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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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The addition of oxidants does not damage Borrelia DNA.A. B. burgdorferi B31A3 cells were grown under microaerobic conditions to a cell density of 5 × 107 cells ml−1, treated with 5 mM H2O2 and plated in the presence and absence of 250 ng ml−1 coumermycin A1. The resistance frequency was calculated as the number of colonies that are CouR per total number of cells plated.B. B. burgdorferi B31A3 and B314 cells were grown in BSK-II to a cell density of 5 × 107 cells ml−1, treated with various oxidants for 1 h, and DNA isolated. The DNA was then mixed with an aldehyde reactive probe (Oxford Biomedical Research) labelled with biotin and detected with an HRP-streptavidin conjugate. The colour development was monitored at 450 nm. The number of aldehyde reactive probe (DNA base lesions) per 105 bp DNA was determined using a standard curve. E. coli TA4315 (ahpCF) (Storz et al., 1989) cells were grown in minimal media to OD600 of 0.4, treated with 100 µM H2O2 for 30 min, and DNA isolated. The number of base lesions was determined as described above.C. B. burgdorferi B31A3 cells were grown in BSK-II to a cell density of 5 × 107 cells ml−1, treated with various oxidants for 1 h, and DNA isolated. The DNA was then converted to single-stranded and digested with P1. The resulting mixture was used in an 8-oxoguanine ELISA assay (Oxford Biomedical Research). The concentration of 8-oxoguanine was determined using a standard curve. E. coli CM1319 (mutM) cells were grown in LB to OD600 of 0.4, treated with 100 µM H2O2 for 1 h and DNA isolated. The concentration of 8-oxoguanine was determined as above.
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fig02: The addition of oxidants does not damage Borrelia DNA.A. B. burgdorferi B31A3 cells were grown under microaerobic conditions to a cell density of 5 × 107 cells ml−1, treated with 5 mM H2O2 and plated in the presence and absence of 250 ng ml−1 coumermycin A1. The resistance frequency was calculated as the number of colonies that are CouR per total number of cells plated.B. B. burgdorferi B31A3 and B314 cells were grown in BSK-II to a cell density of 5 × 107 cells ml−1, treated with various oxidants for 1 h, and DNA isolated. The DNA was then mixed with an aldehyde reactive probe (Oxford Biomedical Research) labelled with biotin and detected with an HRP-streptavidin conjugate. The colour development was monitored at 450 nm. The number of aldehyde reactive probe (DNA base lesions) per 105 bp DNA was determined using a standard curve. E. coli TA4315 (ahpCF) (Storz et al., 1989) cells were grown in minimal media to OD600 of 0.4, treated with 100 µM H2O2 for 30 min, and DNA isolated. The number of base lesions was determined as described above.C. B. burgdorferi B31A3 cells were grown in BSK-II to a cell density of 5 × 107 cells ml−1, treated with various oxidants for 1 h, and DNA isolated. The DNA was then converted to single-stranded and digested with P1. The resulting mixture was used in an 8-oxoguanine ELISA assay (Oxford Biomedical Research). The concentration of 8-oxoguanine was determined using a standard curve. E. coli CM1319 (mutM) cells were grown in LB to OD600 of 0.4, treated with 100 µM H2O2 for 1 h and DNA isolated. The concentration of 8-oxoguanine was determined as above.

Mentions: One reliable indicator of DNA damage by ROS in a cell is an increase in the spontaneous mutation rate. In B. burgdorferi, mutations that confer resistance to coumermycin A1, which targets the β subunit of DNA gyrase, have been mapped to gyrB, the gene encoding DNA gyrase B (Samuels et al., 1994). In each case, a single point mutation correlated with this drug resistance. To determine if exposure to oxidants increases DNA damage by increasing point mutations, B. burgdorferi B31A3 cells grown under microaerobic conditions were treated with 5 mM H2O2 and plated in the presence and absence of 250 ng ml−1 coumermycin A1. The mutation frequency was calculated as the number of colonies that are CouR per total number of cells plated. The spontaneous resistance frequency of treated cells was approximately equivalent to that of untreated cells, 8.8 × 10−8 and 1.33 × 10−7, respectively, indicating no increase in the number of point mutations (Fig. 2A). Also, no increase in point mutations was observed when cells were treated with higher concentrations of H2O2 (10 mM) or when treated with t-butyl hydroperoxide (5 and 10 mM) (data not shown).


