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Borrelia burgdorferi membranes are the primary targets of reactive oxygen species.

Boylan JA, Lawrence KA, Downey JS, Gherardini FC - Mol. Microbiol. (2008)

Bottom Line: Because Borrelia burgdorferi contains no intracellular iron, DNA is most likely not a major target for ROS via Fenton reaction.Fatty acid analysis of cells treated with lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (50-fold) in these cells, with a corresponding increase in the levels of malondialdehyde by-product (fourfold).These data suggest that B. burgdorferi membrane lipids are targets for attack by ROS encountered in the various stages of the infective cycle.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, 903 S 4th Street, Hamilton, MT 59840, USA.

ABSTRACT
Spirochetes living in an oxygen-rich environment or when challenged by host immune cells are exposed to reactive oxygen species (ROS). These species can harm/destroy cysteinyl residues, iron-sulphur clusters, DNA and polyunsaturated lipids, leading to inhibition of growth or cell death. Because Borrelia burgdorferi contains no intracellular iron, DNA is most likely not a major target for ROS via Fenton reaction. In support of this, growth of B. burgdorferi in the presence of 5 mM H(2)O(2) had no effect on the DNA mutation rate (spontaneous coumermycin A1 resistance), and cells treated with 10 mM t-butyl hydroperoxide or 10 mM H(2)O(2) show no increase in DNA damage. Unlike most bacteria, B. burgdorferi incorporates ROS-susceptible polyunsaturated fatty acids from the environment into their membranes. Analysis of lipoxidase-treated B. burgdorferi cells by Electron Microscopy showed significant irregularities indicative of membrane damage. Fatty acid analysis of cells treated with lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (50-fold) in these cells, with a corresponding increase in the levels of malondialdehyde by-product (fourfold). These data suggest that B. burgdorferi membrane lipids are targets for attack by ROS encountered in the various stages of the infective cycle.

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Borrelia cells contain lipid peroxides.A. E. coli Top10 cells were grown to OD600 of 0.4 and cells harvested. The whole cells were visualized with PKH26 Red Fluorescent Cell Linker Dye and the lipid hydroperoxides in the cell membrane were counterstained with DPPP.B. B. burgdorferi strain B31A3 was grown microaerobically to a cell density of 5 × 107 cells ml−1 and stained with PKH26 and DPPP as described above.C. Mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere and stained with PKH22 and DPPP as described above.
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fig05: Borrelia cells contain lipid peroxides.A. E. coli Top10 cells were grown to OD600 of 0.4 and cells harvested. The whole cells were visualized with PKH26 Red Fluorescent Cell Linker Dye and the lipid hydroperoxides in the cell membrane were counterstained with DPPP.B. B. burgdorferi strain B31A3 was grown microaerobically to a cell density of 5 × 107 cells ml−1 and stained with PKH26 and DPPP as described above.C. Mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere and stained with PKH22 and DPPP as described above.

Mentions: The fluorescent probe diphenyl-1-pyrenylphophine (DPPP) has been used for detection of lipid hydroperoxides in cell membranes (Okimoto et al., 2000; Takahashi et al., 2001). In this method, the hydroperoxides are reduced with DPPP, resulting in the formation of the fluorescent arylphosphine oxide. To visualize the lipid hydroperoxides, B. burgdorferi B31A3 cells were labelled with DPPP and observed by fluorescence microscopy (Fig. 5B). E. coli Top10 cells and mouse myeloma SP2 cells were also labelled and visualized to serve as negative and positive controls respectively (Fig. 5A and C). Red Fluorescent dye was used to visualize the cell membranes. A strong fluorescence of DPPP was observed for B31A3 cells (Fig. 5B) and for the mouse myeloma cells (Fig. 5C), but not for the E. coli cells (Fig. 5A). An overlay of the two dyes demonstrates that the DPPP fluorescence of both the Borrelia and myeloma cells corresponds to the areas of red fluorescence. This indicated that lipid hydroperoxides were present on the Borrelia cell membranes and suggested that the membranes were damaged.


