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Keywords:

  • Burkholderia pseudomallei;
  • melioidosis;
  • MyD88-independent pathway;
  • TBK1

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Burkholderia pseudomallei is a facultative intracellular Gram-negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O-antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88-dependent (iκbζ, il-6 and tnf-α), but not of the MyD88-independent (inos, ifn-β and irg-1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant-infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88-independent pathway in B. pseudomallei-infected macrophages, the expression of TBK1, an essential protein in the MyD88-independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant-infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88-independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN-γ upregulated gene expression downstream of the MyD88-independent pathway, and interfered with intracellular survival in both wild type and tbk1-knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88-independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN-γ regulates gene expression through the TBK1-independent pathway.

List of Abbreviations: 
B. pseudomallei

Burkholderia psuedomallei

CFU

colony forming unit

DMM

Dulbecco's modified Eagle's medium

IFN-β

interferon beta

IFN-γ

interferon gamma

IL-1β

interleukin-1 beta

IL-6

interleukin-6

iNOS

inducible nitric oxide synthase

LPS

lipopolysaccharide

MNGC

multinucleated giant cell

MOI

multiplicity of infection

MyD88

myeloid differentiation primary response gene 88

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

PBST

PBS with Tween 20

siRNA

small interfering RNA

TBK1

tank-binding kinase-1

TIR

Toll/interleukin-1 receptor

TLR

Toll-like receptor

TNF-α

tumor necrosis factor-α

TRIF

TIR-domain-containing adapter-inducing interferon-β

B. pseudomallei is the causative agent of melioidosis and is responsible for a large proportion of community-acquired septicemia in South-east Asia and Northern Australia (1). This Gram-negative bacterium can survive and multiply inside both phagocytic and nonphagocytic cells (2). After internalization, B. pseudomallei can escape from membrane-bound phagosomes into the cytoplasm (2). Internalized bacteria can induce cell-to-cell fusion, resulting in MNGC formation (3, 4). This unique phenomenon, which has never been reported in any other bacteria, facilitates the spread of B. pseudomallei from one cell to another (4).

Although macrophages are known to play an essential role in innate immunity against a number of bacterial infections, they fail to kill B. pseudomallei (5). The mechanism by which B. pseudomallei escapes intracellular killing is not fully understood. Previously we have demonstrated that B. pseudomallei fails to stimulate IFN-β production in macrophages, leading to reduced expression of a key enzyme, inducible iNOS, which is needed for intracellular killing (6). Addition of exogenous IFN-β or IFN-γ can restore the ability of macrophages to activate iNOS expression and results in enhanced killing of intracellular B. pseudomallei (7). In contrast to the wild type strain (1026b), B. pseudomallei strain SRM117, an LPS mutant which lacks the O-antigenic polysaccharide moiety, is able to activate IFN-β and iNOS expression in a mouse macrophage cell line (8). These results not only imply that the macrophage signaling pathway for IFN-β production is essential for controlling the fate of intracellular B. pseudomallei, but also demonstrate the ability of this bacterium to modulate macrophage antibacterial responses. This susceptibility to macrophage killing may also contribute to the lack of virulence of the LPS mutant for BALB/c mice as compared to the wild type (9).

TLR are known to have a crucial role in early host defense against invading pathogens (10). Generally, stimulation of TLR triggers activation of the two downstream signaling pathways, the MyD88-dependent and -independent pathways (11). MyD88 is the immediate adaptor molecule that is common to all TLR, except for TLR3. It possesses the TIR domain needed for activation of a universal transcription factor, NF-κB, which is crucial for expression of proinflammatory cytokines, and several other mediators (12). It has been demonstrated, for example, that macrophages from MyD88-deficient mice cannot produce inflammatory cytokines (i.e. IL-6, TNF-α and IL-1β) in response to bacterial components such as LPS, suggesting that MyD88 is required for production of these cytokines (13). However, in MyD88-deficient mice, LPS can still stimulate NF-κB, albeit with delayed kinetics, leading to production of IFN-β and IFN-inducible genes via MyD88-independent pathways (14, 15). TBK1, also a known NF-κB-activating kinase, is an essential molecule required for induction of a type I interferon-dependent antimicrobial effector mechanism (16). This molecule has been suspected to play an important role not only in viral, but also in bacterial, infection (17, 18). In the present study, by comparing macrophages infected with wild type and LPS mutant B. pseudomallei, we have demonstrated that TBK1 is an essential molecule for controlling the fate of this bacterium with regard to intracellular survival.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Cell line and culture condition

The mouse macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (Rockville, MD, USA). If not indicated otherwise, the cells were cultured in advanced DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Hyclone, Logan, UT, US) and 1% L-glutamine (Gibco) at 37°C under 5% CO2 atmosphere.

