Nov 28, 2010 - iNOS and degradation of I B correlated with enhanced macrophage killing capability. These results ... Gram-negative bacteria, B. pseudomallei fails to activate the ... (MAL) protein, TIR domain-containing adaptor-inducing.
INFECTION AND IMMUNITY, July 2011, p. 2921–2927 0019-9567/11/$12.00 doi:10.1128/IAI.01254-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 7
Burkholderia pseudomallei-Induced Expression of a Negative Regulator, Sterile-␣ and Armadillo Motif-Containing Protein, in Mouse Macrophages: a Possible Mechanism for Suppression of the MyD88-Independent Pathway䌤 M. Pudla, K. Limposuwan, and P. Utaisincharoen* Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand Received 28 November 2010/Returned for modification 14 April 2011/Accepted 27 April 2011
Burkholderia pseudomallei, a causative agent of melioidosis, is a Gram-negative facultative intracellular bacterium that can survive and multiply in macrophages. Previously, we demonstrated that B. pseudomallei failed to activate gene expression downstream of the MyD88-independent pathway, particularly the expression of beta interferon (IFN-) and inducible nitric oxide synthase (iNOS), leading to the inability of macrophages to kill this bacterium. In the present report, we extended our study to show that B. pseudomallei was able to activate sterile-␣ and Armadillo motif (SARM)-containing protein, a known negative regulator of the MyD88independent pathway. Both live B. pseudomallei and heat-killed B. pseudomallei were able to upregulate SARM expression in a time-dependent manner in mouse macrophage cell line RAW 264.7. The expression of SARM required bacterial internalization, as it could be inhibited by cytochalasin D. In addition, the intracellular survival of B. pseudomallei was suppressed in SARM-deficient macrophages. Increased expression of IFN- and iNOS and degradation of IB␣ correlated with enhanced macrophage killing capability. These results demonstrated that B. pseudomallei modulated macrophage defense mechanisms by upregulating SARM, thus leading to the suppression of IFN- and iNOS needed for bacterial elimination. of the MyD88-independent pathway, is essential for intracellular killing of B. pseudomallei (29). B. pseudomallei-infected macrophages fail to activate IFN- production, leading to a reduced expression of a key enzyme, inducible nitric oxide synthase (iNOS), needed for antibacterial activity of the macrophages (29). Exogenous IFN- can restore the ability of macrophages to activate iNOS expression and promote the killing of intracellular B. pseudomallei (28, 29). The failure to stimulate IFN- may be due to the fact that, unlike most other Gram-negative bacteria, B. pseudomallei fails to activate the MyD88-independent pathway, which is an essential signaling pathway of Toll-like receptor-4 (TLR4) (24). In contrast to wild-type B. pseudomallei, the lipopolysaccharide (LPS) mutant strain (SRM117) that lacks the O-antigenic polysaccharide moiety was able to activate gene expression downstream of the MyD88-independent pathway, resulting in the inability of the bacteria to survive and replicate inside the macrophages (24). TLRs exert their function via a family of Toll/interleukin 1 (IL-1) receptor (TIR) adaptor proteins. To date, five TIRs have been identified, namely MyD88, MyD88 adaptor-like (MAL) protein, TIR domain-containing adaptor-inducing IFN- (TRIF), TRIF-related adaptor molecule (TRAM), and sterile-alpha and Armadillo motif (SARM)-containing protein (3, 17, 20). Among these, SARM was shown to function as a specific inhibitor of TRIF-dependent TLR signaling (5). Although the molecular mechanism of this inhibition is not known, it has been demonstrated that SARM could directly interact with TRIF, thus leading to suppression of TRIF-dependent activation (5). Overexpression of SARM leads to a decrease in gene expression downstream of TRIF, such as that of ifn-, suggesting that SARM activation may interfere with IFN- production (5). Therefore, SARM may be essential in
Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is responsible for a large proportion of community-acquired septicemia in several tropical areas, including Southeast Asia and Northern Australia (6, 19). Clinical manifestations are extremely variable, ranging from subacute and chronic suppurative infections to acute pneumonias and fulminating septicemias (4, 36). The infection is acquired by percutaneous inoculation, ingestion, or inhalation of B. pseudomallei after an exposure to contaminated water, soil, or aerosols (7, 36). The mortality rate is very high despite prolonged antibiotic treatment (6, 15). Due to the severity of the infection and its potential as a biological threat agent, B. pseudomallei is now listed as a category B agent by the Centers for Disease Control and Prevention (22, 32). At a cellular level, B. pseudomallei is able to invade, survive, and multiply inside both phagocytic and nonphagocytic cells (12). After internalization, the bacterium can escape from a membrane-bound phagosome to the cytoplasm and induce actin-associated membrane protrusion, which may facilitate its spreading from cell to cell (10, 12, 13). Recent studies demonstrated that both type III secretion systems (T3SSs) and the global regulatory factor RpoS are involved in this process (23, 26). Although macrophages play a critical role in innate immunity against a number of bacteria, B. pseudomallei can survive and replicate inside these phagocytes (30). We previously demonstrated that beta interferon (IFN-), an effector downstream
* Corresponding author. Mailing address: Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand, Phone: 662-201-5954. Fax: 662-201-5950. E-mail: scput@mahidol .ac.th. 䌤 Published ahead of print on 9 May 2011. 2921
2922
PUDLA ET AL.
