Burkholderia pseudomallei-induced expression of a negative regulator, sterile-alpha and Armadillo motif-containing protein, in mouse macrophages: a possible mechanism for suppression of the MyD88-independent pathway.

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-ß) 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-α and Armadillo motif (SARM)-containing protein, a known negative regulator of the MyD88-independent 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-ß and iNOS and degradation of IκBα 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-ß and iNOS needed for bacterial elimination.

Involvement of the MyD88-independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7.

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 (ikappabzeta, il-6 and tnf-alpha), but not of the MyD88-independent (inos, ifn-beta 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-gamma 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-gamma regulates gene expression through the TBK1-independent pathway.

Fate of a Burkholderia pseudomallei lipopolysaccharide mutant in the mouse macrophage cell line RAW 264.7: possible role for the O-antigenic polysaccharide moiety of lipopolysaccharide in internalization and intracellular survival.

Burkholderia pseudomallei is a facultative intracellular gram-negative bacterium that can survive and multiply inside macrophages. One of the mechanisms by which B. pseudomallei escapes macrophage killing is by interfering with the expression of inducible nitric oxide synthase (iNOS). However, the bacterial components that modulate antimicrobial activity of the macrophage have not been fully elucidated. In the present study, we demonstrated that B. pseudomallei strain SRM117, a lipopolysaccharide (LPS) mutant that lacks the O-antigenic polysaccharide moiety, was more susceptible to macrophage killing during the early phase of infection than the parental wild-type strain (1026b). Unlike the wild type, the LPS mutant could readily stimulate Y701-STAT-1 phosphorylation (pY701-STAT-1) and interferon-regulatory factor 1 (IRF-1) expression, both of which are essential transcription factors of iNOS. Neutralizing antibody against beta interferon was able to inhibit the phosphorylation of Y701-STAT-1 and the expression of IRF-1 and iNOS, all of which resulted in an increased rate of intracellular replication. These data suggest that the O-antigenic polysaccharide moiety of B. pseudomallei modulates the host cell response, which in turn controls the intracellular fate of B. pseudomallei inside macrophages.

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.

We recently reported that Burkholderia pseudomallei was able to activate the expression of suppressor of cytokine signaling 3 (SOCS3) and cytokine-inducible Src homology 2-containing protein (CIS). In the present study, we presented evidence showing that the induction of these negative regulators was most probably triggered from within rather than at the cell surface of mouse macrophage cell line (RAW264.7) suggesting that macrophage activation most likely requires the interaction of bacteria with a putative host cell cytoplasmic component(s).

Burkholderia pseudomallei RpoS regulates multinucleated giant cell formation and inducible nitric oxide synthase expression in mouse macrophage cell line (RAW 264.7).

Burkholderia pseudomallei is the causative agent of melioidosis. This bacterium can invade and survive inside the phagocytic and nonphagocytic cells. After internalization, the bacteria can escape from the membrane-bound phagosome into the cytoplasm. Internalised B. pseudomallei can also induce a cell-to-cell fusion, resulting in a multinucleated giant cell (MNGC) formation. In the present study, we demonstrated that B. pseudomallei rpoS null mutant was similar to its wild type parent in its ability to survive and multiply inside the mouse macrophages, but it failed to stimulate MNGC formation. The rpoS mutant was also unable to activate inducible Nitric Oxide Synthase (iNOS) in resting mouse macrophages but in gamma interferon (IFN-gamma)-activated macrophages, the mutant was able to induce significantly higher levels of iNOS and NO when compared with its wild-type counterpart, resulting in a significantly lower number of bacteria inside the infected host cells.

Burkholderia pseudomallei-induced expression of suppressor of cytokine signaling 3 and cytokine-inducible src homology 2-containing protein in mouse macrophages: a possible mechanism for suppression of the response to gamma interferon stimulation.

Burkholderia pseudomallei, the causative agent of melioidosis, is a facultative intracellular gram-negative bacterium that is able to survive and multiply in macrophages. Previously, we reported that B. pseudomallei was able to escape macrophage killing by interfering with the expression of inducible nitric oxide synthase (iNOS). In the present study, we extended this finding and demonstrated that B. pseudomallei was able to activate the expression of suppressor of cytokine signaling 3 (SOCS3) and cytokine-inducible Src homology 2-containing protein (CIS) but not SOCS1 in a mouse macrophage cell line (RAW 264.7). The expression of SOCS3 and CIS in B. pseudomallei-infected macrophages directly correlated with a decreased gamma interferon (IFN-gamma) signaling response, as indicated by a reduction in Y701-STAT-1 phosphorylation (pY701-STAT-1). Moreover, a reduction in the expression of IFN-gamma-induced proteins, such as interferon regulatory factor 1 (IRF-1), was observed in B. pseudomallei-infected macrophages that were treated with IFN-gamma. Since pY701-STAT-1 and IRF-1 are essential transcription factors for regulating iNOS expression, the failure to activate these factors could also result in depression of iNOS expression and a loss of macrophage killing capacity. Taken together, the data indicate that the activation of SOCS3 and CIS expression in B. pseudomallei-infected macrophages interfered with IFN-gamma signaling, thus allowing the bacteria to escape killing by these phagocytic cells.

Burkholderia pseudomallei invasion and activation of epithelial cells requires activation of p38 mitogen-activated protein kinase.

