17 research outputs found

    Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

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    The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host-adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development

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    Anti-inflammatory effect of <i>F</i>. <i>tularensis</i> LPS on mouse PECs is IFN-Ξ³ and IL-10 dependent.

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    <p>PECs from naΓ―ve and IL-10 genetically deficient C57BL/6 mice were obtained and resuspended in cell culture media. PECs were cultured in a 96-well plate at 2 x 10<sup>5</sup> cells/well with either <i>Ft</i>-LPS or <i>E</i>. <i>coli</i>-LPS at 1 ng/mL in the presence or absence of recombinant IFN-Ξ³ at 100 U/ml. Cells cultured with PBS were used as a control. The cytokine production was measured using BD Biosciences Cytometric Bead Array (CBA) following vendor instructions. Results are representative of three independent experiments. (*) P-value < 0.1, (**) P-value < 0.05, bars represent SD.</p

    FcΞ³R targeting drives polarization of mouse macrophages towards the AM1 phenotype at the early stages of LVS infection.

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    <p>Lungs of immunized mice were harvested 24, 48 and 96 hours post-infection. For cell surface marker staining, cells were fluorescently labeled with antibodies against CD11b, F4/80, MHC class II, B7.1, B7.2, CCR7, or their corresponding isotype controls were added. Cells were then analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences). Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.</p

    Immunization with mAb-i<i>Ft</i> immune complexes increases the activation of PECs following LVS challenge.

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    <p>C57BL/6 mice were immunized i.n. with PBS, i<i>Ft</i> (2x10<sup>7</sup> CFUs), or mAb-i<i>Ft</i>, boosted on day 21 and challenged on day 35 with 10,000 CFUs of <i>Ft</i> LVS. On day 2 post-infection the peritoneal exudate cells of immunized mice were harvested and the expression of F4/80, MHC class II, B7.1 (CD80), and B7.2 (CD86) were analyzed by flow cytometry. Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.</p

    Administration of mAb-i<i>Ft</i> immune complexes reverses the anti-inflammatory properties of LVS in the lungs of immunized mice.

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    <p>C57BL/6 mice were immunized i.n. with PBS, i<i>Ft</i> (2x10<sup>7</sup> CFUs), or mAb-i<i>Ft</i>, boosted on day 21 and challenged on day 35 with 10,000 CFUs of <i>Ft</i> LVS. Lung tissue homogenates were obtained from immunized mice 24, 48 and 96 hours post-infection as indicated above and spun down at 15,000g for 30 minutes at room temperature to remove tissue debris. Cytokine levels were detected by using the IL-6, IL-10, TNF-Ξ± and IFN-Ξ³ ELISA kits and following vendor instructions (Biolegend). Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.</p
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