Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College School of Medicine, Armstrong Road, London SW7 2AZ, UK1
Author for correspondence: David W. Holden. Tel: +44 20 7594 3073. Fax: +44 20 7594 3076. e-mail: d.holden{at}ic.ac.uk
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ABSTRACT |
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Keywords: intracellular replication, type III secretion system, antimicrobial activity, host defence, intracellular pathogen
Abbreviations: FCS, fetal calf serum; GFP, green fluorescent protein; i.p., intraperitoneal(ly); PFA, paraformaldehyde; SCV, Salmonella-containing vacuole; SPI-2, Salmonella pathogenicity island 2; TRSC, Texas red sulfonyl chloride; TTSS, type III secretion system
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INTRODUCTION |
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Several studies have analysed bacterial replication inside the host cell cytosol, in an attempt to determine whether the cytosol is inhibitory or permissive for the growth of bacteria that normally remain in the vacuole, such as Salmonella, or extracellular bacteria such as Bacillus subtilis and non-pathogenic Escherichia coli (Bielecki et al., 1990 ; Gentschev et al., 1995
; Goebel & Kuhn, 2000
; Goetz et al., 2001
).
Two approaches have been used to get bacteria into the host cell cytosol. First, bacterial strains have been engineered to express and secrete listeriolysin, a protein partly responsible for the escape of Listeria monocytogenes into the host cell cytosol (Bielecki et al., 1990 ; Gentschev et al., 1995
). Only a limited proportion of the bacterial population was released into the cytosol by this method, probably because other proteins, in addition to listeriolysin, are also required for the efficient release of Listeria from the vacuole. In macrophages, listeriolysin-expressing B. subtilis and non-pathogenic E. coli seemed capable of replication within the macrophage cytosol, whereas no growth was detected for Salmonella dublin (reviewed by Goebel & Kuhn, 2000
). A second approach relies on the delivery of individual bacteria directly into the host cell cytosol by microinjection (Goetz et al., 2001
). Although this method guarantees the delivery to the cytosol of every bacterial cell, it may inflict mechanical damage upon the bacteria that could affect their ability to survive and replicate. Furthermore, as bacteria are grown in vitro prior to their microinjection, the growth conditions used may have an effect on the set of bacterial genes expressed at the moment of their introduction into the cytosol, and therefore on the bacterial response. This second method has been applied to deliver S. typhimurium, Yersinia enterocolitica and non-pathogenic E. coli into the cytosol of epithelial cells, where none displayed any replication (Goetz et al., 2001
).
We have identified the Salmonella gene sifA as necessary to maintain the integrity of the Salmonella-containing vacuole (SCV) (Beuzón et al., 2000 ). Bacteria carrying a mutation in this gene are released into the host cell cytosol several hours after uptake by macrophages (Beuzón et al., 2000
). SifA is secreted by a type III secretion system (TTSS), encoded in the Salmonella pathogenicity island 2 (SPI-2) (Brumell et al., 2002a
; Hansen-Wester et al., 2002
). The use of a sifA mutant and wild-type strains allows us to compare the replication of isogenic strains that differ in their intracellular sublocalization, and therefore to address the question of the ability of Salmonella to replicate within the cytosol of different host cell types.
We find that in epithelial cells Salmonella can replicate much more proficiently in the cytosol than when enclosed in a vacuole. However, bacterial replication is strongly inhibited when the bacteria are released into the cytosol of fibroblasts or macrophages. Using an aroC purD double mutant strain which is incapable of replication in host cells (Fields et al., 1986 ), we show that the bacteria encounter a killing activity within the cytosol of macrophages. In vitro experiments using cytosol extracted from either infected or uninfected macrophages suggest that this killing activity is activated upon Salmonella infection.
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METHODS |
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Cell culture.
