.6% by 60 min p.i. This decrease in colocalization with LAMP-1 was likely due to phagosomal disruption, since the percentage of cytoplasmic bacteria significantly increased during the same time period (see Fig. Fig.3),3), as previously shown for the LVS strain (3). Taken together, these results demonstrate that early phagosomes containing virulent Francisella undergo normal maturation along the endocytic pathway prior to phagosomal membrane disruption and bacterial escape into the cytoplasm.

FIG. 1.
Early intracellular trafficking of Schu S4 in BMMs and effect of inhibition of endosomal acidification. BMMs were left untreated or were treated with either BAF or ConA for 1 h prior to infection and infected with Schu S4. At various times p.i., samples ...
FIG. 3.
Inhibition of endosomal acidification delays Schu S4 phagosomal escape. (A) Inhibition of endosomal acidification blocks early phagosomal disruption. BMMs were left untreated or were treated with either DMSO, BAF, or ConA for 1 h prior to infection, infected ...

FCPs are acidified prior to bacterial escape.

A critical aspect of phagosomal maturation is the progressive acidification of the phagosome luminal space via the activity of the vacuolar ATPase, which is both required for and a consequence of maturation events (14). Given that early FCPs display transient interactions with the endocytic pathway, we examined whether they also became acidic prior to phagosomal escape, using live cell imaging of the acidotropic probe Lysotracker Red DND-99. In BMMs infected with GFP-expressing Schu S4, by 20 min p.i., the majority of live bacteria clearly colocalized with Lysotracker (74% ± 0.4%) (Fig. 2A and B), indicating that bacteria reside within an acidified compartment at this early stage, consistent with LAMP-1 accumulation on its membrane (Fig. 1C and D). Thereafter, the percentage of bacterial colocalization with Lysotracker decreased to 26% ± 6.7% at 60 min, indicating that bacteria had reached a nonacidic compartment, consistent with the phagosomal escape kinetics of the LVS strain (3). This was dependent upon bacterial viability, because paraformaldehyde (PFA)-killed Schu S4 remained within a Lysotracker-positive, acidic compartment (Fig. 2A and B). Taken together, these results demonstrate that early FCPs sufficiently mature along the endocytic pathway to become acidic.

FIG. 2.
Early FCPs are acidified prior to phagosomal escape. BMMs were infected with either live or PFA-killed, GFP-expressing Schu S4 for various time periods and loaded with Lysotracker Red DND-99 before live cell imaging analysis. (A) Quantification of Lysotracker-positive ...

To confirm that the transition of intracellular Schu S4 from an acidic, LAMP-1-positive to a nonacidic, LAMP-1-negative compartment was the result of phagosomal disruption and bacterial escape into the cytosol, BMMs were infected with either live or PFA-killed Schu S4 and subjected to a phagosomal integrity assay, as described in Materials and Methods. While PFA-killed bacteria remained within a phagosome (Fig. 3A and B), the percentage of cytoplasmic live Schu S4 increased from 37% ± 4.2% at 20 min p.i. to 78% ± 2.3% at 60 min p.i. (Fig. 3A and B), indicating efficient phagosomal escape. At all time points analyzed, the number of cytoplasmic bacteria inversely correlated with colocalization of bacteria with Lysotracker Red and LAMP-1 (Fig. (Fig.1C,1C, ,2A,2A, and and3A),3A), confirming that the transition of bacteria toward a nonacidic compartment was the result of phagosomal escape. Taken together, these results demonstrate that early FCPs become acidic through interactions with the endocytic pathway prior to phagosomal escape.

Phagosomal escape is impaired by inhibition of phagosome acidification.

