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Infection and Immunity, April 2009, p. 1589-1595, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.01257-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Caspase-1 Mediates Resistance in Murine Melioidosis

Katrin Breitbach,¹ Guang Wen Sun,² Jens Köhler,¹ Kristin Eske,¹ Patimaporn Wongprompitak,^1,3 Gladys Tan,⁴ Yichun Liu,⁴ Yunn-Hwen Gan,² and Ivo Steinmetz^1*

Friedrich Loeffler Institute of Medical Microbiology, Ernst Moritz Arndt University Greifswald, Greifswald, Germany,¹ Department of Biochemistry, National University of Singapore, Singapore, Republic of Singapore,² Department of Immunology, Faculty of Medicine Siriraj, Mahidol University, Bangkok, Thailand,³ Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore, Republic of Singapore⁴

Received 14 October 2008/ Returned for modification 19 December 2008/ Accepted 18 January 2009

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The gram-negative rod Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease which is endemic in tropical and subtropical areas. The bacterium multiplies intracellularly within the cytosol, induces the formation of actin tails, and can spread directly from cell to cell. Recently, it has been shown that B. pseudomallei can induce caspase-1-dependent cell death in macrophages. The aim of the present study was to further elucidate the role of caspase-1 during B. pseudomallei infection. In vivo experiments with caspase-1^–/– mice revealed a high susceptibility to B. pseudomallei challenge. This phenotype was associated with a significantly higher bacterial burden 2 days after infection and decreased gamma interferon (IFN-) and interleukin-18 cytokine levels 24 h after infection compared to control animals. caspase-1^–/– bone marrow-derived macrophages (BMM) exhibited strong caspase-3 expression and reduced cell damage compared to wild-type (WT) cells during early B. pseudomallei infection, indicating "classical" apoptosis, whereas WT BMM showed signs of rapid caspase-1-dependent cell death. Moreover, we found that caspase-1^–/– BMM had a strongly increased bacterial burden compared to WT cells 3 h after infection under conditions where no difference in cell death could be observed between both cell populations at this time point. We therefore suggest that caspase-1-dependent rapid cell death might contribute to resistance by reducing the intracellular niche for B. pseudomallei, but, in addition, caspase-1 might also have a role in controlling intracellular replication of B. pseudomallei in macrophages. Moreover, caspase-1-dependent IFN- production is likely to contribute to resistance in murine melioidosis.

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Burkholderia pseudomallei is a gram-negative rod and the causative agent of melioidosis, an infectious disease of humans and animals in certain areas of the tropics (36). In areas of endemicity, B. pseudomallei is known to be a major cause of morbidity and mortality, and clinical manifestations are extremely variable, ranging from nonapparent to localized subacute or chronic infections and fulminant septicemias with abscesses in multiple organs (24). The infection is acquired by percutaneous inoculation, ingestion, or inhalation after contact with contaminated water, soil, or aerosols (8, 37). Underlying diseases such as diabetes and renal failure are known risk factors for an acute and fulminant course of infection (36), but the underlying immunological mechanisms responsible for the variable outcomes after B. pseudomallei infection are still unclear (6, 9, 37).

B. pseudomallei is able to invade, survive, and replicate inside phagocytic and nonphagocytic cells and furthermore induces the formation of actin tails, which enable the bacterium to spread directly from cell to cell (4, 18, 19). Several studies have shown that gamma interferon (IFN-) is essential for the early control of B. pseudomallei infection in mice (3, 14, 31). Recently, we provided evidence for an essential role of macrophages in controlling the early phase of B. pseudomallei infection. In this context we found that macrophages derived from resistant C57BL/6 mice exhibit an enhanced bactericidal activity after B. pseudomallei infection compared to macrophages from susceptible BALB/c mice (3). Moreover, we could show that NADPH oxidase-mediated mechanisms contribute to early resistance in C57BL/6 mice whereas inducible nitric oxide synthase is not essential in the early phase (3).