RAW 264.7 cells were obtained from ECACC (ECACC 91062702). HeLa (clone HtTA1) cells and Swiss 3T3 murine fibroblast cells were kindly provided by S. Méresse (Centre dImmunologie de Marseille-Luminy, Marseille, France) and E. Caron (Imperial College, London, UK) respectively. Cells were grown in Dulbeccos modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 2 mM glutamine at 37 °C in 5% CO2.
Bacterial infection of HeLa cells and Swiss 3T3 fibroblasts, and survival assays.
Host cells were seeded onto glass coverslips (12 mm diameter) in 24-well plates at a density of 5x105 cells per well, 24 h before infection. Bacteria were incubated for 16 h at 37 °C with shaking, diluted 1:33 in fresh LB broth and incubated in the same conditions for 3·5 h. The cultures were diluted in Earles buffered salt solution (EBSS) pH 7·4 and added to the cells at an m.o.i. of approximately 100:1. The infection was allowed to proceed for 15 min at 37 °C in 5% CO2. The monolayers were washed once with DMEM containing FCS and 100 µg gentamicin ml-1 and incubated in this medium for 1 h, after which the gentamicin concentration was decreased to 16 µg ml-1. For enumeration of intracellular bacteria (gentamicin-protected), cells were washed three times with PBS, lysed with 0·1% Triton X-100 for 10 min, and dilution series were plated onto LB agar, at different time points after bacterial entry. For microscopic examination, cell monolayers were fixed in 3·7% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) pH 7·4 for 15 min at room temperature, and washed three times in PBS.
Bacterial infection of RAW 264.7 macrophages and survival assays.
Macrophages were seeded at a density of 5x105 cells per well in 24-well tissue culture plates, 24 h before use. Bacteria were cultured at 37 °C with shaking until they reached an OD600 of 2·0. The cultures were diluted to an OD600 of 1·0 and opsonized in DMEM containing FCS and 10% normal mouse serum for 20 min. Bacteria were added to the monolayers at an m.o.i. of 100:1, centrifuged at 170 g for 5 min at room temperature and incubated for 25 min at 37 °C in 5% CO2. These conditions render bacteria non-invasive and thus avoid cytotoxic effect in macrophages. Differences in the way of entry have no significant effect on SCV trafficking or on the intracellular fate of Salmonella (Buchmeier & Heffron, 1991 ; Rathman et al., 1997
; S. G. Garvis & D. W. Holden, unpublished results). The macrophages were washed once with DMEM containing FCS and 100 µg gentamicin ml-1 and incubated in this medium for 1 h. The medium was replaced with DMEM containing FCS and 16 µg gentamicin ml-1 for the remainder of the experiment. For enumeration of intracellular bacteria, macrophages were washed three times with PBS, lysed with 0·1% Triton X-100 for 10 min and a dilution series was plated onto LB agar. For microscopic examination, cell monolayers were fixed in 3·7% PFA in PBS pH 7·4 for 15 min at room temperature, and washed three times in PBS.
Preparation of spleen-derived cell suspensions.
Mice were inoculated intraperitoneally (i.p.) with 105 c.f.u. per mouse (wild-type strain) or 106 c.f.u. per mouse (ssaV or sifA mutant strains), as described previously (Beuzón et al., 2000 ). Spleens were removed aseptically 3 days after inoculation, and placed in 2 ml ice-cold PBS. Cell suspensions were obtained as described previously (Salcedo et al., 2001
). Briefly, cell suspensions were obtained by gentle mechanical disruption with a bent needle, filtered through a 70 µm nylon cell strainer (Becton Dickinson), and centrifuged at 400 g for 5 min. Red blood cells were subjected to an ammonium chloride lysis and the rest of the cells were fixed in 1% PFA for 10 min on ice, washed twice and resuspended in PBS.
Antibodies and reagents.