Because phagosomal acidification constitutes an intracellular signal which various parasites respond to by inducing or activating specific pathogenic mechanisms (2, 4, 30), we next examined whether the early acidification of FCPs plays a role in phagosomal escape. For this purpose, we inhibited endosomal acidification prior to infection by treating BMMs with the vacuolar ATPase (v-ATPase) inhibitor BAF or ConA, and examined early trafficking events and phagosomal escape of Schu S4. Compared with results with the vehicle control (DMSO), treatment with BAF or ConA slightly delayed the exclusion of EEA-1 from early FCPs (Fig. (Fig.1A)1A) but did not affect the acquisition of LAMP-1 on FCPs during the first 20 min p.i. (Fig. (Fig.1C).1C). This indicates that inhibition of endosome acidification does not dramatically affect early maturation steps of the FCPs, a finding consistent with the fact that endosomal acidification is important for fusion with late endocytic compartments (5). However, bacteria within either BAF- or ConA-treated BMMs remained colocalized with LAMP-1 until 60 min p.i. (Fig. 1C and D), suggesting they remained enclosed within a phagosome. To confirm this, phagosomal escape of Schu S4 was analyzed under the same experimental conditions. Compared to results with the DMSO control, with which the extent and timing of phagosomal escape were indistinguishable from those in untreated cells (Fig. (Fig.3A),3A), pretreatment of BMMs with either BAF or ConA prevented Schu S4 from reaching the cytoplasm at 20 to 60 min p.i., with levels of cytoplasmic bacteria comparable to those observed with PFA-killed bacteria (Fig. 3A and B). This demonstrates that acidification of early FCPs is required for phagosomal disruption and indicates that the early intraphagosomal environment determines Francisella intracellular fate.

Blocking of phagosomal escape by inhibition of endosomal acidification is transient.

To further investigate the long-term effects of blocking early phagosomal disruption on the intracellular fate of Schu S4, we examined the trafficking of bacteria at later stages. Surprisingly, after 1 h p.i., the percentage of cytoplasmic bacteria started to increase in cells pretreated with either BAF (Fig. (Fig.3C)3C) or ConA (Fig. (Fig.3D),3D), even though the inhibitors were maintained up to 4 or 8 h p.i. Under these conditions, where inhibitors were replenished at 4 h p.i., no acidic compartments could be detected in either BAF- or ConA-treated BMMs using LysoTracker Red DND-99 labeling (data not shown), confirming that endosomal acidification remained blocked all throughout our analysis. While the majority of bacteria were cytoplasmic by 4 h p.i. in untreated cells, more than 50% of intracellular bacteria were cytoplasmic in either BAF- or ConA-treated BMMs (Fig. 3C and D). By 8 h p.i., levels of cytoplasmic bacteria were similar to those in untreated cells (Fig. 3C and D). The timing of BAF or ConA addition was important for dictating effects on phagosomal escape, since inhibitor addition from 2 to 8 h p.i. did not affect phagosomal escape (Fig. 3C and D). This indicates that the early inhibition of phagosomal disruption by BAF or ConA treatment is transient and eventually overcome, suggesting that Francisella uses acidification-dependent and -independent mechanisms of phagosomal disruption to achieve a rapid and efficient escape into the cytosol. Alternatively, acidification of early FCPs may enhance expression and/or activation of bacterial factors required for phagosomal escape, yet these expression or activation processes still occur but less efficiently in the absence of phagosomal acidification.

Inhibition of phagosome maturation delays disruption of the phagosomal membrane.

To further characterize how inhibition of phagosomal acidification/maturation affects phagosomal escape, we examined the ultrastructure of early FCPs in untreated, BAF-treated, and ConA-treated BMMs by electron microscopy. While bacteria in untreated cells had disrupted the phagosomal membrane by 30 min p.i. (Fig. (Fig.4A),4A), bacteria in either BAF- or ConA-treated cells remained within phagosomes displaying distinct surrounding membranes (Fig. 4B and C). At 1 h p.i., bacteria in untreated cells were typically free in the cytosol (Fig. (Fig.4D)4D) whereas bacteria in either BAF- or ConA-treated cells were mostly surrounded by phagosomal membranes (Fig. 4E and F). However, discrete foci of obvious membrane degradation were detectable in either BAF- or ConA-treated cells (Fig. 4E and F, insets), indicating that some phagosomal disruption events were occurring but with slower kinetics. By 2 h p.i., bacteria free in the cytosol were detected under all conditions (Fig. 4G to I), consistent with the increasing percentages of cytosolic bacteria determined by the phagosomal integrity assay (Fig. 3C and D). This confirms that inhibition of phagosomal acidification/maturation does not block but rather delays the onset of phagosomal membrane disruption and reduces the efficiency of Francisella phagosomal escape.

FIG. 4.
Inhibition of phagosomal acidification delays degradation of the phagosomal membrane. BMMs were left untreated or were treated with either BAF or ConA for 1 h prior to infection, infected with Schu S4, and processed for transmission electron microscopy ...

Inhibition of phagosome maturation delays cytosolic replication of Francisella.

