Limits...
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.

Show MeSH

Related in: MedlinePlus

The amount of the lipid peroxide intermediate MDA increases with increasing concentrations of O2 and with exposure to oxidants.A. B. burgdorferi strain B31A3 was grown under microaerobic conditions, the culture split and treated with either 5 mM AAPH or 250 mg of lipoxidase for 4 h at 34°C. To measure the amount of MDA, the cells were derivatized with thiobarbituric acid and analysed by HPLC. MDA standards were prepared in methanol for comparison. The calculated P-values indicated that the two treated samples were significantly different from the untreated sample.B. Mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere, treated with 1 mM AAPH at 37°C for 4 h (Chotimarkorn et al., 2005) and MDA measured as above. The calculated P-value indicated that the treated sample was significantly different from the untreated sample.C. B. burgdorferi strain B31A3 was grown aerobically, microaerobically and anaerobically to a cell density of 5 × 107 cells ml−1 and MDA measured as above. The calculated P-values indicated that both the anaerobic and microaerobic samples were significantly different from the aerobic sample.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2327290&req=5

fig03: The amount of the lipid peroxide intermediate MDA increases with increasing concentrations of O2 and with exposure to oxidants.A. B. burgdorferi strain B31A3 was grown under microaerobic conditions, the culture split and treated with either 5 mM AAPH or 250 mg of lipoxidase for 4 h at 34°C. To measure the amount of MDA, the cells were derivatized with thiobarbituric acid and analysed by HPLC. MDA standards were prepared in methanol for comparison. The calculated P-values indicated that the two treated samples were significantly different from the untreated sample.B. Mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere, treated with 1 mM AAPH at 37°C for 4 h (Chotimarkorn et al., 2005) and MDA measured as above. The calculated P-value indicated that the treated sample was significantly different from the untreated sample.C. B. burgdorferi strain B31A3 was grown aerobically, microaerobically and anaerobically to a cell density of 5 × 107 cells ml−1 and MDA measured as above. The calculated P-values indicated that both the anaerobic and microaerobic samples were significantly different from the aerobic sample.

Mentions: Malondialdehyde (MDA) is generated as a relatively stable end-product from the oxidative degradation of polyunsaturated fatty acids. MDA has thus been used as an indicator of lipid peroxidation (Gutteridge and Halliwell, 1990; Esterbauer et al., 1991). To further demonstrate that Borrelia lipids can undergo lipid peroxidation, B31A3 cells grown microaerobically were treated with 5 mM AAPH (free radical generator) or 250 mg of lipoxidase and MDA measured (Seljeskog et al., 2006). The results are shown in Fig. 3. Untreated cells contained ∼16.5 µM of MDA per mg of protein. When the cells were treated with AAPH or lipoxidase, the amount of MDA increased ∼1.5-fold (27.3 µM of MDA per mg of protein) and approximately twofold (33 µM of MDA per mg of protein) respectively (Fig. 3A). As a control, eukaryotic cells (mouse myeloma cells SP2) were treated with AAPH and MDA measured (Fig. 3B). Mouse myeloma cells have been used as a model system for the determination of phospholipid hydroperoxides (Chotimarkorn et al., 2005). In this case, the amount of MDA increased approximately threefold (36.3 µM of MDA per mg of protein) in the treated cells versus untreated cells (11.7 µM of MDA per mg of protein). Taken together, these data suggested that, like eukaryotic membranes, Borrelia membrane lipids were capable of undergoing lipid peroxidation.


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

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

The amount of the lipid peroxide intermediate MDA increases with increasing concentrations of O2 and with exposure to oxidants.A. B. burgdorferi strain B31A3 was grown under microaerobic conditions, the culture split and treated with either 5 mM AAPH or 250 mg of lipoxidase for 4 h at 34°C. To measure the amount of MDA, the cells were derivatized with thiobarbituric acid and analysed by HPLC. MDA standards were prepared in methanol for comparison. The calculated P-values indicated that the two treated samples were significantly different from the untreated sample.B. Mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere, treated with 1 mM AAPH at 37°C for 4 h (Chotimarkorn et al., 2005) and MDA measured as above. The calculated P-value indicated that the treated sample was significantly different from the untreated sample.C. B. burgdorferi strain B31A3 was grown aerobically, microaerobically and anaerobically to a cell density of 5 × 107 cells ml−1 and MDA measured as above. The calculated P-values indicated that both the anaerobic and microaerobic samples were significantly different from the aerobic sample.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2327290&req=5

fig03: The amount of the lipid peroxide intermediate MDA increases with increasing concentrations of O2 and with exposure to oxidants.A. B. burgdorferi strain B31A3 was grown under microaerobic conditions, the culture split and treated with either 5 mM AAPH or 250 mg of lipoxidase for 4 h at 34°C. To measure the amount of MDA, the cells were derivatized with thiobarbituric acid and analysed by HPLC. MDA standards were prepared in methanol for comparison. The calculated P-values indicated that the two treated samples were significantly different from the untreated sample.B. Mouse myeloma SP2/O cells were cultured with HYQ-CCM1 (HyClone) medium at 37°C in a humidified 5% CO2 atmosphere, treated with 1 mM AAPH at 37°C for 4 h (Chotimarkorn et al., 2005) and MDA measured as above. The calculated P-value indicated that the treated sample was significantly different from the untreated sample.C. B. burgdorferi strain B31A3 was grown aerobically, microaerobically and anaerobically to a cell density of 5 × 107 cells ml−1 and MDA measured as above. The calculated P-values indicated that both the anaerobic and microaerobic samples were significantly different from the aerobic sample.
Mentions: Malondialdehyde (MDA) is generated as a relatively stable end-product from the oxidative degradation of polyunsaturated fatty acids. MDA has thus been used as an indicator of lipid peroxidation (Gutteridge and Halliwell, 1990; Esterbauer et al., 1991). To further demonstrate that Borrelia lipids can undergo lipid peroxidation, B31A3 cells grown microaerobically were treated with 5 mM AAPH (free radical generator) or 250 mg of lipoxidase and MDA measured (Seljeskog et al., 2006). The results are shown in Fig. 3. Untreated cells contained ∼16.5 µM of MDA per mg of protein. When the cells were treated with AAPH or lipoxidase, the amount of MDA increased ∼1.5-fold (27.3 µM of MDA per mg of protein) and approximately twofold (33 µM of MDA per mg of protein) respectively (Fig. 3A). As a control, eukaryotic cells (mouse myeloma cells SP2) were treated with AAPH and MDA measured (Fig. 3B). Mouse myeloma cells have been used as a model system for the determination of phospholipid hydroperoxides (Chotimarkorn et al., 2005). In this case, the amount of MDA increased approximately threefold (36.3 µM of MDA per mg of protein) in the treated cells versus untreated cells (11.7 µM of MDA per mg of protein). Taken together, these data suggested that, like eukaryotic membranes, Borrelia membrane lipids were capable of undergoing lipid peroxidation.

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