Intracellular Metal Speciation in Streptococcus sanguinis Establishes SsaACB as Critical for Redox Maintenance.

Streptococcus sanguinis is an oral commensal bacterium, but it can colonize pre-existing heart valve vegetations if introduced into the bloodstream, leading to infective endocarditis. Loss of Mn- or Fe-cofactored virulence determinants are thought to result in weakening of this bacterium. Indeed, intracellular Mn accumulation mediated by the lipoprotein SsaB, a component of the SsaACB transporter complex, has been shown to promote virulence for endocarditis and O2 tolerance. To delineate intracellular metal-ion abundance and redox speciation within S. sanguinis, we developed a protocol exploiting two spectroscopic techniques, Inductively coupled plasma-optical emission spectrometry (ICP-OES) and electron paramagnetic resonance (EPR) spectroscopy, to respectively quantify total intracellular metal concentrations and directly measure redox speciation of Fe and Mn within intact whole-cell samples. Addition of the cell-permeable siderophore deferoxamine shifts the oxidation states of accessible Fe and Mn from reduced-to-oxidized, as verified by magnetic moment calculations, aiding in the characterization of intracellular metal pools and metal sequestration levels for Mn2+ and Fe. We have applied this methodology to S. sanguinis and an SsaACB knockout strain (ΔssaACB), indicating that SsaACB mediates both Mn and Fe uptake, directly influencing the metal-ion pools available for biological inorganic pathways. Mn supplementation of ΔssaACB returns total intracellular Mn to wild-type levels, but it does not restore wild-type redox speciation or distribution of metal cofactor availability for either Mn or Fe. Our results highlight the biochemical basis for S. sanguinis oxidative resistance, revealing a dynamic role for SsaACB in controlling redox homeostasis by managing the intracellular Fe/Mn composition and distribution.

Iron Redox Chemistry Promotes Antiparallel Oligomerization of α-Synuclein.

Brain metal dyshomeostasis and altered structural dynamics of the presynaptic protein α-synuclein (αS) are both implicated in the pathology of Parkinson's disease (PD), yet a mechanistic understanding of disease progression in the context of αS structure and metal interactions remains elusive. In this Communication, we detail the influence of iron, a prevalent redox-active brain biometal, on the aggregation propensity and secondary structure of N-terminally acetylated αS (NAcαS), the physiologically relevant form in humans. We demonstrate that under aerobic conditions, Fe(II) commits NAcαS to a PD-relevant oligomeric assembly, verified by the oligomer-selective A11 antibody, that does not have any parallel ß-sheet character but contains a substantial right-twisted antiparallel ß-sheet component based on CD analyses and descriptive deconvolution of the secondary structure. This NAcαS-FeII oligomer does not develop into the ß-sheet fibrils that have become hallmarks of PD, even after extended incubation, as verified by TEM imaging and the fibril-specific OC antibody. Thioflavin T (ThT), a fluorescent probe for ß-sheet fibril formation, also lacks coordination to this antiparallel conformer. We further show that this oligomeric state is not observed when O2 is excluded, indicating a role for iron(II)-mediated O2 chemistry in locking this dynamic protein into a conformation that may have physiological or pathological implications.