Burkholderia pseudomallei, the etiological agent of melioidosis, is a facultative intracellular pathogen. As B. pseudomallei is a gram-negative bacterium, its outer membrane contains lipopolysaccharide (LPS) molecules, which have been shown to have low-level immunological activities in vitro. In this study, the biological activities of B. pseudomallei LPS were compared to those of Burkholderia thailandensis LPS, and it was found that both murine and human macrophages produced levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10 in response to B. pseudomallei LPS that were lower than those in response to B. thailandensis LPS in vitro. In order to elucidate the molecular mechanisms underlying the low-level immunological activities of B. pseudomallei LPS, its lipid A moiety was characterized using mass spectrometry. The major lipid A species identified in B. pseudomallei consists of a biphosphorylated disaccharide backbone, which is modified with 4-amino-4-deoxy-arabinose (Ara4N) at both phosphates and penta-acylated with fatty acids (FA) C 14:0 (3-OH), C 16:0 (3-OH), and either C 14:0 or C 14:0 (2-OH). In contrast, the major lipid A species identified in B. thailandensis was a mixture of tetra-and penta-acylated structures with differing amounts of Ara4N and FA C 14:0 (3-OH). Lipid A species acylated with FA C 14:0 (2-OH) were unique to B. pseudomallei and not found in B. thailandensis. Our data thus indicate that B. pseudomallei synthesizes lipid A species with long-chain FA C 14:0 (2-OH) and Ara4N-modified phosphate groups, allowing it to evade innate immune recognition.

Background Burkholderia pseudomallei, a facultative intracellular pathogen, causes systemic infection in humans with high mortality especially when infection occurs through an infectious aerosol. Previous studies indicated that the epithelial cells in the lung are an active participant in host immunity. In this study, we aimed to investigate the innate immune responses of lung epithelial cells against B. pseudomallei.Methodology and Principal FindingsUsing a murine lung epithelial cell line, primary lung epithelial cells and an inhalational murine infection model, we characterized the types of innate immunity proteins and peptides produced upon B. pseudomallei infection. Among a wide panel of immune components studied, increased levels of major pro-inflammatory cytokines IL-6 and TNFα, chemokine MCP-1, and up-regulation of secretory leukocyte protease inhibitor (SLPI) and chemokine (C-C motif) ligand 20 (CCL20) were observed. Inhibition assays using specific inhibitors suggested that NF-κB and p38 MAPK pathways were responsible for these B. pseudomallei-induced antimicrobial peptides.ConclusionsOur findings indicate that the respiratory epithelial cells, which form the majority of the cells lining the epithelial tract and the lung, have important roles in the innate immune response against B. pseudomallei infection.

Melioidosis, an infection caused by Gram-negative Burkholderia pseudomallei (Bp), has high clinical recurrence and mortality rates associated with pneumonia and sepsis. With the limitations in current therapeutic options and the lack of available human vaccines, development of novel countermeasures against Bp infection is vital. In this study, we evaluated the efficacy of an aminoalkyl glucosaminide 4-phosphate (AGP), a synthetic toll like receptor 4 agonist (CRX-527), in conferring protection against melioidosis in a murine model. Survival data showed 66% of mice treated with AGP prior to lethal intranasal Bp challenge survived and presented no signs of illness over a 3 months period. In contrast, all control mice succumbed to infection within 4 days. Kinetic study on organ bacterial burden demonstrated mice treated with AGP had dramatically reduced bacterial loads in both the lungs and spleens as compared to control mice. Notably, all but one AGP-treated mouse had no Bp growth in the blood as compared to overwhelming bacteraemia found in all control mice. The protective effect of CRX-527 was associated with a transient increase in pulmonary cytokine/ chemokine levels, which boosted the host’s innate immunity. This enabled rapid clearance of the pulmonary and systemic bacterial burden and prevented the development of sepsis. This study demonstrated the potential use of TLR4 agonist as a prophylactic immunotherapy in preventing melioidosis.

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