regulating infection outcomes. In the present study, we investigated a possible involvement of SARM in B. pseudomallei infection by using the mouse macrophage cell line as a model.
INFECT. IMMUN. Statistical analysis. Unless indicated otherwise, all experiments in this study were conducted at least three times. Experimental values were expressed as means ⫾ standard errors of the means. Statistical significance of differences between two means was evaluated by Student’s t test. A P value of ⬍0.05 was considered significant.
MATERIALS AND METHODS Cell line and culture condition. RAW 264.7 macrophages (ATCC) were cultured in advanced Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (HyClone) and 1% L-glutamine (Gibco Labs, Grand Island, NY) at 37°C under a 5% CO2 atmosphere. Bacterial strain. B. pseudomallei parental wild-type strain (1026b) was previously described (8). In some experiments, the LPS mutant (SRM117) that lacks the O-antigenic polysaccharide moiety was used for comparison (8). Bacteria were cultured in Luria-Bertani (LB) broth at 37°C with agitation at 150 rpm. Overnight cultures were washed twice in phosphate-buffered saline (PBS) and adjusted to a desired concentration by measuring the optical density at 650 nm, and the CFU was calculated from the precalibrated standard curve. Heat-killed bacteria. Nonviable B. pseudomallei was prepared by heating the bacteria (108 CFU/ml in PBS) in a boiling-water bath for 15 min. The heattreated bacteria were washed three times with PBS, and complete killing was confirmed by inoculating the suspension on tryptic soy agar and observing growth after 48 h. SARM depletion in macrophage cell line (RAW 264.7). The mouse macrophage cell line RAW 264.7 was transfected with SARM small interfering RNAs (siRNAs) (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. In brief, macrophages (1.5 ⫻ 105 cells/well) were seeded overnight in a 6-well plate. Cells were then transfected with control or SARM siRNAs (375 ng) using HiPerFect (Qiagen). After 24 h of incubation, the expression of SARM mRNA and protein was determined by reverse transcriptase PCR (RT-PCR) and immunoblotting. The siRNA sequences used were 5⬘ GGA GAU UGU GAC UGC UUU ATT 3⬘ (sense) and 5⬘ UAA AGC AGU CAC AAU CUC CTT 3⬘ (antisense). As a control, we used AllStars negative control siRNA (Qiagen). Infection of mouse macrophage cell line (RAW 264.7). An overnight culture of mouse macrophages in a 6-well plate was infected with bacteria at a multiplicity of infection (MOI) of 2 for 1 h. To remove extracellular bacteria, the cells were washed twice with 1 ml of PBS, and residual bacteria were killed by incubating the cells in DMEM containing 250 g/ml kanamycin (Gibco) for 2 h. Thereafter, the infection was allowed to continue in the medium containing 20 g/ml of kanamycin until the experiment was terminated (13). Quantification of intracellular bacteria. Intracellular bacterial survival and replication were determined using a standard antibiotic protection assay (13). In brief, at the times indicated in the figures, infected cells were washed with PBS, and intracellular bacteria were liberated by lysing the macrophages with 0.1% Triton X-100. The number of released bacteria was determined by plating CFU. Reverse transcriptase PCR. Total RNA was extracted from infected cells according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany) and used for cDNA synthesis (AMV RT; Promega). PCR was then performed using primer pairs specific for SARM, inos, ifn-, tnf-␣, and -actin. The sequences were as follows: for SARM, 5⬘-GGAGCTCAGTGCATAGGA G-3⬘ (sense) and 5⬘-CAGGTCTGGACCTCAGCTTC-3⬘ (antisense); for inos, 5⬘-GCAGAATGTGACCATCATGG-3⬘ (sense) and 5⬘-ACAACCTTGGTGTT GAAGGC-3⬘ (antisense); for ifn-, 5⬘-TCCAAGAAAGGACGAACATTCG-3⬘ (sense) and 5⬘-TGAGGACATCTCCCACGTCAA-3⬘ (antisense); for tnf-␣, 5⬘GTAGCCCACGTCGTAGCAAA-3⬘ (sense) and 5⬘-CCCTTCTCCAGCTGGG AGAC-3⬘ (antisense); for -actin, 5⬘-CCAGAGCAAGAGAGGTATCC-3⬘ (sense) and 5⬘-CTGTGGTGGTGAAGCTGTAG-3⬘ (antisense). The amplified products were electrophoresed using 1.5% agarose gel and stained with ethidium bromide before visualization under a UV lamp. Immunoblotting. The cells were lysed in lysis buffer containing 20 mM Tris, 100 mM NaCl, and 1% NP-40. The lysates were separated on 8% SDS-PAGE gels. Proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences, Dassel, Germany). The nonspecific binding sites on the membrane were blocked with 5% blocking solution (Roche Diagnostics) for 1 h before proteins were allowed to react with specific primary antibodies against SARM, iNOS, and IB␣ (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. The membrane was washed three times with 0.1% PBS-Tween 20 (PBST) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce, Rockford, IL) for 1 h at room temperature. Thereafter, the membrane was washed four times with 0.1% PBST before a chemiluminescence substrate (Roche Diagnostics) was added, and proteins were detected by enhanced chemiluminescence.
