title: Activation of Toll-like receptors by Burkholderia pseudomallei.
abstract: BACKGROUND
  Melioidosis, a lethal tropical infection that is endemic in southeast Asia and northern Australia, is caused by the saprophytic Gram-negative bacterium Burkholderia pseudomallei. Overall mortality approaches 40% yet little is known about mechanisms of host defense. Toll-like receptors (TLRs) are host transmembrane receptors that recognize conserved pathogen molecular patterns and induce an inflammatory response. The lipopolysaccharide (LPS) of Gram-negative bacteria is a potent inducer of the host innate immune system. TLR4, in association with MD-2, is the archetype receptor for LPS although B. pseudomallei LPS has been previously identified as a TLR2 agonist. We examined TLR signaling induced by B. pseudomallei, B. pseudomallei LPS, and B. pseudomallei lipid A using gain-of-function transfection assays of NF-kappaB activation and studies of TLR-deficient macrophages.
  
  
  RESULTS
  In HEK293 cells transfected with murine or human TLRs, CD14, and MD-2, heat-killed B. pseudomallei activated TLR2 (in combination with TLR1 or TLR6) and TLR4. B. pseudomallei LPS and lipid A activated TLR4 and this TLR4-mediated signaling required MD-2. In TLR2-/- macrophages, stimulation with heat-killed B. pseudomallei augmented TNF-alpha and MIP-2 production whereas in TLR4-/- cells, TNF-alpha, MIP-2, and IL-10 production was reduced. Cytokine production by macrophages stimulated with B. pseudomallei LPS or lipid A was entirely dependent on TLR4 but was increased in the absence of TLR2. TLR adaptor molecule MyD88 strongly regulated TNF-alpha production in response to heat-killed B. pseudomallei.
  
  
  CONCLUSIONS
  B. pseudomallei activates TLR2 and TLR4. In the presence of MD-2, B. pseudomallei LPS and lipid A are TLR4 ligands. Although the macrophage cytokine response to B. pseudomallei LPS or lipid A is completely dependent on TLR4, in TLR2-/- macrophages stimulated with B. pseudomallei, B. pseudomallei LPS or lipid A, cytokine production is augmented. Other MyD88-dependent signaling pathways may also be important in the host response to B. pseudomallei infection. These findings provide new insights into critical mechanisms of host defense in melioidosis.
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington, USA. tewest@u.washington.edu
authors.forename: T Eoin
authors.initials: TE
authors.lastname: West
authors.forename: Robert K
authors.initials: RK
authors.lastname: Ernst
authors.forename: Malinka J
authors.initials: MJ
authors.lastname: Jansson-Hutson
authors.forename: Shawn J
authors.initials: SJ
authors.lastname: Skerrett
mesh_terms: D000818:Animals; D016957:Burkholderia pseudomallei / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D054426:Chemokine CXCL2 / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D008050:Lipid A / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008100:Liver Abscess / Q000378:metabolism / Q000382:microbiology; D051218:Lymphocyte Antigen 96 / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D008554:Melioidosis / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D051195:Toll-Like Receptor 2 / Q000172:deficiency / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D054426:Chemokine CXCL2; D008050:Lipid A; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D053594:Myeloid Differentiation Factor 88; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha