Redox-Sensing Under Hypochlorite Stress and Infection Conditions by the Rrf2-Family Repressor HypR inStaphylococcus aureus

Abstract

Aims: Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections, which requires efficient protection mechanisms to avoid destruction. Here, we have investigated the changes in the RNA-seq transcriptome by the strong oxidant sodium hypochlorite (NaOCl) in S. aureus USA300 to identify novel redox-sensing mechanisms that provide protection under infection conditions.Results: NaOCl stress caused an oxidative stress response in S. aureus as indicated by the induction of the PerR, QsrR, HrcA, and SigmaB regulons in the RNA-seq transcriptome. The hypR-merA (USA300HOU_0588-87) operon was most strongly upregulated under NaOCl stress, which encodes for the Rrf2-family regulator HypR and the pyridine nucleotide disulfide reductase MerA. We have characterized HypR as a novel redox-sensitive repressor that controls MerA expression and directly senses and responds to NaOCl and diamide stress via a thiol-based mechanism in S. aureus. Mutational analysis identified Cys33 and the conserved Cys99 as essential for NaOCl sensing, while Cys99 is also important for repressor activity of HypR in vivo. The redox-sensing mechanism of HypR involves Cys33-Cys99 intersubunit disulfide formation by NaOCl stress both in vitro and in vivo. Moreover, the HypR-controlled flavin disulfide reductase MerA was shown to protect S. aureus against NaOCl stress and increased survival in J774A.1 macrophage infection assays.Conclusion and Innovation: Here, we identified a new member of the widespread Rrf2 family as redox sensor of NaOCl stress in S. aureus that uses a thiol/disulfide switch to regulate defense mechanisms against the oxidative burst under infections in S. aureus. Antioxid. Redox Signal. 29, 615–636.

Staphylococcus aureususes the bacilliredoxin (BrxAB)/ bacillithiol disulfide reductase (YpdA) redox pathway to defend against oxidative stress under infections

“…About 2-4 fold increased transcription of ypdA, brxA and brxB was previously found under H 2O2, diamide, NaOCl, AGXX ® and after exposure to azurophilic granule proteins in S. aureus (Palazzolo-Ballance et al, 2008;Posada et al, 2014;Mäder et al, 2016;Loi et al, 2018a;. The elevated transcription of brxA, brxB and ypdA under disulfide stress correlated with the up-regulation of the bshA, bshB and bshC genes for bacillithiol biosynthesis in S. aureus and B. subtilis (Chi et al, 2011;Nicolas et al, 2012;Loi et al, 2018a;Loi et al, 2018b).…”

“…The co-regulation of BrxA/B and YpdA under disulfide stress points to their function in the same pathway in S. aureus. HOCl, diamide and AGXX ® were shown to cause a strong disulfide stress response in the transcriptome and protein S-bacillithiolation in the proteome of S. aureus (Imber et al, 2018a;Loi et al, 2018a;Loi et al, 2018b). Thus, the BrxA/B and YpdA redox enzymes are up-regulated under conditions of protein S-bacillithiolations, connecting their functions to the de-bacillithiolation pathway.…”

“…For cloning and genetic manipulation, Escherichia coli was cultivated in LB medium. For stress experiments, S. aureus COL wild type and mutant strains were cultivated in LB, RPMI or Belitsky minimal medium and exposed to the different compounds during the exponential growth as described previously (Loi et al, 2017;Loi et al, 2018b). Sodium hypochlorite, methylglyoxal, diamide, methylhydroquinone, dithiothreitol, cumene hydroperoxide (80% w/v), H2O2 (35% w/v), and monobromobimane were purchased from Sigma Aldrich.…”

“…Construction of S. aureus COL ypdA, brxAB and brxAB ypdA deletion mutants and complemented mutant strains. S. aureus COL ypdA (SACOL1520), brxA (SACOL1464) and brxB (SACOL1558) single deletion mutants as well as the brxA brxB double and brxA brxB ypdA triple mutants were constructed using pMAD as described (Arnaud et al, 2004;Loi et al, 2018b). Briefly, the 500 bp up-and downstream regions of ypdA, brxA and brxB were amplified using gene-specific primers (Table S3), fused by overlap extension PCR and ligated into the BglII and SalI sites of plasmid pMAD.…”

“…S. aureus has several defense mechanisms against ROS and HOCl, such as the MarR/SarA family redox regulators SarZ and MgrA, the HOCl-specific Rrf2-family regulator HypR and the peroxide-specific PerR repressor (Horsburgh et al, 2001;Chen et al, 2006;Chen et al, 2009;Chen et al, 2011;Hillion and Antelmann, 2015;Ji et al, 2015;Chandrangsu et al, 2018;Loi et al, 2018b). In addition, low molecular weight (LMW) thiols play important roles for survival and pathogenicity in bacterial pathogens (Loi et al, 2015;Tung et al, 2018).…”