In a recent study we showed that B. pseudomallei is capable of inducing caspase-1-dependent cell death in monocytes and macrophages (34), a phenomenon that has also been reported for other bacterial pathogens such as Salmonella and Listeria (5, 29). Several in vivo studies have shown that caspase-1 is crucial for resistance against a variety of intracellular pathogens (28-30, 35). Therefore, the aim of the present study was to examine the in vivo relevance of caspase-1 in a pulmonary murine B. pseudomallei infection model. Moreover, we investigated the course of B. pseudomallei infection in caspase-1^–/– and wild-type (WT) bone marrow-derived macrophages (BMM).

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Bacterial strains. B. pseudomallei strain E8 comprises a soil isolate from the surrounding area of Ubon Ratchathani in northeast Thailand (40) and was obtained from N. J. White (Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand). B. pseudomallei KHW is a clinical isolate from a patient who died from melioidosis (26). Prior to the experiments, bacteria were cultured either in Luria-Bertani broth or on blood agar plates and adjusted to the desired concentration in phosphate-buffered saline (PBS) or the respective cell culture medium.

Animals. Age- and sex-matched caspase-1^–/– mice on a C57BL/6 background (20) and control animals were purchased from the Laboratory Animals Centre (National University of Singapore, Singapore) and the Department of Laboratory Animal Science (University of Greifswald, Germany), respectively. Animals were housed under specific-pathogen-free conditions, and all in vivo studies were approved by the local authorities.

In vivo infection and determination of bacterial burden. For in vivo experiments mice received approximately 100 CFU of B. pseudomallei intranasally per animal. Mice were monitored daily for survival after infection. To evaluate the bacterial burden of internal organs, mice were sacrificed on day 1 or 2 after infection, and their lungs, spleens, and livers were homogenized and plated onto Ashdown agar plates in appropriate dilutions. Data are presented as the total bacterial count per organ.

Generation and cultivation of BMM. BMM were generated and cultivated in a serum-free cell culture system as recently described (11). Briefly, tibias and femurs were aseptically removed, and bone marrow cells were flushed with sterile PBS and then centrifuged at 150 x g for 10 min. Cells were resuspended in RPMI medium containing 5% Panexin BMM (PAN Biotech, Aidenbach, Germany), 2 ng per ml of recombinant murine granulocyte-macrophage colony-stimulating factor (PAN Biotech), and 50 µM mercaptoethanol and cultivated for at least 10 days at 37°C and 5% CO₂. BMM were positive for the expression of F4/80, Mac-1, and MOMA-2 as determined by fluorescence-activated cell sorter analysis. Less than 0.5% of mature BMM were positive for the lymphoid markers CD3 and DX5. Twenty-four hours prior to infection experiments, BMM were seeded in 6-, 48-, or 96-well plates as indicated below. In some experiments, cells were stimulated with 300 units per ml of recombinant murine IFN- (PAN Biotech) 24 h prior to infection.

LDH assay. To quantify the extent of cell damage after infection, release of lactate dehydrogenase (LDH) in cell culture supernatants was determined. BMM were seeded in 96-well plates (2.5 x 10⁴ cells per well) and infected at the indicated multiplicity of infection (MOI) (see respective figure) with B. pseudomallei E8 for 30 min. Cells were washed twice with PBS, and 100 µl of medium containing 100 µg per ml of kanamycin was added to each well to eliminate extracellular bacteria. At the indicated time points (see respective figure), cell culture supernatant was collected, and LDH activity was detected by using a CytoTox-One homogeneous membrane integrity assay (Promega Corp., Madison, WI) according to the manufacturer's instructions. Briefly, 50 µl of supernatant was added to the kit reagent and incubated for 10 min. After addition of stopping solution, the fluorescence intensity was measured using a microplate reader (1420 Victor 2; LKB-Wallac) (excitation wavelength, 560 nm; emission wavelength, 590 nm).

Caspase-3 assay. To determine caspase-3 activity in WT and caspase-1^–/– BMM, cells were seeded in six-well plates (1 x 10⁶ cells per well) and infected with B. pseudomallei strain E8 for 30 min. Cells were washed twice with PBS, and medium containing 100 µg/ml kanamycin was added to each well. Four hours after incubation, cells were resuspended in caspase lysis buffer (R&D Systems, Inc. Minneapolis, MN), and protein concentration was determined by using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL) according to the manufacturer's instructions. Protein content was adjusted to 100 µg of protein per sample, and caspase-3 activity was determined using a caspase-3 colorimetric assay system (R&D Systems) according to the manufacturer's instructions. Optical density was measured with a microplate reader at 405 nm (Multiskan EX; Thermo Scientific).

