Abstract
The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNγ can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNγ, IL-1β, and TNFα is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNγ-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.
Highlights
The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an emerging infectious disease of humans and animals in certain areas of the tropics and subtropics
A recent paper proposed that B. pseudomallei-induced cell fusion and the Abbreviations: AG, aminoguanidine hemisulfate; Apocynin, 4-hydroxy-3methoxyacetophenone; BMM, bone marrow-derived macrophages; CAT, catalase; CFU, colony forming units; COX, cyclooxygenase; EPS, exopolysaccharide; IFNγ, interferon γ; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; LAMP-1, lysosomal-associated membrane protein-1; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MOI, multiplicity of infection; NAC, N -acetyl-cysteine; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase; NO, nitric oxide; ROS, reactive oxygen species; SOD, superoxide dismutase; T3SS, type three secretion system; T6SS, type six secretion system; TNFα, tumor necrosis factor
Catalase–polyethylene glycol (CAT– PEG), superoxide dismutase–polyethylene glycol (SOD–PEG), N -acetyl-cysteine (NAC), aminoguanidine (AG), and colchicine (Col) were from Sigma Aldrich (Taufkirchen, Germany). hIFNγ was purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany), and both mIL-1β and mTNFα were from Roche (Mannheim, Germany)
Summary
The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an emerging infectious disease of humans and animals in certain areas of the tropics and subtropics. B. pseudomallei is an intracellular pathogen that can invade a variety of host cells (Jones et al, 1996). Within the cytosol bacteria can multiply and induce the BimA-dependent formation of actin tails, facilitating intracellular motility as well as spreading of B. pseudomallei into neighboring cells (Kespichayawattana et al, 2000; Breitbach et al, 2003; Stevens et al, 2005). Several reports have shown that interferon γ (IFNγ) is essential for the early control of B. pseudomallei infection in mice (Santanirand et al, 1999; Breitbach et al, 2006). We recently demonstrated that the downstream effector molecule of IFNγ, nitric oxide (NO), has a dual role among resistant and susceptible mouse strains after B. pseudomallei infection. In a previous study we revealed that NADPH oxidase-mediated mechanisms contribute to early resistance in bone marrow-derived macrophages and C57BL/6 mice (Breitbach et al, 2006)
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