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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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HPLC chromatograms with three corresponding dimensional diode array spectra scanned during elution of the peak with a retention time corresponding to the MDA standard.A. TBA-treated, authentic MDA standard with an absorbance maximum of 532 nm. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.B. TBA-treated sample from B. burgdorferi strain B31A3 grown under anaerobic conditions. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.C. TBA-treated sample from B. burgdorferi strain B31A3 grown under microaerobic conditions. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.
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fig04: HPLC chromatograms with three corresponding dimensional diode array spectra scanned during elution of the peak with a retention time corresponding to the MDA standard.A. TBA-treated, authentic MDA standard with an absorbance maximum of 532 nm. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.B. TBA-treated sample from B. burgdorferi strain B31A3 grown under anaerobic conditions. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.C. TBA-treated sample from B. burgdorferi strain B31A3 grown under microaerobic conditions. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.

Mentions: Our High Performance Liquid Chromatography analyses of the MDA present in anaerobically grown B. burgdorferi cells showed a small peak with a retention time similar to that of the MDA standard. This was puzzling as little or no lipid peroxidation should occur under these conditions. To further characterize this ‘MDA’ peak, a three-dimensional diode array spectra was generated by scanning each sample during the elution of the peak (Fig. 4, lower sections). An authentic MDA standard was also scanned as a control (Fig. 4A, lower section). In the anaerobically grown cells, the spectrum shows that two compounds with different absorbance maximums (Fig. 4B, lower section) comprised the single retention time peak from the HPLC chromatogram (Fig. 4B, upper section). Based on this spectrum, the amount of actual MDA contributed < 15% of the total amount of material detected in the HPLC peak while the second contaminating peak contributed > 85%. Therefore, the amount of MDA in untreated anaerobically grown cells was considerably less than the 7.6 µM of MDA per mg of protein actually measured. However, in cells grown under microaerobic conditions, the MDA peak contributed more to the overall retention time peak when compared with the anaerobic spectrum, while the second contaminating peak stays relatively constant (Fig. 4C, lower section). These spectra demonstrated that the increase in the MDA retention time peak between anaerobically and aerobically grown cells was due to the increase in the amount of authentic MDA present.


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

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

HPLC chromatograms with three corresponding dimensional diode array spectra scanned during elution of the peak with a retention time corresponding to the MDA standard.A. TBA-treated, authentic MDA standard with an absorbance maximum of 532 nm. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.B. TBA-treated sample from B. burgdorferi strain B31A3 grown under anaerobic conditions. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.C. TBA-treated sample from B. burgdorferi strain B31A3 grown under microaerobic conditions. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.
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Related In: Results  -  Collection

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fig04: HPLC chromatograms with three corresponding dimensional diode array spectra scanned during elution of the peak with a retention time corresponding to the MDA standard.A. TBA-treated, authentic MDA standard with an absorbance maximum of 532 nm. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.B. TBA-treated sample from B. burgdorferi strain B31A3 grown under anaerobic conditions. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.C. TBA-treated sample from B. burgdorferi strain B31A3 grown under microaerobic conditions. Upper section, HPLC chromatogram; lower section, 3D diode array spectra.
Mentions: Our High Performance Liquid Chromatography analyses of the MDA present in anaerobically grown B. burgdorferi cells showed a small peak with a retention time similar to that of the MDA standard. This was puzzling as little or no lipid peroxidation should occur under these conditions. To further characterize this ‘MDA’ peak, a three-dimensional diode array spectra was generated by scanning each sample during the elution of the peak (Fig. 4, lower sections). An authentic MDA standard was also scanned as a control (Fig. 4A, lower section). In the anaerobically grown cells, the spectrum shows that two compounds with different absorbance maximums (Fig. 4B, lower section) comprised the single retention time peak from the HPLC chromatogram (Fig. 4B, upper section). Based on this spectrum, the amount of actual MDA contributed < 15% of the total amount of material detected in the HPLC peak while the second contaminating peak contributed > 85%. Therefore, the amount of MDA in untreated anaerobically grown cells was considerably less than the 7.6 µM of MDA per mg of protein actually measured. However, in cells grown under microaerobic conditions, the MDA peak contributed more to the overall retention time peak when compared with the anaerobic spectrum, while the second contaminating peak stays relatively constant (Fig. 4C, lower section). These spectra demonstrated that the increase in the MDA retention time peak between anaerobically and aerobically grown cells was due to the increase in the amount of authentic MDA present.

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