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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

Immunoblot analysis of oxidized soluble proteins of parental and sodA mutant strains of B. burgdorferi.Soluble proteins from the parental strain (wt) and sodA mutant (mt) were obtained before (Control) and after treatment with 20 mM MV (Treated) and subjected to two-dimensional gel electrophoresis after derivatization with dinitrophenylhydrazine (DNPH) as described in the material and methods. The proteins were transferred to PVDF membranes and probed with anti-DNP antiserum and blots developed using Enhanced Chemiluminescence Plus Detection reagents. Lines and numbers represent the protein spots oxidized in the sodA mutant compared with the parental strain. Spots were excised from the SYPRO stained gels as shown in Fig 1 and analyzed by MALDI-TOF MS. Molecular mass markers (MW) are represented on the left side of each gel in kilodaltons, OxyBlot Protein standard was used in this assay (Chemicon International, Inc.). Representative blots are presented in this figure.
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pone.0136707.g002: Immunoblot analysis of oxidized soluble proteins of parental and sodA mutant strains of B. burgdorferi.Soluble proteins from the parental strain (wt) and sodA mutant (mt) were obtained before (Control) and after treatment with 20 mM MV (Treated) and subjected to two-dimensional gel electrophoresis after derivatization with dinitrophenylhydrazine (DNPH) as described in the material and methods. The proteins were transferred to PVDF membranes and probed with anti-DNP antiserum and blots developed using Enhanced Chemiluminescence Plus Detection reagents. Lines and numbers represent the protein spots oxidized in the sodA mutant compared with the parental strain. Spots were excised from the SYPRO stained gels as shown in Fig 1 and analyzed by MALDI-TOF MS. Molecular mass markers (MW) are represented on the left side of each gel in kilodaltons, OxyBlot Protein standard was used in this assay (Chemicon International, Inc.). Representative blots are presented in this figure.

Mentions: We hypothesized that lack of sodA will impact the functions of cytosolic proteins critical for multiple metabolic pathways of B. burgdorferi. Therefore, we exposed the parental and mutant strains to methyl viologen and determined the identity of soluble borrelial proteins that were oxidized using anti-DNP antibodies following derivatization of oxidized proteins using dinitrophenylhydrazine (DNPH) [36]. As shown in Fig 1, there were no significant differences in the levels of proteins stained with SYPRO Ruby between the parental and mutant strains before and after treatment with methyl viologen. On the other hand, as shown in Fig 2, immunoblot analysis using anti-DNP antibodies showed a collection of proteins with significant levels of oxidation in the sodA deficient strain compared to the parental control strain. Furthermore, even in the absence of MV treatment, the sodA mutant exhibited increased levels of oxidation in select proteins that were not observed in the parental strain. Five proteins that were preferentially oxidized in the sodA mutant were identified by mass spectrometry (Table 1). Three of the five identified proteins [glyceraldehyde 6-phosphate dehydrogenase (BB0057, GAPDH), pyrophophate phospho fructo kinase (BB0020, Pfk) and pyruvate kinase (BB0348, Pky)] are part of the glycolytic pathway (Fig 3). One heat shock protein (hsp90, HtpG, BB0590) involved in protein folding and the outer surface protein A (OspA, BBA15) known to play a key role in the attachment of the spirochetes to the tick midgut were also observed to have increased levels of oxidation in the sodA negative mutant [48]. We also analyzed the putative target residues of the enzymes that were identified by mass spectrophotometric analysis and determined that the active sites of several of these enzymes had residues that could serve as targets of oxidation (Table 2). These observations demonstrated that oxidation of key residues of cytosolic proteins are increased in the absence of sodA and that these changes could lead to alterations in the survival capabilities of B. burgdorferi in the presence of oxidative stressors. We focused on identifying the effects of oxidation of soluble, cytosolic proteins on the central metabolic pathways of B. burgdorferi although determination of levels of oxidation of membrane proteins may provide additional information on the phenotype of the sodA mutant. In addition, the availability of specific antibody reagents to major lipoproteins of B. burgdorferi also allowed us to determine if there are significant changes in the levels of expression of select borrelial proteins as described below.


