Abstract
Staphylococcus aureus 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 S. aureus 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 S-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 S. aureus COL under oxidative stress and macrophage infection conditions in vivo and in biochemical assays in vitro. Using HPLC thiol metabolomics, a strongly enhanced BSSB level and a decreased BSH/BSSB ratio were measured in the S. aureus COL ΔypdA 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 (EBSH) upon recovery from oxidative stress. In addition, the ΔypdA 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 in vitro and inside murine J-774A.1 macrophages in infection assays in vivo. 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 S-bacillithiolated GapDH. In conclusion, our results point to a major role of the BrxA/BSH/YpdA pathway in BSH redox homeostasis in S. aureus during recovery from oxidative stress and under infections.
Highlights
Staphylococcus aureus is an important human pathogen, which can cause many diseases, ranging from local soft-tissue and wound infections to life-threatening systemic and chronic infections, such as endocarditis, septicaemia, bacteraemia, pneumonia or osteomyelitis (Archer, 1998; Lowy, 1998; Boucher and Corey, 2008)
The cysteine levels could be restored to wild type level in the ypdA complemented strain. These results indicate that bacillithiol disulfide reductase (YpdA) is important to maintain the reduced level of BSH under control and sodium hypochlorite (NaOCl) stress, supporting previous results (Mikheyeva et al, 2019), while the bacilliredoxins bacilliredoxin A/bacilliredoxin B (BrxA/B) are dispensible for the cellular BSH/bacillithiol disulfide (BSSB) redox balance during the growth and under oxidative stress in S. aureus
The putative disulfide reductase YpdA was previously shown to be phylogenetically associated with the BSH biosynthesis enzymes and bacilliredoxins (Supplementary Figure S2), providing evidence for a functional Brx/BSH/YpdA pathway in BSH-producing bacteria (Gaballa et al, 2010)
Summary
Staphylococcus aureus is an important human pathogen, which can cause many diseases, ranging from local soft-tissue and wound infections to life-threatening systemic and chronic infections, such as endocarditis, septicaemia, bacteraemia, pneumonia or osteomyelitis (Archer, 1998; Lowy, 1998; Boucher and Corey, 2008). During infections, activated macrophages and neutrophils produce reactive oxygen and chlorine species (ROS, RCS) in large quantities, including H2O2 and HOCl with the aim to kill invading pathogens (Winterbourn and Kettle, 2013; Hillion and Antelmann, 2015; Beavers and Skaar, 2016; Winterbourn et al, 2016). Many firmicutes utilize BSH as alternative LMW thiol (Figure 1A), which is essential for virulence of S. aureus in macrophage infection assays (Newton et al, 2012; Pöther et al, 2013; Posada et al, 2014; Chandrangsu et al, 2018). In S. aureus and Bacillus subtilis, BSH was characterized as cofactor of thiolS-transferases (e.g., FosB), glyoxalases, peroxidases, and other redox enzymes that are involved in detoxification of ROS, HOCl, methylglyoxal, toxins, and antibiotics (Chandrangsu et al, 2018). BSH participates in post-translational thiolmodifications under HOCl stress by formation of BSH mixed protein disulfides, termed as protein S-bacillithiolations (Chi et al, 2011, 2013; Imber et al, 2018a,c)
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