The mouse monoclonal antibody anti-LAMP-1 H3A4 developed by J. T. August and J. E. K. Hildreth was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA, USA), and was used at a dilution of 1:2000 for LAMP-1 staining in HeLa cells. Anti-LAMP-1 rabbit polyclonal antibody 156 against the 11 amino acid residues of the cytoplasmic domain of LAMP-1 has been described previously (Steele-Mortimer et al., 1999 ); it was used at a dilution of 1:1000 for LAMP-1 staining in Swiss 3T3 fibroblasts. Texas red sulfonyl chloride (TRSC)- and fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse, anti-rabbit and anti-goat antibodies were purchased from Jackson Immunoresearch Laboratories, and used at a dilution of 1:200.
Immunofluorescence and electron microscopy.
For immunofluorescence, cell monolayers were fixed for 15 min at room temperature in 3·7% PFA in PBS pH 7·4, and washed three times in PBS. Antibodies were diluted in 10% horse serum, 1% bovine serum albumin, 0·1% saponin in PBS. Coverslips were washed twice in PBS containing 0·1% saponin, incubated for 30 min with primary antibodies, washed twice with 0·1% saponin in PBS and incubated for 30 min with secondary antibodies. Coverslips were washed twice in 0·1% saponin in PBS, once in PBS and once in H2O, and mounted on Mowiol. Samples were analysed using a fluorescence microscope (BX50; Olympus Optical Company) or a confocal laser scanning microscope (LSM510, Zeiss).
For transmission electron microscopy of infected HeLa cells, cell suspensions were fixed in 3% glutaraldehyde prepared in 0·1 M sodium cacodylate pH 7·3. Fixation was for 12 h at room temperature, after which the cells were washed in fresh buffer before post-fixing in 1% osmium tetroxide in the same buffer. The cells were encased in agar (Ryder & MacKenzie, 1981 ), dehydrated through a graded series of alcohols and embedded in Araldite epoxy resin. Ultrathin sections were cut on a diamond knife and stained in alcoholic uranyl acetate and lead citrate before examination in a transmission electron microscope operated at 75 kV.
For transmission electron microscopy of splenocytes, spleens were fixed in 2·5% glutaraldehyde and 4% PFA in PBS on ice for 1 h, rinsed in 0·1 M sodium cacodylate pH 7·3 and post-fixed in 1% osmium tetroxide in the same buffer at room temperature for 1 h. After rinsing in buffer, 1% tannic acid was added for 30 min. Cells were rinsed for 5 min in 1% sodium sulfate and then dehydrated in an ethanol series followed by propylene oxide, adding 1% uranyl acetate at the 30% stage, embedded in Epon/Araldite 502 and finally polymerized at 60 °C for 24 h. Sections were cut on a Leica Ultracut ultramicrotome at 60 nm using a Diatome knife, contrasted with uranyl acetate and lead citrate, and examined in a Philips CM100 transmission electron microscope.
For immuno-electron microscopy, HeLa cells were infected with either wild-type or sifA mutant strains. At 10 h after bacterial entry, cells were fixed with 8% PFA in PBS for 5 min at room temperature, scraped, pelleted at 100 g for 3 min, and further fixed for 1 h on ice. Cells were rinsed in PBS three times without disturbing the pellet, and infiltrated with 2·3 M sucrose in PBS three times for 5 min at room temperature. The samples were then frozen by immersion in liquid nitrogen, where they were stored until sectioning.
For labelling, 70 nm sections were cut on a Leica FCS ultramicrotome and transferred to copper grids on PBS containing 0·02 M glycine, blocked with 5% FCS for 30 min and incubated with mouse monoclonal anti-LAMP-1 primary antibody at a 1:50 dilution, for 1 h. Sections were rinsed three times in PBS for 5 min and incubated for 1 h with goat f(ab')2 anti-mouse IgG secondary antibody conjugated to 10 nm gold particles, at a 1:20 dilution, for 1 h. Sections were rinsed twice in PBS for 2 min, fixed in 2·5% glutaraldehyde in PBS for 5 min, and rinsed in water. Sections were counterstained with uranyl acetate in methyl cellulose for 10 min on ice, picked up on copper loops to air-dry, and examined in a Philips CM100 transmission electron microscope.
Cytosol extraction and growth assays.