BMM bactericidal assay and in vitro caspase-1 inhibition. To determine the bactericidal capacity of BMM, cells were seeded in 48-well plates (1.5 x 10⁵ cells per well) and infected with B. pseudomallei strain E8 at the indicated MOI (see respective figure) for 30 min. Cells were washed twice with PBS, and medium containing 100 µg/ml kanamycin was added to each well. At the indicated time points (time zero was taken 20 min after incubation under antibiotic-containing medium), the number of intracellular CFU was determined as previously described (3). For in vitro caspase-1 inhibition, BMM were treated with the specific caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk) (Calbiochem), dissolved in dimethyl sulfoxide, at a final concentration of 50 µM starting 1 h prior to infection and during the course of infection. Control cells were incubated with an equivalent volume of dimethyl sulfoxide.

Determination of cytokines. Serum samples from infected mice were taken at 24 h postinfection and stored at –20°C. Production of interleukin 1β (IL-1β) (IBL, Hamburg, Germany) and IL-18 (Medical and Biological Laboratories, Nagoya, Japan) were measured by enzyme-linked immunosorbent assay; release of IFN- was determined by using a Cytometric bead assay mouse inflammation kit (Becton Dickinson) according to the manufacturer's instructions. For the detection of IL-1β in the cell culture supernatant, BMM were seeded and infected in 48-well plates as described above. Supernatants were taken 6 h after infection and stored at –20°C until IL-1β detection was performed. Cell culture supernatant was sterilized by UV light treatment prior to the cytokine measurements.

Statistical analysis. Survival curves were compared using the log rank Kaplan-Meier test. To determine statistically significant differences between groups, either a Student's t test, Mann-Whitney test, or two-way analysis of variance was used as indicated for each experiment. Statistical analyses were performed using GraphPad Prism, version 4.0.

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C57BL/6 caspase-1^–/– mice are highly susceptible to B. pseudomallei challenge. To assess the importance of caspase-1 in vivo, we challenged C57BL/6 mice lacking caspase-1. C57BL/6 mice are, per se, relatively resistant to B. pseudomallei challenge (16, 23, 26), and knockout mice with this genetic background were previously used to identify genes important to the defense against B. pseudomallei (2, 3, 38). Since inhalation is considered to be an important route of infection in humans (8), we challenged animals via the intranasal route with the clinical B. pseudomallei isolate KHW. This bacterial strain was shown to induce caspase-1-dependent cell death in macrophages in our previous study (34). As demonstrated in Fig. 1A, C57BL/6 caspase-1^–/– mice died within 72 h after intranasal infection with B. pseudomallei KHW, whereas all control animals survived the observation period. To exclude a bacterial strain-specific effect, we also challenged C57BL/6 caspase-1^–/– mice with the environmental B. pseudomallei isolate E8 and could confirm the high susceptibility of C57BL/6 caspase-1^–/– mice (Fig. 1B). The enhanced mortality rate of caspase-1^–/– animals was associated with a significantly increased bacterial burden in spleens, livers, and lungs 48 h after challenge with B. pseudomallei E8 (Fig. 1C). Furthermore, caspase-1^–/– mice exhibited significantly decreased serum levels of IFN- (Fig. 2A) and IL-18 (Fig. 2B) at 24 h after infection, whereas no differences could be observed in the production of IL-12, tumor necrosis factor alpha, monocyte chemotactic protein 1, and IL-6 at this time point (data not shown).

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FIG. 1. (A and B) Survival curves of C57BL/6 WT and caspase-1–/– mice after intranasal infection with B. pseudomallei KHW (A) and E8 (B). Mice received 100 CFU per animal. All WT mice survived for at least 2 weeks (data not shown). Statistical analyses were performed by using a Kaplan-Meier test. (C) Bacterial loads in spleens, livers, and lungs of B. pseudomallei E8-infected mice were determined 24 h and 48 h after infection. Each dot represents the bacterial count of the respective organ of a single animal. The horizontal line represents the median of each group. Statistical analyses were performed by using a Mann-Whitney test. For all panels, data were obtained and pooled from two independent experiments. n.s., nonsignificant. *, P < 0.05; **, P < 0.01.

