Staphylococcus aureus Uses the Bacilliredoxin (BrxAB)/Bacillithiol Disulfide Reductase (YpdA) Redox Pathway to Defend Against Oxidative Stress Under Infections

Abstract

<i>Staphylococcus aureus</i> is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections. The low molecular weight thiol bacillithiol (BSH) is an important defense mechanism of <i>S. aureus</i> for detoxification of ROS and HOCl stress to maintain the reduced state of the cytoplasm. Under HOCl stress, BSH forms mixed disulfides with proteins, termed as <i>S</i>-bacillithiolations, which are reduced by bacilliredoxins (BrxA and BrxB). The NADPH-dependent flavin disulfide reductase YpdA is phylogenetically associated with the BSH synthesis and BrxA/B enzymes and was recently suggested to function as BSSB reductase (Mikheyeva et al., 2019). Here, we investigated the role of the complete bacilliredoxin BrxAB/BSH/YpdA pathway in <i>S. aureus</i> COL under oxidative stress and macrophage infection conditions <i>in vivo</i> and in biochemical assays <i>in vitro</i>. Using HPLC thiol metabolomics, a strongly enhanced BSSB level and a decreased BSH/BSSB ratio were measured in the <i>S. aureus</i> COL Δ<i>ypdA</i> deletion mutant under control and NaOCl stress. Monitoring the oxidation degree (OxD) of the Brx-roGFP2 biosensor revealed that YpdA is required for regeneration of the reduced BSH redox potential (<i>E</i> _BSH) upon recovery from oxidative stress. In addition, the Δ<i>ypdA</i> mutant was impaired in H₂O₂ detoxification as measured with the novel H₂O₂-specific Tpx-roGFP2 biosensor. Phenotype analyses further showed that BrxA and YpdA are required for survival under NaOCl and H₂O₂ stress <i>in vitro</i> and inside murine J-774A.1 macrophages in infection assays <i>in vivo</i>. Finally, NADPH-coupled electron transfer assays provide evidence for the function of YpdA in BSSB reduction, which depends on the conserved Cys14 residue. YpdA acts together with BrxA and BSH in de-bacillithiolation of <i>S</i>-bacillithiolated GapDH. In conclusion, our results point to a major role of the BrxA/BSH/YpdA pathway in BSH redox homeostasis in <i>S. aureus</i> during recovery from oxidative stress and under infections.