Because early inhibition of phagosomal acidification/maturation impairs Francisella phagosomal escape, we examined whether it also affected cytosolic replication. For this purpose, viable intracellular Schu S4 bacteria were enumerated at various time points p.i. in either untreated BMMs or BMMs treated with either BAF or ConA for various times. In untreated BMMs, intracellular viable numbers of Schu S4 clearly increased after 4 h p.i. (Fig. 5A and B), indicating an onset of replication after a lag period of 2 to 3 h in the cytosol (Fig. 3C and D). Treatments with BAF or ConA from 2 to 12 h p.i. did not affect cytosolic replication of Schu S4, since bacteria multiplied to the same extent as in untreated cells over a 12-h time period (Fig. 5A and B) and displayed similar replication patterns at 8 h p.i. (Fig. (Fig.5C).5C). In contrast, intracellular multiplication of Schu S4 was delayed in BMMs that were pretreated with either BAF or ConA, with a clear delay in the onset of replication of cytosolic bacteria to 8 h p.i. (Fig. (Fig.5C).5C). Thereafter, the growth rate of Schu S4 was similar to that observed in untreated cells, since intracellular doubling times were not significantly different among all conditions tested (Fig. 5A, B, and D). Taken together, these results demonstrate that inhibition of phagosomal acidification/maturation delays the onset of Francisella cytosolic replication, consistent with the observed delay in phagosomal escape, but does not affect its intracellular growth rate. Hence, conditions encountered in the early FCP are required for efficient intracellular proliferation of Francisella.

FIG. 5.
Inhibition of phagosomal acidification delays cytosolic replication of Schu S4. BMMs were left untreated or were treated with either BAF or ConA for various time periods (−1 to 4 h p.i., green curve; −1 to 12 h p.i., red curve; 2 to 12 ...

Early FCPs provide cues for expression of FPI proteins.

Since the perturbation of the FCP pH affects the ability of Francisella to undergo phagosomal escape and initiate replication, we postulated that the phagosomal environment is conducive to a rapid expression and/or activation of bacterial factors required for phagosomal escape. In support of our hypothesis, transcriptional profiling of intracellular Schu S4 has shown significant upregulation of a large subset of genes during the early phagosomal stage (J. Celli, unpublished results). To date, the FPI-encoded IglC protein and the acid phosphatases AcpA, AcpB, AcpC and Hap have been linked to phagosomal escape in either F. tularensis subsp. novicida or strain LVS, since bacteria carrying mutations in the corresponding genes are affected in their ability to disrupt phagosomal membranes (17, 19, 20, 28). However, only genes within the FPI showed significant upregulation during the early phagosomal stage (J. Celli, unpublished results), which prompted us to focus on FPI-associated functions and examine whether phagosomal pH influences FPI-dependent cytosolic release. To this end, we first compared the kinetics of phagosomal escape of wild-type F. tularensis subsp. novicida and an iglC::ermC mutant in the absence or presence of BAF. We used F. tularensis subsp. novicida in these experiments due to the availability of an iglC mutant in this subspecies and the comparable intracellular trafficking of F. tularensis subsp. novicida and F. tularensis subsp. tularensis strains in macrophages (6, 28). Because of its insertional nature, we first verified whether the disruption of iglC displays a polar effect on the expression of iglD, which lies immediately downstream. While IglC could not be detected in the iglC mutant, expression of IglD was significantly reduced (Fig. (Fig.6A),6A), suggesting that this mutant is deficient for both IglC and IglD-dependent functions. Over an 8-h time course in untreated BMMs, the wild-type U112 strain displayed phagosomal escape kinetics similar to those of Schu S4, although slightly slower, with the percentage of cytoplasmic bacteria reaching ~90% by 4 h p.i. (Fig. (Fig.6B).6B). Consistent with a previous report on human blood-derived macrophages using ultrastructural analysis of phagosomes (29), the iglC::ermC mutant of U112 showed a significant defect in phagosomal escape, since only 37% ± 7.5% of mutant bacteria were cytoplasmic at 8 h p.i., compared to 94% ± 3.0% for the wild-type strain (Fig. 6B and C). This defect was significantly restored upon expression of both IglC and IglD in the ΔiglC mutant (Fig. 6A and B), demonstrating that phagosomal escape is IglCD dependent. Because of the polar effect of the iglC insertional mutation on IglD expression (Fig. (Fig.6A),6A), conclusions on specific roles of IglC drawn from the use of this particular strain in the absence of complementation studies (29) should be reconsidered.

