Intracellular Metal Speciation in <i>Streptococcus sanguinis</i> Establishes SsaACB as Critical for Redox Maintenance

Abstract

<i>Streptococcus sanguinis</i> 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 <i>S. sanguinis</i>, 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 <i>S. sanguinis</i> and an SsaACB knockout strain (Δ<i>ssaACB</i>), indicating that SsaACB mediates both Mn and Fe uptake, directly influencing the metal-ion pools available for biological inorganic pathways. Mn supplementation of Δ<i>ssaACB</i> 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 <i>S. sanguinis</i> oxidative resistance, revealing a dynamic role for SsaACB in controlling redox homeostasis by managing the intracellular Fe/Mn composition and distribution.