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

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

The addition of oxidants does not damage Borrelia DNA.A. B. burgdorferi B31A3 cells were grown under microaerobic conditions to a cell density of 5 × 107 cells ml−1, treated with 5 mM H2O2 and plated in the presence and absence of 250 ng ml−1 coumermycin A1. The resistance frequency was calculated as the number of colonies that are CouR per total number of cells plated.B. B. burgdorferi B31A3 and B314 cells were grown in BSK-II to a cell density of 5 × 107 cells ml−1, treated with various oxidants for 1 h, and DNA isolated. The DNA was then mixed with an aldehyde reactive probe (Oxford Biomedical Research) labelled with biotin and detected with an HRP-streptavidin conjugate. The colour development was monitored at 450 nm. The number of aldehyde reactive probe (DNA base lesions) per 105 bp DNA was determined using a standard curve. E. coli TA4315 (ahpCF) (Storz et al., 1989) cells were grown in minimal media to OD600 of 0.4, treated with 100 µM H2O2 for 30 min, and DNA isolated. The number of base lesions was determined as described above.C. B. burgdorferi B31A3 cells were grown in BSK-II to a cell density of 5 × 107 cells ml−1, treated with various oxidants for 1 h, and DNA isolated. The DNA was then converted to single-stranded and digested with P1. The resulting mixture was used in an 8-oxoguanine ELISA assay (Oxford Biomedical Research). The concentration of 8-oxoguanine was determined using a standard curve. E. coli CM1319 (mutM) cells were grown in LB to OD600 of 0.4, treated with 100 µM H2O2 for 1 h and DNA isolated. The concentration of 8-oxoguanine was determined as above.
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Related In: Results  -  Collection

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fig02: The addition of oxidants does not damage Borrelia DNA.A. B. burgdorferi B31A3 cells were grown under microaerobic conditions to a cell density of 5 × 107 cells ml−1, treated with 5 mM H2O2 and plated in the presence and absence of 250 ng ml−1 coumermycin A1. The resistance frequency was calculated as the number of colonies that are CouR per total number of cells plated.B. B. burgdorferi B31A3 and B314 cells were grown in BSK-II to a cell density of 5 × 107 cells ml−1, treated with various oxidants for 1 h, and DNA isolated. The DNA was then mixed with an aldehyde reactive probe (Oxford Biomedical Research) labelled with biotin and detected with an HRP-streptavidin conjugate. The colour development was monitored at 450 nm. The number of aldehyde reactive probe (DNA base lesions) per 105 bp DNA was determined using a standard curve. E. coli TA4315 (ahpCF) (Storz et al., 1989) cells were grown in minimal media to OD600 of 0.4, treated with 100 µM H2O2 for 30 min, and DNA isolated. The number of base lesions was determined as described above.C. B. burgdorferi B31A3 cells were grown in BSK-II to a cell density of 5 × 107 cells ml−1, treated with various oxidants for 1 h, and DNA isolated. The DNA was then converted to single-stranded and digested with P1. The resulting mixture was used in an 8-oxoguanine ELISA assay (Oxford Biomedical Research). The concentration of 8-oxoguanine was determined using a standard curve. E. coli CM1319 (mutM) cells were grown in LB to OD600 of 0.4, treated with 100 µM H2O2 for 1 h and DNA isolated. The concentration of 8-oxoguanine was determined as above.
Mentions: One reliable indicator of DNA damage by ROS in a cell is an increase in the spontaneous mutation rate. In B. burgdorferi, mutations that confer resistance to coumermycin A1, which targets the β subunit of DNA gyrase, have been mapped to gyrB, the gene encoding DNA gyrase B (Samuels et al., 1994). In each case, a single point mutation correlated with this drug resistance. To determine if exposure to oxidants increases DNA damage by increasing point mutations, B. burgdorferi B31A3 cells grown under microaerobic conditions were treated with 5 mM H2O2 and plated in the presence and absence of 250 ng ml−1 coumermycin A1. The mutation frequency was calculated as the number of colonies that are CouR per total number of cells plated. The spontaneous resistance frequency of treated cells was approximately equivalent to that of untreated cells, 8.8 × 10−8 and 1.33 × 10−7, respectively, indicating no increase in the number of point mutations (Fig. 2A). Also, no increase in point mutations was observed when cells were treated with higher concentrations of H2O2 (10 mM) or when treated with t-butyl hydroperoxide (5 and 10 mM) (data not shown).

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