Borrelia burgdorferi membranes are the primary targets of reactive oxygen species.

Boylan JA, Lawrence KA, Downey JS, Gherardini FC - Mol. Microbiol. (2008)

Borrelia cells contain lipid peroxides.A. E. coli Top10 cells were grown to OD600 of 0.4 and cells harvested. The whole cells were visualized with PKH26 Red Fluorescent Cell Linker Dye and the lipid hydroperoxides in the cell membrane were counterstained with DPPP.B. B. burgdorferi strain B31A3 was grown microaerobically to a cell density of 5 × 107 cells ml−1 and stained with PKH26 and DPPP as described above.C. Mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere and stained with PKH22 and DPPP as described above.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2327290&req=5

fig05: Borrelia cells contain lipid peroxides.A. E. coli Top10 cells were grown to OD600 of 0.4 and cells harvested. The whole cells were visualized with PKH26 Red Fluorescent Cell Linker Dye and the lipid hydroperoxides in the cell membrane were counterstained with DPPP.B. B. burgdorferi strain B31A3 was grown microaerobically to a cell density of 5 × 107 cells ml−1 and stained with PKH26 and DPPP as described above.C. Mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere and stained with PKH22 and DPPP as described above.
Mentions: The fluorescent probe diphenyl-1-pyrenylphophine (DPPP) has been used for detection of lipid hydroperoxides in cell membranes (Okimoto et al., 2000; Takahashi et al., 2001). In this method, the hydroperoxides are reduced with DPPP, resulting in the formation of the fluorescent arylphosphine oxide. To visualize the lipid hydroperoxides, B. burgdorferi B31A3 cells were labelled with DPPP and observed by fluorescence microscopy (Fig. 5B). E. coli Top10 cells and mouse myeloma SP2 cells were also labelled and visualized to serve as negative and positive controls respectively (Fig. 5A and C). Red Fluorescent dye was used to visualize the cell membranes. A strong fluorescence of DPPP was observed for B31A3 cells (Fig. 5B) and for the mouse myeloma cells (Fig. 5C), but not for the E. coli cells (Fig. 5A). An overlay of the two dyes demonstrates that the DPPP fluorescence of both the Borrelia and myeloma cells corresponds to the areas of red fluorescence. This indicated that lipid hydroperoxides were present on the Borrelia cell membranes and suggested that the membranes were damaged.

Bottom Line: Because Borrelia burgdorferi contains no intracellular iron, DNA is most likely not a major target for ROS via Fenton reaction.Fatty acid analysis of cells treated with lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (50-fold) in these cells, with a corresponding increase in the levels of malondialdehyde by-product (fourfold).These data suggest that B. burgdorferi membrane lipids are targets for attack by ROS encountered in the various stages of the infective cycle.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, 903 S 4th Street, Hamilton, MT 59840, USA.

ABSTRACT
Spirochetes living in an oxygen-rich environment or when challenged by host immune cells are exposed to reactive oxygen species (ROS). These species can harm/destroy cysteinyl residues, iron-sulphur clusters, DNA and polyunsaturated lipids, leading to inhibition of growth or cell death. Because Borrelia burgdorferi contains no intracellular iron, DNA is most likely not a major target for ROS via Fenton reaction. In support of this, growth of B. burgdorferi in the presence of 5 mM H(2)O(2) had no effect on the DNA mutation rate (spontaneous coumermycin A1 resistance), and cells treated with 10 mM t-butyl hydroperoxide or 10 mM H(2)O(2) show no increase in DNA damage. Unlike most bacteria, B. burgdorferi incorporates ROS-susceptible polyunsaturated fatty acids from the environment into their membranes. Analysis of lipoxidase-treated B. burgdorferi cells by Electron Microscopy showed significant irregularities indicative of membrane damage. Fatty acid analysis of cells treated with lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (50-fold) in these cells, with a corresponding increase in the levels of malondialdehyde by-product (fourfold). These data suggest that B. burgdorferi membrane lipids are targets for attack by ROS encountered in the various stages of the infective cycle.

Show MeSH
Related in: MedlinePlus