FIG. 6.
Inhibition of phagosomal acidification delays phagosomal escape of F. tularensis subsp. novicida. (A) Western blot analysis of IglC and IglD expression in F. tularensis subsp. novicida U112, U112 ΔiglC::ermC, and U112 ΔiglC::ermC(piglCD ...

Interestingly, and in conflict with previous results (17, 27), the number of cytoplasmic ΔiglC bacteria was significantly higher than that for PFA-killed U112 at all times after 1 h p.i. (Fig. (Fig.6B).6B). Consistently, we observed localized foci of membrane degradation on phagosomes containing iglC mutants by electron microscopy (Fig. (Fig.6D).6D). This indicates that mutation of IglCD functions does not completely prevent disruption of the FCP membrane, as previously concluded (17, 27, 29), but significantly decreases it. This could be explained by IglCD potentiation of other membrane degradation functions or because some IglCD-independent mechanisms also contribute to FCP disruption.

In BMMs pretreated with BAF, phagosomal escape of F. tularensis subsp. novicida U112 was delayed but not blocked (Fig. (Fig.6C),6C), like that of Schu S4 (Fig. (Fig.3).3). As a control, BAF treatment after 2 h p.i. did not affect phagosomal escape (Fig. 6B and C). Levels of cytoplasmic bacteria in BAF-treated cells were comparable to those of PFA-killed U112 at 1 h p.i., yet by 8 h p.i., they had reached levels similar to that observed for U112 in untreated BMMs (Fig. 6B and C). This is in contrast with a recent report in which BAF pretreatment of human macrophages inhibited phagosomal escape of U112 up to 6 h p.i. (27). Whether bacteria eventually escaped and replicated in the cytosol was not examined in this study, however. The proportion of cytoplasmic U112 in BAF-treated BMMs was also comparable to that of the ΔiglC mutant in untreated cells at 1 h p.i. (Fig. 6B and C), suggesting that the defect in early phagosomal disruption caused by the iglCD mutation can be mimicked by inhibition of phagosomal acidification. Moreover, inhibition of phagosomal acidification did not have an additive effect on the iglCD mutation, since BAF treatment of BMMs did not significantly alter the levels of cytoplasmically accessible ΔiglC mutants at any time analyzed (Fig. 6B and C). Complementation of the ΔiglC mutant restored not only phagosomal escape (Fig. (Fig.6B)6B) but also its sensitivity to acidification, since the percentages of cytoplasmically accessible ΔiglC (piglCD) bacteria were lower in BAF-treated BMMs than in untreated BMMs (Fig. 6B and C). Altogether, these results demonstrate that acidification is required for the IglCD-dependent phagosomal disruption events. At later time points, however, phagosomal escape of the iglC mutant remained limited while wild-type bacteria in BAF-treated BMMs eventually escaped in the cytoplasm. Hence, IglCD-associated functions are required for both early acidification-dependent and late acidification-independent phagosomal disruption events.

From these data, we postulated that early FCP acidification induces expression of FPI proteins. To test this hypothesis, we examined the intrabacterial accumulation of two FPI proteins, IglC and PdpC, during the first 4 h of the Schu S4 intracellular cycle. In untreated BMMs, a 4.6-fold ± 1.3-fold increase in IglC and a 6.7-fold ± 2.0-fold increase in PdpC were detected between 0 and 2 h p.i. (Fig. 7A and B), demonstrating that FPI protein expression is increased upon entry into macrophages. In BMMs pretreated with BAF, a similar pattern of IglC and PdpC expression was observed (Fig. 7A and B), with IglC and PdpC intrabacterial levels increasing 3.8-fold ± 2.0-fold and 7.9-fold ± 4.8-fold, respectively, between 0 and 2 h p.i., indicating that early FCP acidification is not required for the induction of FPI protein expression. Since BAF treatment prevents phagosomal acidification and alters the kinetics of phagosomal escape (Fig. (Fig.33 and and4)4) but FPI induction kinetics remained unchanged, we conclude that intracellular FPI protein expression does not depend upon cytosolic release of bacteria or phagosomal pH. In spite of this, our data demonstrate that the early FCP does provide the necessary signals to induce FPI expression.

FIG. 7.
FPI protein expression is induced early but does not depend upon FCP acidification. Untreated or BAF-treated BMMs were infected with Schu S4, and samples were processed for CFU enumeration and Western blotting at 0, 1, 2, and 4 h p.i., as described in ...