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)

Immunoblot analysis of oxidized soluble proteins of parental and sodA mutant strains of B. burgdorferi.Soluble proteins from the parental strain (wt) and sodA mutant (mt) were obtained before (Control) and after treatment with 20 mM MV (Treated) and subjected to two-dimensional gel electrophoresis after derivatization with dinitrophenylhydrazine (DNPH) as described in the material and methods. The proteins were transferred to PVDF membranes and probed with anti-DNP antiserum and blots developed using Enhanced Chemiluminescence Plus Detection reagents. Lines and numbers represent the protein spots oxidized in the sodA mutant compared with the parental strain. Spots were excised from the SYPRO stained gels as shown in Fig 1 and analyzed by MALDI-TOF MS. Molecular mass markers (MW) are represented on the left side of each gel in kilodaltons, OxyBlot Protein standard was used in this assay (Chemicon International, Inc.). Representative blots are presented in this figure.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556403&req=5

pone.0136707.g002: Immunoblot analysis of oxidized soluble proteins of parental and sodA mutant strains of B. burgdorferi.Soluble proteins from the parental strain (wt) and sodA mutant (mt) were obtained before (Control) and after treatment with 20 mM MV (Treated) and subjected to two-dimensional gel electrophoresis after derivatization with dinitrophenylhydrazine (DNPH) as described in the material and methods. The proteins were transferred to PVDF membranes and probed with anti-DNP antiserum and blots developed using Enhanced Chemiluminescence Plus Detection reagents. Lines and numbers represent the protein spots oxidized in the sodA mutant compared with the parental strain. Spots were excised from the SYPRO stained gels as shown in Fig 1 and analyzed by MALDI-TOF MS. Molecular mass markers (MW) are represented on the left side of each gel in kilodaltons, OxyBlot Protein standard was used in this assay (Chemicon International, Inc.). Representative blots are presented in this figure.
Mentions: We hypothesized that lack of sodA will impact the functions of cytosolic proteins critical for multiple metabolic pathways of B. burgdorferi. Therefore, we exposed the parental and mutant strains to methyl viologen and determined the identity of soluble borrelial proteins that were oxidized using anti-DNP antibodies following derivatization of oxidized proteins using dinitrophenylhydrazine (DNPH) [36]. As shown in Fig 1, there were no significant differences in the levels of proteins stained with SYPRO Ruby between the parental and mutant strains before and after treatment with methyl viologen. On the other hand, as shown in Fig 2, immunoblot analysis using anti-DNP antibodies showed a collection of proteins with significant levels of oxidation in the sodA deficient strain compared to the parental control strain. Furthermore, even in the absence of MV treatment, the sodA mutant exhibited increased levels of oxidation in select proteins that were not observed in the parental strain. Five proteins that were preferentially oxidized in the sodA mutant were identified by mass spectrometry (Table 1). Three of the five identified proteins [glyceraldehyde 6-phosphate dehydrogenase (BB0057, GAPDH), pyrophophate phospho fructo kinase (BB0020, Pfk) and pyruvate kinase (BB0348, Pky)] are part of the glycolytic pathway (Fig 3). One heat shock protein (hsp90, HtpG, BB0590) involved in protein folding and the outer surface protein A (OspA, BBA15) known to play a key role in the attachment of the spirochetes to the tick midgut were also observed to have increased levels of oxidation in the sodA negative mutant [48]. We also analyzed the putative target residues of the enzymes that were identified by mass spectrophotometric analysis and determined that the active sites of several of these enzymes had residues that could serve as targets of oxidation (Table 2). These observations demonstrated that oxidation of key residues of cytosolic proteins are increased in the absence of sodA and that these changes could lead to alterations in the survival capabilities of B. burgdorferi in the presence of oxidative stressors. We focused on identifying the effects of oxidation of soluble, cytosolic proteins on the central metabolic pathways of B. burgdorferi although determination of levels of oxidation of membrane proteins may provide additional information on the phenotype of the sodA mutant. In addition, the availability of specific antibody reagents to major lipoproteins of B. burgdorferi also allowed us to determine if there are significant changes in the levels of expression of select borrelial proteins as described below.

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