RESULTS B. pseudomallei induces SARM expression in a mouse macrophage cell line (RAW 264.7). To investigate SARM expression in B. pseudomallei-infected cells, mouse macrophages were cocultured with the bacteria at an MOI of 2. After 15, 30, 60, and 120 min of infection, the cells were harvested and the levels of SARM were analyzed by RT-PCR. Expression of SARM protein was determined by immunoblotting at 15, 30, 60, 120, and 360 min of infection. The results showed that B. pseudomallei was able to induce SARM expression within 30 min after infection and gradually increased with time (Fig. 1A). Previously, we demonstrated that B. pseudomallei lacking the O-antigenic polysaccharide moiety (LPS mutant) was more susceptible to macrophage killing during the early phase of infection; therefore, this LPS mutant strain was used for comparison (2). In contrast to the wild-type B. pseudomallei, the LPS mutant failed to upregulate SARM expression (Fig. 1A). To determine if the expression of SARM by B. pseudomalleiinfected macrophages requires viable bacteria, heat-killed bacilli were cocultured with macrophages at an MOI of 10. After different time intervals, the infected cells were harvested, and the levels of SARM mRNA were analyzed by RT-PCR and immunoblotting. The results (Fig. 1B) showed that, similarly to live bacteria, heat-killed B. pseudomallei but not the LPS mutant was able to induce SARM expression in a time-dependent manner. It should be mentioned that the enhanced SARM expression continued to persist after 8 h of infection (data not shown). In order to rule out the possibility that SARM expression induced by B. pseudomallei was mediated through other mediators, including cytokines, a similar experiment was performed in the presence of a protein synthesis inhibitor. The cells were pretreated with cycloheximide (5 g/ml) for 1 h before infection. As shown in Fig. 2, the levels of SARM mRNA expression were not affected by cycloheximide. In contrast, as to be expected, the SARM protein level was markedly reduced in the presence of this inhibitor. It should be mentioned that cycloheximide at the concentration used did not interfere with cell viability (data not shown). These results suggested that the expression of SARM was induced directly by B. pseudomallei. SARM depletion enhances ifn- and inos expression in B. pseudomallei-infected macrophages. SARM is known to interfere with gene expression downstream of the TRIF-dependent pathway, including that of ifn- (5). To elucidate the function of SARM in B. pseudomallei-infected macrophages, the cells were first transfected with SARM siRNA 24 h before infection. The levels of SARM mRNA and protein in SARM-depleted macrophages were markedly decreased compared with those of wild-type macrophages (Fig. 3). To determine the inhibition of gene expression downstream of TRIF, the level of ifn- mRNA was analyzed 4 h after infection. As shown in Fig. 3A, the ifn- mRNA expression was markedly increased in SARMdepleted macrophages compared to that of the wild-type or control siRNA-transfected cells. Because it was previously
VOL. 79, 2011
B. PSEUDOMALLEI INHIBITS THE MyD88-INDEPENDENT PATHWAY
2923
FIG. 1. B. pseudomallei-induced SARM expression in the mouse macrophage cell line RAW 264.7. Macrophages were infected with either wild-type or LPS mutant bacteria at an MOI of 2. (A) At different time intervals, the infected cells were lysed and the kinetics of SARM mRNA and protein levels were determined by RT-PCR and immunoblotting. (B) Heat-killed bacteria were cocultured with macrophages at a ratio equivalent to an MOI of 10. The expression levels of SARM mRNA and protein were determined as described above. -Actin mRNA and protein were used as internal loading controls. Data are representative of results from three independent experiments.
