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

Abstract

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 O 2 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 Mn 2+ 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.

Time-course analysis ofStreptococcus sanguinisafter manganese depletion reveals changes in glycolytic, nucleotide, and redox metabolites

“…This transporter and orthologs in related species have been shown to be important for manganese (Mn) transport and virulence (Colomer-Winter et al, 2018;Crump et al, 2014;Dintilhac et al, 1997;Kehl-Fie et al, 2013). Previous studies utilizing a Δ ssaACB strain of S. sanguinis revealed that this mutant is significantly deficient in cellular Mn levels (Murgas et al, 2020) and virulence in our rabbit model of IE (Baker et al, 2019). These studies also suggested that the reduced virulence of Mn-deficient cells is due to growth arrest in the aerobic, low-Mn environment characteristic of an aortic valve infection, implying the existence of one or more Mn-dependent metabolic pathways that are essential for aerobic growth.…”

Manganese depletion leads to multisystem changes in the transcriptome of the opportunistic pathogenStreptococcus sanguinis

“…All plates were incubated for 24 h at 37°C under anaerobic conditions, where atmospheric composition was adjusted using a programmable Anoxomat™ Mark II jar-filling system (AIG, Inc.) and a palladium catalyst was included in the jars. The Δ ssaACB::aphA-3 mutation was nonpolar, as confirmed by complementation (Murgas et al, 2020). All mutants were confirmed to have the expected composition by sequence analysis of the DNA flanking the insertion sites.…”

“…1, cellular Zn levels in Δ ssaACB were not significantly altered by EDTA addition, despite the high affinity of this chelator for Zn (Perrin and Dempsey, 1974). Zn level maintenance may be due to the higher levels of Zn than Mn in BHI (1.7 ± 0.02 vs. 0.02 ± 0.003 µg ml -1 , respectively) (Murgas et al, 2020) or the regulation of Zn transporter genes. In S. pneumoniae, transcription of the operon encoding the Zn ABC transporter AdcCBA (Dintilhac and Claverys, 1997) is repressed by a Zndependent, MarR-family regulator called AdcR (Shafeeq et al, 2011;Manzoor et al, 2015).…”

“…3). This is unsurprising, as levels of Mg in BHI are very high (15.0 ± 1.5 µg ml -1 ) (Murgas et al, 2020), and EDTA has a lower affinity for Mg than many other metals (8.7 log β 1 for Mg vs. 14.1 log β 1 for Mn) (Perrin and Dempsey, 1974). For reasons that are unclear, expression of genes for two other putative Mg transporters, mgtE and mgtB (Groisman et al, 2013), was significantly upregulated post-EDTA (Fig.…”

Manganese Depletion Leads to Multisystem Changes in the Transcriptome of the Opportunistic Pathogen Streptococcus sanguinis

“…Previous work from our lab established that the ABC transporter SsaACB is important for Mn transport and essential for virulence in a rabbit model of IE (Crump et al, 2014;Baker et al, 2019;Murgas et al, 2020). In S. sanguinis, Mn acts as a cofactor for superoxide dismutase (SodA) (Parker and Blake, 1988;Poyart et al, 1988) and the aerobic class 1b ribonucleotide reductase (NrdEF) (Makhlynets et al, 2014;Rhodes et al, 2014).…”