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FIG. 2. Detection of IFN- (n = 4) (A) and IL-18 (n = 7) (B) release in the serum of caspase-1–/– and C57BL/6 WT mice 24 h after intranasal infection with B. pseudomallei E8. Uninfected mice revealed either no detectable or negligible amounts of IFN- and IL-18 release (data not shown). Values are the means ± standard deviations. Statistical analysis was performed by using a Student's t test. **, P < 0.01; ***, P < 0.001.

Course of cell death in caspase-1^–/– BMM after B. pseudomallei infection is MOI dependent. Macrophages play a crucial role in determining resistance against murine melioidosis (3). We therefore examined the kinetics of cell death of caspase-1^–/– and WT BMM after infection with B. pseudomallei under different experimental conditions. Our previous study revealed strongly enhanced cell damage of C57BL/6 peritoneal macrophages compared to macrophages lacking caspase-1 within 8 h after infection with B. pseudomallei at a high MOI of 100 (34). To further characterize the cell damage in caspase-1^–/– macrophages, we infected caspase-1^–/– BMM and WT cells with B. pseudomallei at different MOIs (Fig. 3). When a low MOI (3) was used, we detected no differences in LDH release within 3 h after infection and slight but already significantly reduced cell damage 6 h postinfection in caspase-1^–/– BMM compared to WT cells (Fig. 3A). In contrast, 24 h after infection the cell damage was significantly more enhanced in caspase-1^–/– BMM (Fig. 3A). Similar results were obtained when cells were infected at a moderate MOI of 30 (data not shown). By using a high MOI (200), we found much more pronounced cell damage in WT BMM than in caspase-1^–/– cells within the early stages of infection (Fig. 3B), which is in agreement with results from our previous study (34). However, 24 h after infection, caspase-1^–/– BMM exhibited strongly increased LDH release, and a difference in the extent of cell damage was no longer observed compared to WT BMM at this time point (Fig. 3B).

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FIG. 3. Course of cell damage in C57BL/6 WT and caspase-1–/– BMM after B. pseudomallei E8 infection at the indicated MOIs. Values are the means ± standard deviations from triplicate determinations. One out of at least three independent experiments with similar results is shown. Statistical analyses were performed by using a Student's t test. n.s., not significant. **, P < 0.01; ***, P < 0.001.

Caspase-3 activation and IL-1β release of caspase-1^–/– and WT BMM after B. pseudomallei infection. To further analyze the underlying mechanism of the different courses of cell death in caspase-1^–/– and WT BMM, we determined caspase-1 and caspase-3 activation after B. pseudomallei infection. It has previously been shown that caspase-1 activation is crucial for sufficient IL-1β release of macrophages infected with various pathogens (25, 28, 41). Therefore, we examined whether caspase-1-dependent IL-1β release might occur in B. pseudomallei-infected BMM. We were able to detect significant amounts of mature IL-1β in the supernatant of B. pseudomallei-infected WT BMM and only negligible amounts of IL-1β in caspase-1^–/– BMM supernatant 6 h after infection (Fig. 4A), which argues for rapid caspase-1-dependent cell death in WT cells. Conversely, caspase-1^–/– BMM exhibited caspase-3 activation, as illustrated in Fig. 4B, within 4 h after infection, whereas no significant activation of caspase-3 could be detected in WT cells at this time point. We suggest that the delayed onset of caspase-3-dependent cell damage in caspase-1^–/– BMM finally led to a strong LDH release 24 h after infection (Fig. 3).

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FIG. 4. (A) Release of IL-1β from C57BL/6 WT and caspase-1–/– BMM 6 h after B. pseudomallei E8 infection (MOI of 30). Values are the means ± standard deviations from triplicate determinations. One out of three independent experiments with similar results is shown. Statistical analysis was performed by using a Student's t test. (B) Caspase-3 activity of caspase-1–/– and C57BL/6 WT macrophages 4 h after B. pseudomallei E8 infection (MOI of 30). Data were obtained from three independent experiments. Statistical analysis was performed by two-way analysis of variance. **, P < 0.01; ***, P < 0.001.