demonstrated that IFN- was required for iNOS induction (2, 29), we assessed iNOS expression in SARM-depleted cells. As shown in Fig. 3, iNOS was highly upregulated in infected SARM-deficient macrophages. These results suggested that SARM upregulation interfered with IFN- and iNOS expression in B. pseudomallei-infected macrophages. Depletion of SARM increases the rate of IB␣ degradation. In order to determine the effect of SARM on IB␣ degradation, SARM-deficient or wild-type macrophages were infected
with bacteria at an MOI of 2. At 15, 30, and 60 min after infection, the levels of IB␣ expression were determined by immunoblotting. The rate of IB␣ degradation in B. pseudomallei-infected macrophages was lower than that of the LPS mutant-infected cells (Fig. 4). However, increased IB␣ degradation was observed in B. pseudomallei-infected SARM-depleted macrophages compared to that of control cells. These results suggested that SARM decreased the rate of IB␣ degradation in B. pseudomallei-infected macrophages.
2924
INFECT. IMMUN.
PUDLA ET AL.
FIG. 2. B. pseudomallei-induced SARM expression in the presence of cycloheximide. Macrophages were pretreated with cycloheximide (5 g/ml) for 1 h prior to infection. The pretreated cells were infected with B. pseudomallei at an MOI of 2. The levels of SARM mRNA and protein expression in the infected macrophages were determined at 4 h after infection by RT-PCR and immunoblotting, respectively. Data are representative of results from three independent experiments.
Cytochalasin D inhibits SARM expression in B. pseudomallei-infected macrophages. To examine a possible relationship between bacterial internalization and SARM expression, the macrophages were pretreated with cytochalasin D (2 g/ml) for 1 h immediately before exposure to B. pseudomallei. This condition of cytochalasin D treatment was selected, as it was previously shown to significantly reduce the number of intracellular B. pseudomallei cells in mouse macrophages without interfering with cell viability (9). The level of SARM was markedly reduced in cytochalasin D-treated macrophages, as judged by a decrease in the band intensity (Fig. 5). In contrast, inhibition of bacterial internalization did not interfere with tnf-␣ expression. Since suppression of SARM directly correlated with an increase in iNOS expression (Fig. 3B), the level of iNOS protein was also analyzed by immunoblotting. As shown in Fig. 5, cytochalasin D markedly enhanced iNOS expression. These results implied that upregulation of SARM might require intracellular receptors. Depletion of SARM suppresses B. pseudomallei intracellular survival. Our previous findings are consistent with the prediction that B. pseudomallei escapes macrophage killing by interfering with IFN- production, resulting in suppression of iNOS expression (2, 29). In the present study, we showed that SARM depletion led to an increase in iNOS expression (Fig. 3). Because this process might affect the intracellular fate of B. pseudomallei, additional experiments were performed. In order to demonstrate this relationship, SARM-depleted macrophages were infected with B. pseudomallei at an MOI of 2, and after 2, 4, 6, and 8 h of infection, the infected cells were lysed and the number of intracellular bacteria was analyzed. As shown in Fig. 6, at 2 h after infection, the numbers of intracellular bacteria were similar among these three groups. These results suggested that SARM did not interfere with B. pseudomallei in-
FIG. 3. Alteration of mRNA and protein expression profiles in SARM-deficient mouse macrophages. Mouse macrophages (1.5 ⫻ 105 cells) were transfected with siRNAs against SARM prior to infection with B. pseudomallei at an MOI of 2. The infected cells were lysed after 4 h and 8 h of infection for mRNA (A) and protein (B) analyses, respectively. Data are representative of results from three independent experiments.
