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.
Highlights
Borrelia burgdorferi, the causative agent of Lyme disease is transmitted to humans by the bite of infected Ixodes spp. ticks [1]
These observations demonstrated that oxidation of key residues of cytosolic proteins are increased in the absence of superoxide dismutase gene (sodA) and that these changes could lead to alterations in the survival capabilities of B. burgdorferi in the presence of oxidative stressors
The ability of B. burgdorferi to survive in the mammalian host requires a coordinated regulation of expression of a variety of determinants to overcome the deleterious effects of the adaptive immune system, and mechanisms to survive and colonize in microenvironments doi:10.1371/journal.pone.0136707.g005
Summary
The causative agent of Lyme disease is transmitted to humans by the bite of infected Ixodes spp. ticks [1]. We previously reported that the inactivation of the gene encoding superoxide dismutase A (sodA) in B. burgdorferi resulted in a mutant strain that could not be re-isolated from infected tissues following intradermal needle inoculation in C3H/HeN mice at 21 days post-infection [19] Additional studies employing these strains have expanded the significance of sodA within the context of borrelial physiology While the sodA mutant did not have a significant growth defect under in vitro growth conditions, we analyzed the contributions of the levels of oxidation and synthesis of select borrelial proteins that contributed to reduced survival capabilities of the sodA mutant under in vivo conditions
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