Bacterial strains

B. pseudomallei parental wild type strain (1026b) and LPS mutant (SRM117) were used in the experiments (19).

TBK1 gene knockdown in the macrophage cell line RAW 264.7

Mouse macrophage cells (2.5 × 105 cells) were cultured in a 6-well plate overnight. SiRNA (375 ng) was dissolved in DMEM containing a transfection reagent (Qiagen, Hilden, Germany) before adding to the cells, and the cells were then incubated at 37°C and in 5% CO2 for 24 hr. The siRNA sequences are as follows: sense 5′CCA CAA AUU UGA UAA GCA A 3′ and antisense 5′UUG CUU AUC AAA UUU GUG G 3′. Expression of tbk1 gene and protein was determined by PCR and immunoblotting, respectively.

Infection of mouse macrophages (RAW 264.7)

An overnight culture of mouse macrophages (1 × 106 cells) in a 6-well plate was co-cultured with bacteria at an MOI of 2 for 1 hr. To remove extracellular bacteria, the cells were washed twice with 1 ml of PBS, and residual bacteria were killed by incubating in DMEM containing 250 μg/ml kanamycin (Gibco) for 2 hr. Thereafter, infection was allowed to continue in the medium containing 20 μg/ml of kanamycin until the experiment was terminated (4, 5).

Quantification of intracellular bacteria

To determine intracellular survival and multiplication of the bacteria, a standard antibiotic protection assay was performed as previously described (7). In brief, at the times indicated, the infected cells were washed three times with PBS as above, intracellular bacteria liberated by lysing the macrophages with 0.1% Triton X-100 and the released bacteria plated on tryptic soy agar. The number of intracellular bacteria, expressed as CFU, was determined by bacterial colony counting.

Reverse transcriptase PCR

Total RNA was extracted from infected cells according to the manufacturer's instructions (Roche, Mannheim, Germany) before being used for cDNA synthesis using avian myeloblastosis virus RT (Promega, Madison, WI, USA). PCR was performed using Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) and cDNA as the template. Primer pairs specific for IκBζ, IL-6, TNF-α, SOCS3, iNOS, IFN-β, IRG1, SOCS1 and β-actin used in this study are shown in Table 1. The amplified products were electrophoresed on 1.5% and 4% agarose gels and stained with ethidium bromide before being visualized under an ultraviolet lamp.

Table 1.  Primer specifications for RT-PCR
GenePrimerSequence (5′[RIGHTWARDS ARROW]3′)
il-6ForwardGTT CTC TGG GAA ATC GTG GA
ReverseGCT GAC CCT AGA GCA TCC TG
iκb-ζForwardTGT TGC CTT CTC ACT TCG TG
ReverseTGG TCC ATC ATC TGT GGA GA
irg1ForwardGGT ATC ATT CGG AGG AGC AA
ReverseACA GAG GGA GGG TGG AAT CT
ifn-βForwardTCC AAG AAA GGA CGA ACA TTC G
ReverseTGA GGA CAT CTC CCA CGT CAA
inosForwardGCA GAA TGT GAC CAT CAT GG
ReverseAC A ACC TTG GTG TTG AAG GC
tbk1ForwardCTT CAG GCA CTG CTT ACC C
ReverseCGG CTC GTG ACA AAG ATA GGA
tnf-αForwardGTA GCC CAC GTC GTA GCA AA
ReverseCCC TTC TCC AGC TGG GAG AC
β-actinForwardCCA GAG CAA GAG AGG TAT CC
ReverseCTG TGG TGG TGA AGC TGT AG

Immunoblotting

The cells were lysed in lysis buffer containing 20 mM Tris, 100 mM NaCl and 1% NP40. The lysates were separated in 8% SDS-PAGE gel by electrophoresis in SDS-PAGE buffer. The protein on SDS-PAGE gel was transferred to a nitrocellulose membrane (Amersham Bioscience, Dassel, Germany). The membrane was blocked in 10% blocking solution (Roche Diagnostics, Mannheim, Germany) in PBS for 1 hr before incubating overnight at 4°C with specific primary antibody against TBK1 (Epitomics, Burlingame, CA, USA) or iNOS (Santa Cruz, Santa Cruz, CA, USA). The membrane was washed three times with 0.01% PBST and incubated with horseradish peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL, USA) for 1 hr at room temperature. Thereafter, the membrane was washed four times with 0.01% PBST before a chemiluminescence substrate (Roche Diagnostics, Mannheim, Germany) was added, and protein bands detected by enhanced chemiluminescence.