ternalization. However, depletion of SARM resulted in a partial enhancement of the macrophage’s ability to kill B. pseudomallei (Fig. 6), as judged by a significant reduction in the number of viable intracellular bacteria at 6 and 8 h after infection, thus compatible with the prediction that SARM expression suppresses antibactericidal activity of macrophages. In order to prove that the decrease of intracellular bacteria was not a consequence of decreasing infected macrophage viability, the viability of the infected cells in the presence of control and SARM siRNA was analyzed by trypan blue staining. The results showed that at 2, 4, 6, and 8 h after infection, the viability of infected macrophages treated with control siRNA was 97%, 94%, 92%, and 76%, respectively. These results are comparable to those of cells treated with SARM siRNA (98%, 93%, 90%, and 72%, respectively). Of note, the survival of these infected cells from both treatments dramatically drops to 30% and less than 10% at 10 h and 12 h, respectively, after the infection. DISCUSSION TLRs are known to play essential roles in innate immune defense against invading pathogens. Upon infection, the signal initiated from membrane-associated TLRs can activate medi-
VOL. 79, 2011
B. PSEUDOMALLEI INHIBITS THE MyD88-INDEPENDENT PATHWAY
2925
FIG. 4. Increased IB␣ degradation in B. pseudomallei-infected SARM-deficient mouse macrophages. Mouse macrophages (1.5 ⫻ 105 cells) were transfected with siRNAs against SARM prior to infection with either the wild type or the LPS mutant at an MOI of 2. The infected cells were harvested at 15, 30, and 60 min of infection, and IB␣ levels were analyzed by immunoblotting. Data are representative of results from three independent experiments.
ators like iNOS (1, 16). Most Gram-negative bacteria activate cells by the interaction of their LPS with TLR4. However, the LPS isolated from Porphyromonas gingivalis has been shown to activate host cells via TLR2 (11, 25). Although the roles of TLRs have been extensively investigated in B. pseudomallei infection, it is still controversial whether TLR2 or TLR4 or the combination of both is involved in triggering host cells. Wiersinga et al. used HEK293 cells stably transfected with TLR2/ CD14 or TLR4/CD14 to show that the LPS isolated from B. pseudomallei signals only through TLR2 (35). Moreover, TLR2-deficient mice infected with B. pseudomallei display decreased bacterial loads and prolonged survival, suggesting that TLR2 is essential in B. pseudomallei infection (35). In contrast, West et al. showed that tumor necrosis factor alpha (TNF-␣) production was decreased in LPS-treated TLR4⫺/⫺ primary macrophages, indicating that TLR4 is involved in proinflammatory cytokine production (34). TLR signaling pathways require either MyD88 (MyD88dependent pathway) or TRIF (MyD88-independent pathway) as an initial adaptor molecule (37). With the exception of TLR3, all TLRs recruit MyD88 to initiate signaling (1). TRIF is involved only with TLR3 and TLR4 signaling. Although both the MyD88-dependent and MyD88-independent pathways can activate NF-B, the gene expression downstream of each pathway is different. In MyD88-deficient mice, the animals fail to produce proinflammatory cytokines, such as TNF-␣, IL-6, and IL-1, but retain their capacity to produce IFN- (1). On the contrary, TRIF expression leads to the induction of IFN-, indicating that TRIF is an essential adaptor molecule for regulating IFN- production (18, 38). Although the mechanism underlying macrophage activation by TLR signaling in B. pseudomallei infection has not been fully elucidated, we previously
demonstrated that this bacterium fails to activate IFN- production, leading to the suppression of iNOS expression and the failure to inhibit intracellular growth of B. pseudomallei (29). However, the addition of exogenous IFN- or IFN-␥ could restore the macrophage’s ability to activate iNOS expression and result in enhanced killing of intracellular B. pseudomallei (28–30). In contrast, an LPS mutant lacking the O-antigenic polysaccharide moiety is more susceptible to macrophage killing, because this mutant is able to activate IFN- production (2). Moreover, knocking down tbk1 does not interfere with the replication of wild-type B. pseudomallei but allows the LPS mutant to replicate intracellularly (24). These results suggest that the MyD88-independent pathway is essential for controlling the intracellular fate of B. pseudomallei. Because the inability of B. pseudomallei to activate IFN- production may relate to the upregulation of negative regulator TRIF, in our present study, we designed additional experiments to demonstrate that B. pseudomallei is able to activate SARM expression in a time-dependent manner (Fig. 1A). This adaptor molecule has been shown to inhibit signaling pathways downstream of the MyD88-independent pathway, including that of ifn- (5). Our conclusion was reached by the fact that increased ifn- and inos mRNA expressions were observed in the B. pseudomallei-infected SARM knockdown macrophages (Fig. 3). These results are also consistent with the finding that both the living and heat-killed LPS mutants do not activate SARM expression (Fig. 1), resulting in the upregulation of ifn- and inos expression levels previously observed (2, 24). The inability of both the living and heat-killed LPS mutants to induce SARM expression also implied that O-polysaccharide may play an essential role in SARM activation. It is well documented that IkB␣ regulates NF-B activation
2926
PUDLA ET AL.
