EPR spectroscopy reveals antioxidant manganese defenses in the Lyme disease pathogen <i>Borrelia burgdorferi</i>

Oxidative stress defense in aerobic bacteria relies on Mn-superoxide dismutase (MnSOD) and antioxidant Mn-metabolite complexes (H-Mn) to quench superoxide radicals (O₂^•-). We investigated these antioxidant systems in <i>Borrelia burgdorferi</i>, the Mn-accumulating, Fe-independent Lyme disease pathogen. Using electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopies, we tracked Mn²⁺ partitioning between enzyme-bound (L-Mn) and metabolite-bound (H-Mn) pools in spirochetes at exponential and stationary phases. Results show that MnSOD neutralizes extracellular O₂^•- generated by γ-irradiation (a model for host immune attack); H-Mn neutralizes cytoplasmic O₂^•- and is a reservoir of labile Mn²⁺ for metalating Mn-dependent enzymes. MnCl₂ supplementation in log phase <i>B. burgdorferi</i> restored radioresistance in ΔMnSOD mutants via H-Mn hyperaccumulation but induced toxicity in older, stationary phase cells as metabolites became depleted. These findings support an expanded oxidative-stress model in which H-Mn complements MnSOD and positions Mn homeostasis as a therapeutic target. Our approach highlights the utility of EPR and ENDOR in studying Mn-dependent pathogens.IMPORTANCEWe employed electron paramagnetic resonance and electron-nuclear double resonance spectroscopies of Mn²⁺ in intact <i>Borrelia burgdorferi</i> supplemented with MnCl₂ to track changes in the amounts of enzyme-bound Mn and substitutionally labile, antioxidant Mn-metabolite complexes. We measured the spirochete's survivability to acute γ-irradiation, which simulates the respiratory burst of O₂^•- deployed as a critical weapon in the host's innate immune response. While Mn-superoxide dismutase (MnSOD) has classically been viewed as the main defense against oxidative damage in <i>B. burgdorferi</i>, our study demonstrates that antioxidant Mn²⁺ complexes with the metabolite components of H-Mn play a crucial antioxidant role, particularly when MnSOD is deficient. However, <i>B. burgdorferi's</i> inability to safely store excess Mn in metabolite-depleted cells highlights novel metabolic vulnerabilities that could be exploited for managing Lyme disease.