Burkholderia pseudomallei is a causative agent of melioidosis. This gram-negative bacterium is able to survive inside the macrophages and also able to invade non-phagocytic cells including epithelial cells. Interaction of pathogenic bacteria to the host cells is frequently associated with activation of mitogen-activated protein (MAP) kinases signaling activity. In this study, we demonstrated that B. pseudomallei stimulated p38 MAP kinase of human alveolar lung epithelial cell line (A549). Phosphorylation of p38 was observed after 15 min, attained a maximal level at 60 min after the infection. A specific inhibitor of p38 phosphorylation, SB 203580, was able to inhibit invasion of this bacterium into the cells suggesting that invasion of B. pseudomallei required activation of p38. In contrast, wortmannin which is a specific inhibitor of phosphoinositide 3-kinase (PI3-kinase) failed to inhibit the invasion. Moreover, SB 203580 can also interfere with IkappaBalpha degradation and IL-8 mRNA expression, indicating that the phosphorylation of p38 occurred upstream of NF-kappaB activation. Cytochalasin D, an inhibitor of actin polymerization needed for internalisation of bacteria, did not have any effect on the phosphorylation of p38. These results indicate that B. pseudomallei stimulate phosphorylation of p38 making by initial contact with the cell surface components and do not require internalisation and interaction with intracellular cytoplasmic components of the cells.

Burkholderia pseudomallei stimulates low interleukin-8 production in the human lung epithelial cell line A549.

Melioidosis is a life-threatening disease caused by Burkholderia pseudomallei. The lung is the most commonly affected organ, resulting in abscess formation in patients with chronic melioidosis. Previous study has shown that B. pseudomallei was able to invade and multiply in epithelial cells. In the present study, we have demonstrated that B. pseudomallei is able to stimulate interleukin 8 (IL-8) production from the human alveolar lung epithelium cell line A549. However, the level of IL-8 production was significantly lower than when the cells were infected with other Gram-negative bacteria such as Salmonella enterica serovar Typhi (S. typhi) which were used for comparison. The degree of IkappaBalpha degradation in the B. pseudomallei-infected cells was lower than that of the S. typhi-infected cells, suggesting that B. pseudomallei is also a poorer cell activator. Inhibition of B. pseudomallei invasion by cytochalasin D did not interfere with either IL-8 production or IkappaBalpha degradation, indicating that bacterial uptake is not required for the production of this chemokine. Thus, it appears that the signalling initiated by the interaction of B. pseudomallei with the epithelial cell surface is sufficient for epithelial cell activation.

Induction of iNOS expression and antimicrobial activity by interferon (IFN)-beta is distinct from IFN-gamma in Burkholderia pseudomallei-infected mouse macrophages.

Burkholderia pseudomallei is a causative agent of melioidosis. This Gram-negative bacterium is able to survive and multiple inside both phagocytic and nonphagocytic cells. We previously reported that exogenous interferons (both type I and type II) enhanced antimicrobial activity of the macrophages infected with B. pseudomallei by up-regulating inducible nitric oxide synthase (iNOS). This enzyme thus plays an essential role in controlling intracellular growth of bacteria. In the present study we extended our investigation, analysing the mechanism(s) by which the two types of interferons (IFNs) regulate antimicrobial activity in the B. pseudomallei-infected macrophages. Mouse macrophage cell line (RAW 264.7) that was exposed simultaneously to B. pseudomallei and type I IFN (IFN-beta) expressed high levels of iNOS, leading to enhanced intracellular killing of the bacteria. However, neither enhanced iNOS expression nor intracellular bacterial killing was observed when the macrophages were preactivated with IFN-beta prior to being infected with B. pseudomallei. On the contrary, the timing of exposure was not critical for the type II IFN (IFN-gamma) because when the cells were either prestimulated or co-stimulated with IFN-gamma, both iNOS expression and intracellular killing capacity were enhanced. The differences by which these two IFNs regulate antimicrobial activity may be related to the fact that IFN-gamma was able to induce more sustained interferon regulatory factor-1 (IRF-1) expression compared with the cells activated with IFN-beta.

Involvement of beta interferon in enhancing inducible nitric oxide synthase production and antimicrobial activity of Burkholderia pseudomallei-infected macrophages.

Burkholderia pseudomallei is the causative agent of melioidosis, a life-threatening disease that affects both humans and animals. This bacterium is able to survive and multiply inside both phagocytic and nonphagocytic cells. We recently reported that mouse macrophages infected with B. pseudomallei fail to produce a significant level of inducible nitric oxide synthase (iNOS), a crucial enzyme needed for the cells to control the intracellular growth of this bacterium. In the present study, we extended our investigation to demonstrate that, unlike other gram-negative bacteria that have been investigated, B. pseudomallei only minimally activates beta interferon (IFN-beta) production; this minimal activation leads to a low level of interferon regulating factor 1 (IRF-1) in the macrophages, in parallel with poor iNOS expression. Adding exogenous IFN-beta to the system could upregulate IRF-1 production, which in turn could enhance iNOS expression in the B. pseudomallei-infected macrophages and lead to suppression of the intracellular growth of this bacterium. Taken together, these results imply that the failure of macrophages to successfully control the growth and survival of intracellular B. pseudomallei is related, at least in part, to the defective production of IFN-beta, which modulates the ability of macrophages to synthesize iNOS.