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Absence of sodA Increases the Levels of Oxidation of Key Metabolic Determinants of Borrelia burgdorferi.

Esteve-Gassent MD, Smith TC, Small CM, Thomas DP, Seshu J - PLoS ONE (2015)

Bottom Line: Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway.Viable sodA mutant spirochetes could not be recovered from both gp91/phox-⁄- and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain.Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease.

View Article: PubMed Central - PubMed

Affiliation: South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX-78249, United States of America; Department of Biology, The University of Texas at San Antonio, San Antonio, TX-78249, United States of America.

ABSTRACT
Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA) capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE) and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat). HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348) exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560), and outer surface protein A (OspA, BBA15) were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC), Decorin binding protein A (DbpA), fibronectin binding protein (BBK32), RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox-⁄- and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease. This study, utilizing the sodA mutant, has provided insights into adaptive capabilities critical for survival of B. burgdorferi in its hosts.

No MeSH data available.


Related in: MedlinePlus

Levels of ATP, NAD/NADH and GAPDH in parental and sodA mutant strains of B. burgdorferi.Soluble proteins from the parental (wt) and sodA mutant (mt) of B. burgdorferi before (-) and after (+) treatment with 20 mM MV were prepared for measuring the ATP levels using ATP-Bioluminescent Assay Kit as per manufacturer’s instructions and the luminescence was measured using Synergy HT (BioTek) (A). A similar procedure was adopted for the isolation of soluble proteins for measurement of NAD/NADH using Fluoro NAD/NADH kit (Cell Technology) (B and C). The data represented were from 4 separate experiments and statistical analysis was carried out using one-way ANOVA followed by the Bonferroni’s multiple comparison test. * Denotes P value < 0.05, ** P value < 0.01 and *** P value < 0.001. The soluble proteins were from the parental (wy) and the sodA mutant (mt) were isolated as described above before (-) and after (+) treatment with 20 mM MV and separated on SDS-12% PAGE gels and stained with Coommassie blue (D) or transferred to PVDF membrane, probed with anti-GAPDH serum and blots developed using Enhanced Chemiluminescence system (E). Molecular masses in kilodaltons are indicated to the left of panel D and E.
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pone.0136707.g004: Levels of ATP, NAD/NADH and GAPDH in parental and sodA mutant strains of B. burgdorferi.Soluble proteins from the parental (wt) and sodA mutant (mt) of B. burgdorferi before (-) and after (+) treatment with 20 mM MV were prepared for measuring the ATP levels using ATP-Bioluminescent Assay Kit as per manufacturer’s instructions and the luminescence was measured using Synergy HT (BioTek) (A). A similar procedure was adopted for the isolation of soluble proteins for measurement of NAD/NADH using Fluoro NAD/NADH kit (Cell Technology) (B and C). The data represented were from 4 separate experiments and statistical analysis was carried out using one-way ANOVA followed by the Bonferroni’s multiple comparison test. * Denotes P value < 0.05, ** P value < 0.01 and *** P value < 0.001. The soluble proteins were from the parental (wy) and the sodA mutant (mt) were isolated as described above before (-) and after (+) treatment with 20 mM MV and separated on SDS-12% PAGE gels and stained with Coommassie blue (D) or transferred to PVDF membrane, probed with anti-GAPDH serum and blots developed using Enhanced Chemiluminescence system (E). Molecular masses in kilodaltons are indicated to the left of panel D and E.

Mentions: GAPDH, Pfk and Pky are key enzymes of the glycolytic pathway (Fig 3). Oxidation of these proteins might alter their function and consequently, there is a possibility that the cellular levels of ATP could be reduced rendering the sodA mutant more susceptible under conditions of limited nutrient availability or in microenvironments with limited energy sources [49–54]. To test this hypothesis, we measured the levels of ATP before and after treatment with 20mM MV in the parental and sodA negative strains. As shown in Fig 4A, in the presence of MV, the levels of ATP in the sodA negative strain were significantly reduced when compared with those of the untreated sample (P < 0.001), and were also lower than the levels observed in the parental strain after treatment (P < 0.001). On the other hand, the ATP levels in the parental strain were not significantly reduced in the presence of stressor. Furthermore, we hypothesized that if levels of ATP are reduced, levels of NADH will also be affected by the increased oxidation of GAPDH, Pfk and Pky in the sodA mutant with an increase in the levels of accumulated NAD+ (Fig 3). As shown in Fig 4B, the parental strain had the same levels of NAD+ in the presence or absence of MV, while the sodA mutant strain had significantly more NAD+ compared to the parental strain independent of the levels of oxidative stress. This accumulation of NAD+ is consistent with the observation that GAPDH—a critical enzyme involved in generating NADH from NAD+—presumably has reduced enzymatic functions due to oxidation of critical residues in its active site. We observed a reduction in the levels of NADH in the parental strain after treatment with MV (P < 0.01) as a potential outcome of oxidation of GAPDH in wild type strain (Fig 4C). Surprisingly, the levels of NADH in the sodA negative strain were significantly higher than those observed in the parental strain (P < 0.001) prior to treatment with MV. However, the levels of NADH in the sodA mutant after treatment with MV dropped to levels significantly lower than those observed in the untreated sample (P < 0.001) as well as that observed in the treated parental strain. These observations suggest that oxidative damage of key enzymes, notably GAPDH, involved in the glycolytic pathway, result in a significant reduction of the energy levels of the spirochetes and could translate into reduced survival capability of the sodA mutant in select microenvironments. The survival capabilities of sodA mutant of B. burgdorferi could be significantly compromised under conditions of increased oxidative stress due to a reduced energy flux and contribute to a defect in the colonization of the mouse model of Lyme disease [19].