caspase-1^–/– BMM exhibit impaired bactericidal activity compared to WT BMM. We next assessed whether caspase-1-dependent mechanisms might contribute to the bactericidal capacity of macrophages, examining the course of the intracellular bacterial burden in caspase-1^–/– and WT BMM at low (MOI of 3) and moderate (MOI of 30) B. pseudomallei infection doses (Fig. 5). Although the uptake of bacteria was comparable, deficiency of caspase-1 was associated with an approximately 10-fold increase in bacterial burden 3 h after infection at an MOI of 3 (Fig. 5A). At this time point no differences in cell damage could be observed between caspase-1^–/– and WT BMM (Fig. 3A). The bacterial load was even more pronounced 6 h after infection and also when cells were infected at an MOI of 30 (Fig. 5A and C). To assess whether IFN- might restore the impaired killing potential of caspase-1^–/– BMM, we treated cells with IFN- prior to infection. As illustrated in Fig. 5B and D, IFN--stimulated caspase-1^–/– BMM showed impaired elimination of intracellular B. pseudomallei compared to WT macrophages at different MOIs. A decreased ability to eliminate intracellular B. pseudomallei was also observed in BMM from C57BL/6 and BALB/c BMM after treatment with the specific caspase-1 inhibitor Ac-YVAD-cmk (Fig. 5E). Thus, caspase-1 seems to be essential for the bactericidal function of macrophages to combat B. pseudomallei infection.

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FIG. 5. (A to D) Course of the bacterial burden in C57BL/6 WT and caspase-1–/– BMM after B. pseudomallei infection. (A) Unstimulated BMM infected at an MOI of 3. (B) IFN--stimulated macrophages infected at an MOI of 3. (C) Unstimulated BMM infected at an MOI of 30. (D) IFN--stimulated BMM infected at an MOI of 30. (E) Bacterial load in caspase-1-inhibited (Ac-YVAD-cmk [Ac-YVAD] treated) BMM from BALB/c and C57BL/6 mice 6 h after infection with B. pseudomallei E8 (MOI of 30). Bacterial uptake after infection was comparable for all types of macrophages (data not shown). Values are the means ± standard deviations from triplicate determinations. One out of at least three independent experiments with similar results is shown. Statistical analyses were performed by using a Student's t test. **, P < 0.01; ***, P < 0.001. w/o, without.

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Caspase-1 is the prototypical member of the inflammatory caspases and is involved in the maturation process of proinflammatory cytokines such as IL-1β and IL-18 (22, 27). Its role in controlling intracellular infections has received increased attention in recent years and was shown to vary among different infection models. Whereas caspase-1 contributes to resistance against systemic infections with Salmonella, Shigella, Listeria, and Francisella (28-30, 35), a recent study reported that the bacterial load during Chlamydia trachomatis infection was not affected in caspase-1^–/– mice and, moreover, that these animals showed reduced inflammatory pathologies compared to WT animals (7). In the present study, we describe an essential function of caspase-1 in resistance against infection with B. pseudomallei using a murine pulmonary infection model in which C57BL/6 mice lacking caspase-1 proved to be highly susceptible to infection. Moreover, caspase-1^–/– mice exhibited reduced expression of IFN- and IL-18, which are known to be essential for an effective defense against B. pseudomallei challenge (3, 14, 31, 39). In a different set of experiments we also observed higher mortality and reduced IFN- and IL-18 expression in caspase-1-deficient nonobese diabetic mice after intravenous B. pseudomallei infection (data not shown). Thus, caspase-1-dependent resistance is likely to be at least partly due to IL-18-mediated production of IFN-.