Statistical analysis

If not otherwise indicated, all experiments in this study were conducted at least three times. Experimental values were expressed as means ± standard error of the mean. Statistical significance of differences between two means was evaluated by Student's t test, and a P value < 0.01 was considered significant.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

LPS mutant stimulates gene expression of both MyD88-dependent and -independent pathways in the mouse macrophage cell line RAW 264.7

Previously we demonstrated that the LPS mutant strain SRM117 was more susceptible to macrophage killing during the early phase of infection (8). In the present study, we extended our finding to demonstrate the differences in the profile of gene expression between LPS mutant- and wild type-infected macrophages. To investigate gene expression regulated by the TLR signaling pathway, mouse macrophages (RAW 264.7) were infected with either the LPS mutant or wild type strain of B. pseudomallei at a MOI of 2, and 4 hr later, gene expression was examined by RT-PCR. The results are presented in Figure 1 and show that, in macrophages, the LPS mutant is able to upregulate gene expression downstream of both MyD88-dependent (iκbζ, il-6 and tnf-α) and -independent pathways (inos, ifn-β and irg-1). In contrast, the wild type failed to stimulate the MyD88-independent pathway.

image

Figure 1. Profiles of gene expression in wild type and LPS mutant-infected mouse macrophages. Mouse macrophages (2.5 × 105 cells) were infected with the LPS mutant or the wild type strain of B. pseudomallei at a MOI of 2. Four hr after infection, the infected cells were lysed, and gene expression was determined by RT-PCR.

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Knockdown tbk1 inhibits gene expression in LPS mutant-infected macrophages

TBK1 is known to be constitutively expressed in macrophages, but this can be suppressed in cells transfected with siRNA (Fig. 2). In order to investigate the possible role of TBK1 in controlling the fate of B. pseudomallei, tbk1- knockdown macrophages were infected with the bacteria at a MOI of 2 for 1 hr. Eight hr after infection, the number of intracellular bacteria was determined by a standard antibiotic protection assay. The results, presented in Figure 3, confirm our previous conclusion that the LPS mutant is more susceptible to macrophage killing (8). Further, the data show that the number of intracellular bacteria in tbk1-knockdown macrophages infected with the LPS mutant strain is significantly greater than in wild type macrophages (Fig. 3), suggesting that TBK1 might be involved in suppressing replication of the LPS mutant in macrophages. In contrast, the numbers of intracellular bacteria in tbk1 knockdown and wild type macrophages which had been infected with wild type were not significantly different (Fig. 3). It should be noted that infection with either wild type or LPS mutant did not alter the degree of TBK1 protein expression (data not shown).

image

Figure 2. Expression of TBK1 protein expression in mouse macrophages infected with B. pseudomallei. Mouse macrophages (2.5 × 105 cells) were transfected with siRNA targeting tbk1 prior to infection with the LPS mutant or wild type B. pseudomallei at a MOI of 2. Eight hr after infection, levels of TBK1 were determined by immunoblotting.

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image

Figure 3. Intracellular replication of both wild type and LPS mutant in TBK1 knockdown macrophages. Mouse macrophages (2.5 × 105 cells) transfected with siRNA targeting tbk1 were infected with either the LPS mutant or wild type B. pseudomallei at a MOI of 2. The number of intracellular bacteria was determined 8 hr after infection by a standard antibiotic protection assay. Data represent means and standard errors of three separate experiments, each carried out in duplicate. *P < 0.01 by Student's t test. Wt, wild type.