RAW macrophages (2x108 cells) were washed in ice-cold PBS and scraped into 100 ml ice-cold PBS. Samples were centrifuged at 400 g for 5 min to collect the cells, which were then resuspended in 1 ml ice-cold PMEE (35 mM PIPES pH 7·4, 5 mM MgSO4, 1 mM EGTA, 0·5 mM EDTA and 250 mM sucrose). Samples were passed through a 21 G needle several times, until more than 80% of the cells were lysed. The lysates were cleared of nuclei and other cellular debris by centrifugation at 400 g for 5 min at 4 °C. Cleared lysates were then centrifuged at 150000 g for 1 h at 4 °C, onto a 30% sucrose layer. The supernatant of this centrifugation (cytosol) was either used directly for growth assays or frozen by immersion of tubes in liquid nitrogen. To obtain cytosol from Salmonella-infected macrophages, the cells were first infected with aroC purD double mutant bacteria expressing green fluorescent protein (GFP) at an m.o.i. of approximately 100:1. After 4 h, cells were processed as described above. Samples were taken from each preparation to confirm that at least 50% of the cells were infected. For growth assays, 103 c.f.u. of exponentially growing wild-type bacteria were added to a 100 µl aliquot of cytosol extract and incubated at 37 °C for 8 h. All cytosol extractions were diluted to a final protein concentration of 50 µg ml-1 in PMEE before use. Aliquots of 10 µl were removed immediately after adding the bacteria and 8 h later, diluted, and plated onto LB plates to enumerate bacteria.
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RESULTS AND DISCUSSION |
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It is noteworthy that the replication of the ssaV mutant strain at 8 h after bacterial entry was equivalent to that of the wild-type strain, but was 10 times lower at 16 h (Fig. 1b, c
). This is consistent with results of Brumell et al. (2001)
, who found that SPI-2 is not required for replication in HeLa cells up to 6 h after bacterial entry.
The presence of large numbers of sifA mutant bacteria in the cytosol of HeLa cells 10 h after entry could be explained by either an increased replication inside the vacuole followed by release into the cytosol, or release from the vacuole followed by an increased replication rate within the cytosol. To differentiate between these two possibilities, an experiment was performed in which bacterial numbers per infected cell were counted by microscopy at different time points throughout the infection. To distinguish between vacuolar and cytosolic bacteria, we used LAMP-1 as a marker for the presence of the vacuolar membrane. Immunogold labelling of ultrathin sections of HeLa cells infected with wild-type bacteria with a monoclonal anti-LAMP-1 antibody showed that LAMP-1 is localized on the SCV membrane (Fig. 2), confirming the suitability of LAMP-1 as a marker for the presence of the SCV membrane.
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A striking characteristic of the cytosolic bacteria was their unusually large size. Although these bacteria maintain a normal bacillus shape, approximately 75% of them were 24 µm long, almost double the usual length of the wild-type, which ranges from 1 to 2 µm (data not shown). Such large bacteria were never found in cells infected with wild-type bacteria (n>100 infected cells). A representative example can be seen in Fig. 1(a), where cross-sections of both wild-type and cytosolic sifA mutant bacteria are shown in electron micrographs at the same magnification.
Together, these results indicate that S. typhimurium is capable of replicating within the cytosol of human epithelial cells. These results are in contrast to the results obtained by direct microinjection of Salmonella into epithelial cells (Goetz et al., 2001 ). There are several possible explanations for these differences. We cannot rule out the possibility that the sifA mutation may confer a replication advantage upon bacteria in the cytosol. However, it seems more likely that as a consequence of microinjection, bacteria delivered into the cytosol are not capable of replication, either because of mechanical damage affecting the integrity of the bacterial cell, or because the appropriate set of genes that allow bacterial replication within the cytosol is only activated upon normal bacterial entry and passage through the vacuole.