Many intracellular pathogens such as Salmonella, Shigella, and Listeria are known to induce activation of caspase-1 in macrophages, which can cause rapid death of these cells (5, 15, 29, 30, 35). In this context, caspase-1 is reported to act like a double-edged sword since, on the one hand, cell death might be protective for the host when the intracellular niche of the pathogen can be destroyed, and, on the other hand, caspase-1-dependent inflammatory processes can be harmful to the host. Caspase-1-mediated cell death is also discussed as a mechanism used by pathogens in order to kill, e.g., macrophages before they themselves are killed (17, 34). Recently, we showed that the facultative intracellular gram-negative rod B. pseudomallei can induce caspase-1-dependent cell death in murine peritoneal macrophages (34). This kind of cell death resembled oncosis and pyroptosis (12) rather than classical apoptosis and had also been observed in macrophages after infection with Listeria monocytogenes (5). The present data supplement our former observations (34) since murine WT BMM exhibited caspase-1-dependent IL-1β release but no activation of caspase-3, in contrast to caspase-1^–/– macrophages, which seemed to die as a result of caspase-3-dependent classical apoptosis. This might explain the different kinetics of cell damage after challenge with B. pseudomallei at a high MOI since rapid caspase-1-dependent cell death in WT cells led to high LDH release within the first hours after infection compared to caspase-1^–/– macrophages, which exhibited the slower caspase-3-dependent cell death.

However, the extent as well as the course of cell damage seemed to be strongly dependent on the infection modus. When a relatively low MOI was used, we observed no differences within 3 h after infection and only a minor difference 6 h after infection in LDH release between WT and caspase-1^–/– macrophages. Interestingly, we already detected a 10-fold enhanced bacterial burden in caspase-1^–/– macrophages 3 h after infection under these experimental conditions. Since the cell damage at that time was still negligible, we suggest that caspase-1-dependent effector functions might contribute to control the intracellular replication of B. pseudomallei. Decreased bactericidal activity was also observed in BMM from caspase-1^–/– nonobese diabetic mice after B. pseudomallei infection (data not shown). Thus, absence of caspase-1 might result in not only a prolonged but also a more favorable intracellular niche with a reduced bactericidal capacity for B. pseudomallei. This might eventually lead to increased intracellular replication and subsequent spreading of the pathogen. caspase-1^–/– macrophages have been previously reported to show increased intracellular replication of Legionella due to impaired phagosome-lysosome fusion (1). In this context, the Naip5/Birc1e protein family is considered to promote the fusion of phagosomes with lysosomes to eventually control intracellular Legionella growth, and a possible caspase-1-dependent link is under debate (13, 21, 41). However, in contrast to Legionella, which resides within the vacuole, B. pseudomallei gains access to the cytosol within 3 h postinfection (33). Thus, further studies are necessary to investigate possible caspase-1-dependent antibacterial effector functions that control intracytosolic replication of B. pseudomallei in macrophages. Apart from macrophages, neutrophils have also been shown to be essential in protecting mice from acute fatal disease after B. pseudomallei challenge (10), whereas NK cells as well as T and B cells do not seem to contribute to early resistance in murine melioidosis (14). Since caspase-1 was recently shown to be expressed in neutrophils after infection with Legionella pneumophila (32), the lack of this enzyme in these cells of caspase-1^–/– mice might also contribute to the high in vivo susceptibility to B. pseudomallei observed in this study.

Taken together, our results indicate that caspase-1-dependent immune mechanisms play an essential role in resistance against B. pseudomallei infection. In this context, caspase-1-dependent cell death might reduce a favorable intracellular niche for the pathogen. Furthermore, we suggest that caspase-1-dependent mechanisms contribute to the control of intracellular replication of B. pseudomallei in macrophages. Finally, caspase-1-dependent IL-18-mediated IFN- production is likely to play a role in the defense of B. pseudomallei infections.

 
Y.-H.G. was supported by a grant of the National University Singapore, Office of Life Sciences R-183-000-602-712. P.W. was supported by the Royal Golden Jubilee Ph.D. Program under the Thailand Research Fund. J.K. and K.E. were supported by a grant of the Graduate College 840 (Deutsche Forschungsgemeinschaft) to I.S.

We are grateful to Helga Schalimow and Claudia Cordt for excellent technical assistance.

 
* Corresponding author. Mailing address: Friedrich Loeffler Institute of Medical Microbiology, Ernst Moritz Arndt University Greifswald, Martin Luther Str. 6, 17489 Greifswald, Germany. Phone: 49 (0) 3834 865587. Fax: 49 (0) 3834 865561. E-mail: steinmetz.ivo{at}uni-greifswald.de

Published ahead of print on 29 January 2009.

Editor: S. R. Blanke

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Infection and Immunity, April 2009, p. 1589-1595, Vol. 77, No. 4
0019-9567/09/$08.00+0     doi:10.1128/IAI.01257-08
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