INFECT. IMMUN.
FIG. 6. Intracellular fate of B. pseudomallei in SARM-deficient macrophages. SARM-deficient macrophages were infected with B. pseudomallei at an MOI of 2. At 2, 4, 6, and 8 h after infection, bacterial internalization and intracellular replication were determined by standard antibiotic protection assay as described in Materials and Methods. The data indicate the means and standard deviations of results from three separate experiments, each carried out in duplicate. *, P ⬍ 0.05.
FIG. 5. Cytochalasin D inhibits SARM expression. Cytochalasin D (2 g/ml) was added to macrophages for 1 h prior to infection. Infected cells were lysed after 4 h and 6 h of infection, and SARM mRNA (A) and protein (B) were analyzed. Data are representative of results from three independent experiments.
by forming a complex with NF-B, thereby preventing it from entering into the nucleus (31). In response to stimuli, IkB␣ is degraded by the proteasome, resulting in NF-B nuclear translocation. Previously, we demonstrated that the level of IB␣ degradation in human lung epithelial A549 cells infected with B. pseudomallei is lower than that in cells infected with other Gram-negative bacteria, including Salmonella enterica serovar Typhi (27). A low level of IB␣ degradation was also observed in B. pseudomallei-infected macrophages compared to that observed in LPS mutant (Fig. 4)- or S. Typhi-infected cells (data not shown). However, the IB␣ degradation level was noticeably increased in B. pseudomallei-infected SARM-deficient macrophages (Fig. 4), suggesting that SARM could influence IB␣ processing. These results are also in accord with a recent report which demonstrated a correlation between SARM expression and reduced IB␣ degradation (21). The mechanism by which SARM reduces IB␣ degradation may relate to its ability to inhibit the mitogen-activated protein kinase (MAPK) pathway, such as by suppressing p38 phosphorylation (20, 21). Heat-killed B. pseudomallei is able to upregulate SARM expression, suggesting that the activation of SARM does not require active invasion of the macrophages (Fig. 1B). In addi-
tion, the expression of SARM can be rapidly induced by either live or heat-killed B. pseudomallei (within 30 min), and cycloheximide did not interfere with SARM mRNA (Fig. 2). These results imply that SARM upregulation is initiated directly by the physical contact of bacteria rather than indirectly by mediators released from the host. Furthermore, inhibition of bacterial internalization by cytochalasin D decreases SARM expression in B. pseudomallei-infected cells (Fig. 5). These data suggest that the signal generated by the interaction of B. pseudomallei with the appropriate receptor(s) at the surface of macrophages is sufficient to activate TNF-␣ expression but insufficient for SARM upregulation. It is possible that intracellular pattern recognition receptors may be involved in this process. It is logical to conclude that although depletion of SARM does not interfere with internalization of B. pseudomallei, it influences the bacterial intracellular survival (Fig. 6), which correlates with increased iNOS expression (Fig. 3). However, intracellular suppression of B. pseudomallei observed in SARM-depleted macrophages was not as efficient as IFN-␥ treatment, as previously reported by our group (28, 30). It is possible that regulation of SARM by B. pseudomallei may be only partially involved in inhibition of gene expression downstream of the MyD88-independent pathway. Recent evidence demonstrates that SARM directly interacts with TRIF (5); therefore, it is possible that other negative regulators of TLR signaling downstream of TRIF, such as MAP kinase phosphatase 1 (MKP-1) or signal regulatory protein ␣ (SIRP␣), which negatively regulate phosphorylation of p38 and TBK-1, respectively (14, 33), may also participate in B. pseudomallei infection. The involvement of these molecules is under investigation by our group. All together, the results presented here suggest that B. pseudomallei interferes with the MyD88-independent signaling pathway at least in some part by inducing SARM
