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- MATERIALS AND METHODS
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.
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.
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- MATERIALS AND METHODS
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.