Here we have examined whether the early phagosomal stage of F. tularensis in primary murine macrophages contributes to the intracellular fate of phagocytosed bacteria. We have shown that the early FCP plays an important role in phagosomal escape and intracellular proliferation by providing cues for optimal phagosomal disruption and expression of FPI proteins, thereby ensuring efficient intracellular survival of Francisella. This therefore highlights the importance of the early phagosomal stage in Francisella intracellular pathogenesis, since it determines the efficiency of phagosomal escape and the temporal expression of virulence-related genes. Although it interacts with early and late endocytic compartments very transiently, the early FCP containing virulent F. tularensis becomes acidified before the onset of phagosomal disruption. This is consistent with the recent finding by Santic et al. that early FCPs containing nonpathogenic F. tularensis subsp. novicida acquire v-ATPase and are acidified in human blood-derived macrophages (27) but contrasts with a previous report by Clemens et al., which claimed that phagosomes containing virulent F. tularensis resist acidification. In the latter study, bacteria were likely taken up by opsonic phagocytosis due to the presence of fresh serum during infection, and phagosomal escape was belated (6), compared to results under our nonopsonic experimental conditions (see reference 3 and this study). It is therefore possible that the mode of uptake affects the maturation process of the FCP and the kinetics of phagosomal disruption, but this hypothesis needs to be tested through side-by-side comparisons. Our results also contradict those of Santic et al., who have reported a complete inhibition of phagosomal escape of F. tularensis subsp. novicida in the presence of BAF (27). Instead, we observed only a delayed and inefficient phagosomal disruption process by either virulent F. tularensis or F. tularensis subsp. novicida in the presence of this v-ATPase inhibitor (Fig. (Fig.33 and and6).6). While one could invoke differences between murine and human macrophages to explain these results, the eventual fate of F. tularensis subsp. novicida-containing phagosomes past 6 h p.i. was not examined by Santic et al. Since we observed that F. tularensis subsp. novicida phagosomal escape is slower than that of F. tularensis subsp. tularensis (this study) or F. tularensis subsp. holarctica (3) strains in directly comparable models, it is possible that phagosomal escape of F. tularensis subsp. novicida in BAF-treated human macrophages occurs later than the time frame examined in this previous report.

That inhibition of FCP acidification renders Francisella phagosomal disruption inefficient suggests that the intravacuolar pH potentiates the expression and/or activity of bacterial factors responsible for membrane degradation. Because IglC-dependent functions within the FPI have been associated with phagosomal escape (17, 29), we examined whether expression of FPI proteins required FCP acidification. Although we demonstrated that FPI genes are induced during the early stage of the Francisella intracellular cycle, intravacuolar pH did not appear to be the inducing signal. Since BAF treatment of BMMs prior to infection reduced phagosomal escape levels of a wild-type strain to that of an IglC-deficient strain in untreated BMMs, we surmise that the rapid IglCD-dependent phagosomal disruption requires an acidic pH, likely by activating, rather than inducing the expression of, particular IglCD- or FPI protein-associated functions. It is to be expected that phagosomal escape requires the input of numerous bacterial factors, so it remains possible that FCP acidification is important for the activation and/or expression of other non-FPI-encoded factors. Recent examples include the acid phosphatases AcpA, AcpB, AcpC, and Hap, whose single or combined deletions in F. tularensis subsp. novicida prevent phagosomal escape (19, 20) and whose maximal activity is likely to require an acidic pH, as is the case for AcpA (26). Expression of AcpA, AcpB, or AcpC, which are encoded within the genome of type A strains, was not increased within the early FCP, since transcriptional profiling of intracellular Schu S4 did not reveal any significant upregulation of the corresponding genes (FTT0221, FTT0156, and FTT0620) at this stage (J. Celli, unpublished results). It will therefore be interesting to determine whether early acidification of the FCP is important for their activity. In conclusion, our study reveals the importance of the early phagosomal stage in Francisella intracellular pathogenesis by determining the efficiency of phagosomal escape and the temporal expression of virulence-related genes. Future characterization of both bacterial factors and host processes involved in this early step of the Francisella intracellular cycle is essential to better understand the pathogenesis of this highly virulent bacterium.

Acknowledgments

We are grateful to Anders Sjöstedt and Francis Nano for the gift of strains and antibodies and to Leigh Knodler and Jessica Edwards for critical reading of the manuscript and helpful discussions.

This work was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.

Notes

Editor: A. J. Bäumler

Footnotes

[down-pointing small open triangle]Published ahead of print on 13 October 2008.

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