VOL. 79, 2011
B. PSEUDOMALLEI INHIBITS THE MyD88-INDEPENDENT PATHWAY
expression, which results in the inhibition of the MyD88-independent pathway; therefore, the bacteria can survive inside macrophages. ACKNOWLEDGMENTS Matsayapan Pudla was supported by a grant under the program Strategic Scholarships for Frontier Research Network for the Join Ph.D. program Thai doctoral degree, the Office of the Higher Education Commission, Thailand. This work was supported by a research grant from Thailand Research Fund (grant number BRG5180003) and National Science and Technology Development Agency (Thailand) (grant number P-00-10450). We thank D. E. Woods (Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada) for providing the B. pseudomallei parental wild-type strain (1026b) and the LPS mutant (SRM117). REFERENCES 1. Akira, S., and K. Takeda. 2004. Toll-like receptor signaling. Nat. Rev. Immunol. 4:499–511. 2. Arjcharoen, S., et al. 2007. The fate of a Burkholderia pseudomallei lipopolysaccharide mutant in the mouse macrophage cell line (RAW 264.7): a possible role for the O-antigenic polysaccharide moiety of lipopolysaccharide in internalization and intracellular survival. Infect. Immun. 75:4298–4304. 3. Belinda, L. W., et al. 2008. SARM: a novel Toll-like receptor adaptor, is functionally conserved from arthropod to human. Mol. Immunol. 45:488– 494. 4. Brett, P. J., and D. E. Woods. 2000. Pathogenesis of and immunity to melioidosis. Acta Trop. 74:201–210. 5. Carty, M., et al. 2006. The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling. Nat. Immunol. 7:1074–1081. 6. Cheng, A. C., and B. J. Currie. 2005. Melioidosis: epidemiology, pathophysiology, and management. Clin. Microbiol. Rev. 18:383–416. 7. Currie, B. J., et al. 2000. The epidemiology of melioidosis in Australia and Papua New Guinea. Acta Trop. 74:121–127. 8. DeShazer, D., P. J. Brett, and D. E. Woods. 1998. The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence. Mol. Microbiol. 30:1081–1100. 9. Ekchariyawat, P., et al. 2007. Expression of suppressor of cytokine signaling 3 (SOCS3) and cytokine-inducible Src homology 2-containing protein (CIS) induced in Burkholderia pseudomallei-infected mouse macrophages requires bacterial internalization. Microb. Pathog. 42:104–110. 10. Harley, V. S., D. A. B. Dance, B. J. Drasar, and G. Tovey. 1998. Effects of Burkholderia pseudomallei and other Burkholderia species on eukaryotic cells in tissue culture. Microbios 96:71–93. 11. Hirschfeld, M. J., et al. 2001. Signaling by Toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect. Immun. 69:1477–1482. 12. Jones, A. L., T. J. Beveridge, and D. E. Woods. 1996. Intracellular survival of Burkholderia pseudomallei. Infect. Immun. 64:782–790. 13. Kespichayawattana, W., S. Rattanachetkul, T. Wanun, P. Utaisincharoen, and S. Sirisinha. 2000. Burkholderia pseudomallei induces cell fusion and actin-associated membrane protrusion: a possible mechanism for cell-to-cell spreading. Infect. Immun. 68:5377–5384. 14. Kong, X. N., et al. 2007. LPS-induced downregulation of signal regulatory protein (alpha) contributed to innate immune activation in macrophages. J. Exp. Med. 204:2719–2731. 15. Leelarasamee, A. 2004. Recent development in melioidosis. Curr. Opin. Infect. Dis. 17:131–136. 16. Medzhitov, R. 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1:135–145.