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In order to determine the suppressive function of TBK1, tbk1 knockdown cells were infected with the bacteria at a MOI of 2 for 1 hr. Four hr after infection, expression of genes known to be activated through MyD88-dependent (iκbζ, il-6 and tnf-α) and -independent pathways (inos, ifn-β and irg-1) was determined by RT-PCR. The results showed that, in wild type macrophages, the LPS mutant can activate genes of both MyD88-dependent and -independent pathways (Fig. 4a and b). As was to be expected, in tbk1-knockdown macrophages only the genes known to be associated with the MyD88-independent pathway were suppressed in LPS mutant infected cells. The effect of TBK1 on MyD88-independent genes (e.g., inos, ifn-β and irg-1) in macrophages infected with wild type bacteria could not be evaluated because they were not initially expressed in this system.

image

Figure 4. Gene expression profile in tbk1 knockdown mouse macrophages infected with wild type and LPS mutant strains. Tbk1-knockdown and wild type mouse macrophages (2.5 × 105 cells) were infected with either wild type or the LPS mutant at a MOI of 2. Four hr after infection, the infected cells were lysed and gene expression determined by RT-PCR. Wt, wild type.

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Exogenous IFN-γ enhances gene expression and suppresses intracellular survival of bacteria via the tbk1-independent pathway in B. pseudomallei-infected macrophages

In this set of experiments, tbk1 knockdown and wild type macrophages were pretreated with IFN-γ (10 U/ml) overnight prior to infection with LPS mutant and wild type B. pseudomallei at a MOI of 2. Four hr after infection gene expression of the infected cells was determined by RT-PCR. Without the bacteria, IFN-γ alone slightly upregulated gene expression via the MyD88-independent pathway in both wild type and tbk1 knockdown cells (Fig. 5a, lane 3 and 4). However, IFN-γ was able to markedly enhance gene expression downstream of the MyD88-independent pathway in wild type macrophages that were infected with either LPS mutant (Fig. 5c, lane 3) or wild type bacteria (Fig. 5b, lane 3). A similar observation was noted when the experiment was set up with tbk1 knockdown macrophages that were infected with either wild type or LPS mutant bacteria (lane 4 of Fig. 5b and c). Previously we demonstrated that IFN-β production directly correlates with iNOS expression (6, 8). In order to prove that the gene expression observed by RT-PCR directly correlates with protein expression, an immunoblotting experiment using antibody against iNOS was performed. The results showed that the expression of the iNOS protein profile was similar to the results obtained by RT-PCR, as shown in Figure 6. The number of intracellular LPS mutant and wild type bacteria in these cells (Fig. 7) paralleled the gene expression profile noted above. It should be noted that IFN-γ can also significantly reduce intracellular bacteria in both wild type- and LPS mutant-infected macrophages in the absence of siRNA (data not shown) (5, 7). These results suggest that gene expression that does not depend on TBK-1, and which occurs downstream of the MyD88-independent pathway, is essential in controlling the intracellular fate of B. pseudomallei.

image

Figure 5. Exogenous IFN-γ enhances gene expression downstream of TBK1 in the MyD88-independent pathway of infected macrophages. Tbk1 knockdown and wild type mouse macrophages (2.5 × 105 cells) were pretreated with IFN-γ (10 U/ml) overnight prior to infection with the wild type or LPS mutant at a MOI of 2. Four hr after infection, the infected macrophages were lysed and gene expression determined by RT-PCR. Wt, wild type.

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image

Figure 6. Exogenous IFN-γ enhances iNOS protein expression in B. pseudomallei-infected macrophages. Tbk1 knockdown and wild type mouse macrophages (2.5 × 105 cells) were pretreated with IFN-γ (10 U/ml) overnight prior to infection with the wild type or LPS mutant at a MOI of 2. Eight hr after infection, the infected macrophages were lysed and protein expression determined by immunoblotting. Wt, wild type.

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image

Figure 7. IFN-γ suppresses intracellular replication of B. pseudomallei. Tbk1knockdown and wild type mouse macrophages (2.5 × 105 cells) were pretreated with IFN-γ (10 U/ml) overnight prior to infection with the wild type or LPS mutant at a MOI of 2. The number of intracellular bacteria was determined as described in Materials and Methods. Data represent means and standard errors of three separate experiments, each carried out in duplicate.*P < 0.01 by Student's t test.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