Absence of sodA Increases the Levels of Oxidation of Key Metabolic Determinants of Borrelia burgdorferi.

Esteve-Gassent MD, Smith TC, Small CM, Thomas DP, Seshu J - PLoS ONE (2015)

Levels of ATP, NAD/NADH and GAPDH in parental and sodA mutant strains of B. burgdorferi.Soluble proteins from the parental (wt) and sodA mutant (mt) of B. burgdorferi before (-) and after (+) treatment with 20 mM MV were prepared for measuring the ATP levels using ATP-Bioluminescent Assay Kit as per manufacturer’s instructions and the luminescence was measured using Synergy HT (BioTek) (A). A similar procedure was adopted for the isolation of soluble proteins for measurement of NAD/NADH using Fluoro NAD/NADH kit (Cell Technology) (B and C). The data represented were from 4 separate experiments and statistical analysis was carried out using one-way ANOVA followed by the Bonferroni’s multiple comparison test. * Denotes P value < 0.05, ** P value < 0.01 and *** P value < 0.001. The soluble proteins were from the parental (wy) and the sodA mutant (mt) were isolated as described above before (-) and after (+) treatment with 20 mM MV and separated on SDS-12% PAGE gels and stained with Coommassie blue (D) or transferred to PVDF membrane, probed with anti-GAPDH serum and blots developed using Enhanced Chemiluminescence system (E). Molecular masses in kilodaltons are indicated to the left of panel D and E.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556403&req=5

pone.0136707.g004: Levels of ATP, NAD/NADH and GAPDH in parental and sodA mutant strains of B. burgdorferi.Soluble proteins from the parental (wt) and sodA mutant (mt) of B. burgdorferi before (-) and after (+) treatment with 20 mM MV were prepared for measuring the ATP levels using ATP-Bioluminescent Assay Kit as per manufacturer’s instructions and the luminescence was measured using Synergy HT (BioTek) (A). A similar procedure was adopted for the isolation of soluble proteins for measurement of NAD/NADH using Fluoro NAD/NADH kit (Cell Technology) (B and C). The data represented were from 4 separate experiments and statistical analysis was carried out using one-way ANOVA followed by the Bonferroni’s multiple comparison test. * Denotes P value < 0.05, ** P value < 0.01 and *** P value < 0.001. The soluble proteins were from the parental (wy) and the sodA mutant (mt) were isolated as described above before (-) and after (+) treatment with 20 mM MV and separated on SDS-12% PAGE gels and stained with Coommassie blue (D) or transferred to PVDF membrane, probed with anti-GAPDH serum and blots developed using Enhanced Chemiluminescence system (E). Molecular masses in kilodaltons are indicated to the left of panel D and E.
Mentions: GAPDH, Pfk and Pky are key enzymes of the glycolytic pathway (Fig 3). Oxidation of these proteins might alter their function and consequently, there is a possibility that the cellular levels of ATP could be reduced rendering the sodA mutant more susceptible under conditions of limited nutrient availability or in microenvironments with limited energy sources [49–54]. To test this hypothesis, we measured the levels of ATP before and after treatment with 20mM MV in the parental and sodA negative strains. As shown in Fig 4A, in the presence of MV, the levels of ATP in the sodA negative strain were significantly reduced when compared with those of the untreated sample (P < 0.001), and were also lower than the levels observed in the parental strain after treatment (P < 0.001). On the other hand, the ATP levels in the parental strain were not significantly reduced in the presence of stressor. Furthermore, we hypothesized that if levels of ATP are reduced, levels of NADH will also be affected by the increased oxidation of GAPDH, Pfk and Pky in the sodA mutant with an increase in the levels of accumulated NAD+ (Fig 3). As shown in Fig 4B, the parental strain had the same levels of NAD+ in the presence or absence of MV, while the sodA mutant strain had significantly more NAD+ compared to the parental strain independent of the levels of oxidative stress. This accumulation of NAD+ is consistent with the observation that GAPDH—a critical enzyme involved in generating NADH from NAD+—presumably has reduced enzymatic functions due to oxidation of critical residues in its active site. We observed a reduction in the levels of NADH in the parental strain after treatment with MV (P < 0.01) as a potential outcome of oxidation of GAPDH in wild type strain (Fig 4C). Surprisingly, the levels of NADH in the sodA negative strain were significantly higher than those observed in the parental strain (P < 0.001) prior to treatment with MV. However, the levels of NADH in the sodA mutant after treatment with MV dropped to levels significantly lower than those observed in the untreated sample (P < 0.001) as well as that observed in the treated parental strain. These observations suggest that oxidative damage of key enzymes, notably GAPDH, involved in the glycolytic pathway, result in a significant reduction of the energy levels of the spirochetes and could translate into reduced survival capability of the sodA mutant in select microenvironments. The survival capabilities of sodA mutant of B. burgdorferi could be significantly compromised under conditions of increased oxidative stress due to a reduced energy flux and contribute to a defect in the colonization of the mouse model of Lyme disease [19].

Bottom Line: Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway.Viable sodA mutant spirochetes could not be recovered from both gp91/phox-⁄- and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain.Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease.

View Article: PubMed Central - PubMed

Affiliation: South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX-78249, United States of America; Department of Biology, The University of Texas at San Antonio, San Antonio, TX-78249, United States of America.

ABSTRACT
Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA) capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE) and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat). HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348) exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560), and outer surface protein A (OspA, BBA15) were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC), Decorin binding protein A (DbpA), fibronectin binding protein (BBK32), RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox-⁄- and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease. This study, utilizing the sodA mutant, has provided insights into adaptive capabilities critical for survival of B. burgdorferi in its hosts.

No MeSH data available.


Related in: MedlinePlus