Editor: J. B. Bliska
2927
17. O’Neill, L. A. J., and A. G. Bowie. 2007. The family of five: TIR-domaincontaining adaptors in Toll-like receptor signaling. Nat. Rev. Immunol. 7:353–364. 18. Oshiumi, H., M. Matsumoto, M. Funami, K. Akazawa, and T. Seya. 2003. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon- induction. Nat. Immunol. 4:161–167. 19. Peacock, S. J. 2006. Meliodosis. Curr. Opin. Infect. Dis. 19:421–428. 20. Peng, J., et al. 2010. SARM inhibits both TRIF- and MyD88-mediated AP-1 activation. Eur. J. Immunol. 40:1–10. 21. Piao, W., et al. 2009. Endotoxin tolerance dysregulates MyD88-and Toll/ IL-1R domain-containing adapter inducing IFN--dependent pathways and increases expression of negative regulators of TLR signaling. J. Leukoc. Biol. 86:863–875. 22. Rotz, L. D., A. S. Khan, S. R. Lillibridge, S. M. Ostroff, and J. M. Hughes. 2002. Public health assessment of potential biological terrorism agents. Emerg. Infect. Dis. 8:225–230. 23. Suparak, S., et al. 2005. Multinucleated giant cell formation and apoptosis in infected host cells is mediated by Burkholderia pseudomallei type III secretion protein BipB. J. Bacteriol. 187:6556–6560. 24. Tangsudjai, S., M. Pudla, K. Limposuwan, S. Sirisinha, and P. Utaisincharoen. 2010. Involvement of the MyD88-independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7. Microbiol. Immunol. 54:282–290. 25. ⌻oshchakov, V., et al. 2002. TLR4, but not TLR2, mediates IFN--dependent gene expression in macrophages. Nat. Immunol. 3:392–398. 26. Utaisincharoen, P., S. Arjcharoen, K. Limposuwan, S. Tungpradabkul, and S. Sirisinha. 2006. Burkholderia pseudomallei RpoS regulates multinucleated giant cell formation and inducible nitric oxide synthase expression in mouse macrophage cell line (RAW 264.7). Microb. Pathog. 40:184–189. 27. Utaisincharoen, P., et al. 2004. Burkholderia pseudomallei stimulates low interleukin-8 production in human lung epithelial cell line (A549). Clin. Exp. Immunol. 138:61–65. 28. Utaisincharoen, P., et al. 2004. Induction of iNOS expression and antimicrobial activity by interferon (IFN)- is distinct from IFN-␥ in Burkholderia pseudomallei-infected mouse macrophages. Clin. Exp. Immunol. 136:277– 283. 29. Utaisincharoen, P., N. Anuntagool, K. Limposuwan, P. Chaisuriya, and S. Sirisinha. 2003. Involvement of beta interferon in enhancing inducible nitric oxide synthase production and antimicrobial activity of Burkholderia pseudomallei-infected macrophages. Infect. Immun. 71:3053–3057. 30. Utaisincharoen, P., N. Tangthawornchaikul, W. Kespichayawattana, P. Chaisuriya, and S. Sirisinha. 2001. Burkholderia pseudomallei interferes with inducible nitric oxide synthase (iNOS) production: a possible mechanism of evading macrophage killing. Microbiol. Immunol. 45:307–313. 31. Verma, I., et al. 1995. Ral/NF-kB/IkB family: intimate tales of association and dissociation. Genes Dev. 9:2723–2735. 32. Voskuhl, G. W., P. Cornea, M. S. Bronze, and R. A. Greenfield. 2003. Other bacterial diseases as a potential consequence of bioterrorism: Q fever, brucellosis, glanders, and melioidosis. J. Okla. State Med. Assoc. 96:214–217. 33. Wang, X., and Y. Liu. 2007. Regulation of innate immune response by MAP kinase phosphatase-1. Cell. Signal. 19:1372–1382. 34. West, T. E., R. K. Ernst, M. J. Jansson-Hutson, and S. J. Skerrett. 2008. Activation of Toll-like receptors by Burkholderia pseudomallei. BMC Immunol. 9:46–56. 35. Wiersinga, W. J., et al. 2007. Toll-like receptor 2 impairs host defense in Gram-negative sepsis caused by Burkholderia pseudomallei (melioidosis). PLoS One 4:e248. 36. Wiersinga, W. J., T. Van Der Poll, N. J. White, N. P. Day, and S. J. Peacock. 2006. Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei. Nat. Rev. Microbiol. 4:272–282. 37. Yamamoto, M., K. Takeda, and S. Akira. 2004. TIR domain-containing adaptor defines the specificity of TLR signaling. Mol. Immunol. 40:861–868. 38. Yamamoto, M., et al. 2002. A novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN- promoter in the Toll-like receptor signaling. J. Immunol. 169:6668–6672.