Salmonella replication in the cytosol of non-permissive cell lines is impaired
The ability of the sifA mutant strain to replicate in the cytosol of epithelial cells is in contrast to its replication defect in tissue-cultured macrophages (Beuzón et al., 2000 ; Brumell et al., 2001
). The sifA mutant strain also displayed a severe replication defect in mouse Swiss 3T3 fibroblasts, equivalent to that of the ssaV mutant strain, and approximately a tenth of the replication displayed by the wild-type strain (Fig. 5a
, b
). This cell line has been shown to be restrictive for Salmonella replication (Cano et al., 2001
; García-del Portillo, 2001
; Martínez-Moya et al., 1998
). The presence of the SCV membrane surrounding intracellular bacteria was assessed using LAMP-1 association with either wild-type or sifA mutant bacteria as a marker for the vacuolar membrane, 10 h after bacterial entry. Whereas more than 60% of the wild-type bacteria were clearly associated with LAMP-1, less than 5% of the sifA bacteria were found to associate with the membrane marker (Fig. 5c
). As observed in epithelial cells, in both macrophages and fibroblasts a high proportion of unusually large bacteria could be detected in the LAMP-1-negative population of sifA mutant bacteria (data not shown).
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Evidence of Salmonella killing in the cytosol of macrophages
Although the sifA mutant strain has a replication defect in macrophages, we have consistently observed a small net increase in the number of intracellular mutant bacteria between 2 and 16 h (Beuzón et al., 2000 ; and data not shown). To test if bacterial death is occurring simultaneously with bacterial replication, we followed the same approach as used by Buchmeier & Libby (1997)
. To estimate bacterial death in the absence of bacterial replication, we used an auxotrophic double mutant strain (aroC purD), which is unable to grow in cultured macrophages (Fields et al., 1986
). When assayed in time-course replication assays, the aroC purD mutant strain, which remains within a vacuole throughout the course of the infection (Ruiz-Albert et al., 2002
), displayed a fivefold decrease in numbers between 2 and 16 h, in close agreement with the results reported by Buchmeier & Libby (1997)
(Fig. 7a
). An aroC purD sifA triple mutant strain, which is released into the host cell cytosol at the same rate as a sifA mutant strain (Ruiz-Albert et al., 2002
), also displayed a decrease in bacterial numbers (Fig. 7a
). Furthermore, the growth defect of the aroC purD double mutant strain, but not that of the aroC purD sifA triple mutant, could be completely restored by supplementing the macrophage culture medium with appropriate metabolites to supplement the auxotrophy (data not shown). These results indicate that a fraction of the cytosolic population of Salmonella is being actively killed by an antimicrobial activity. However, in contrast with what happens to vacuole-enclosed Salmonella, the low net bacterial growth in the cytosol of macrophages suggests that bacterial replication, as well as survival, is reduced in this environment. Similar replication assays using the auxotrophic strains in HeLa cells did not reveal any evidence of an antimicrobial activity (data not shown).
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It is interesting to consider the apparent contradiction between the different replication efficiencies of Salmonella and Listeria in the cytosol of macrophages, and their equal sensitivity in vitro to the antimicrobial activity activated by interferon- addition or by Salmonella infection. One possible explanation is that Listeria is only sensitive to this activity(s) in vitro, because it responds to conditions within the vacuole by expressing proteins that allow it to survive and replicate within the cytosol of macrophages. An alternative explanation could be that Salmonella, but not Listeria, infection triggers the onset of this activity(s). This would explain how an extracellular soil micro-organism such as B. subtilis could replicate in the cytosol of macrophages if its uptake does not trigger the onset of this defence mechanism(s). Further work will be necessary to reveal the mechanism(s) that allow cytosolic pathogens to evade this activity(s).
In summary, our results indicate that when S. typhimurium is released into the cytosol of epithelial cells it is able to replicate, whereas in macrophage cytosol it cannot replicate and encounters an anti-Salmonella killing activity. Presumably, this constitutes a strong selective pressure for the maintenance of the integrity of the Salmonella-containing vacuole.
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 May 2002;
revised 30 May 2002;
accepted 31 May 2002.