The mechanisms underlying macrophage activation, particularly those regarding TLR signaling by B. pseudomallei, have not been fully elucidated. We have previously demonstrated that wild type B. pseudomallei fails to stimulate IFN-β production (6). In contrast, the LPS mutant is able to stimulate IFN-β production, leading to enhanced antimicrobial killing within macrophages (8). These results imply that signaling via the MyD88-independent pathway might be essential for controlling the intracellular fate of B. pseudomallei. In the present study, we extended our findings to demonstrate that the MyD88-independent pathway is required for control of intracellular survival of B. pseudomallei. Both wild type and the LPS mutant strains were able to stimulate expression of genes, including tnf-α and iκbζ, downstream of the MyD88-dependent pathway as shown in Figure 1. However, gene expression (irg1, inos and ifn-β) downstream of the MyD88-independent pathway could only be upregulated following infection by the LPS mutant. Moreover, in tbk1 knockdown macrophages, intracellular survival of LPS mutant B. pseudomallei was significantly increased, a result which is in contrast to that observed in infection with wild type bacteria (Fig. 3). The inability of tbk1 knockdown macrophages to control intracellular growth of the LPS mutant strain directly correlates with the downregulation of MyD88-independent gene expression (Figs 4, 5). These results together suggest that the differences in regulation of TLR signaling induced by these two strains, particularly the failure to stimulate MyD88-independent pathway, may facilitate B. pseudomallei survival within macrophages.

Our results are consistent with those reported previously by another group of investigators, who demonstrated that TBK1 knockdown macrophages do not interfere with intracellular survival of this bacterium (20). Moreover, inactivation of TRIF, another adaptor molecule in the MyD88-independent pathway, does not affect survival of infected animals, suggesting that only the MyD88-dependent pathway is needed to control wild type infection (21). In contrast, MyD88 KO mice significantly exhibited increased bacterial loads in blood and enhanced liver damage, suggesting that the MyD88-dependent, but not MyD88-independent, pathway is essential for resistance against B. pseudomallei infection (21). Even though these results suggest that the MyD88-dependent pathway contributes to a protective host response, susceptibility to B. pseudomallei infection in wild type mice expressing the MyD88 molecule suggest that activation of the MyD88-dependent pathway alone is insufficient to eliminate the bacteria. One possible explanation for these differences in the significance of the role of the MyD88-independent pathway may relate to the fact that B. pseudomallei fails to activate this pathway (Fig. 1). Thus, knocking down the molecules downstream of this pathway would have very little effect on intracellular survival within macrophages.

The O-antigenic polysaccharide moiety of B. pseudomallei has been demonstrated to play an essential role in the innate immune response. A B. pseudomallei mutant deficient in O-antigenic polysaccharide production was found to be more susceptible to serum bactericidal activity, particularly via the alternative complement pathway (19, 22). Moreover, internalization of this mutant is significantly greater than occurs with wild type B. pseudomallei, suggesting that O-antigenic polysaccharide may also reduce the ability of macrophages to take up this bacterium (8). In the present study, we have also demonstrated that the O-antigenic polysaccharide of B. pseudomallei can prevent this bacterium from activating gene expression downstream of the MyD88-independent pathway (Fig. 1). Therefore exogenous O-polysaccharide of B. pseudomallei may also play an essential role in evading macrophage killing by altering the macrophage signaling pathway, which would then facilitate survival of bacteria within macrophages. However, the components and the mechanism by which LPS mutants activate the MyD88-independent pathway remained to be investigated.

Previously, we demonstrated that, in the presence of IFN-β or IFN-γ, macrophages are able to kill intracellular B. pseudomallei (7). The mechanisms by which the IFN enhance bacterial killing are most likely related to the fact that both types of IFN provide interferon regulatory factor-1, which is an essential transcription factor for iNOS expression (5, 6). In the present study, the finding that, even in knockdown tbk1 wild type infected-macrophages, IFN-γ is able to markedly enhance gene expression downstream of the MyD88-independent pathway, is consistent with the conclusion that gene expression downstream of the MyD88-independent pathway can be activated through the TBK1-independent pathway (Fig. 5). Moreover, the expression of genes downstream of the MyD88-independent pathway was found to be directly correlated with the ability of macrophages to suppress intracellular survival of both the wild type and LPS mutant strains. This suggests that the MyD88-independent pathway plays an essential role in macrophage killing of B. pseudomallei. It was also demonstrated that the O-polysaccharide moiety of B. pseudomallei LPS may interfere with TLR signaling by preventing the bacteria from activating the MyD88-independent pathway, thus resulting in the inability of macrophages to activate expression of antimicrobial agents, iNOS in particular. It is also possible that different types of TLR are activated in LPS mutant- infected macrophages. Altogether, these results suggest that the MyD88-independent pathway plays an essential role in regulating the fate of intracellular B. pseudomallei.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

This work was supported by a research grant from the Office of Higher Education Commission.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
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