YpdA, a putative bacillithiol disulfide reductase, contributes to cellular redox homeostasis and virulence in Staphylococcus aureus

Abstract

Summary The intracellular redox environment of Staphylococcus aureus is mainly buffered by bacillithiol (BSH), a low molecular weight thiol. The identity of enzymes responsible for the recycling of oxidized bacillithiol disulfide (BSSB) to the reduced form (BSH) remains elusive. We examined YpdA, a putative bacillithiol reductase, for its role in maintaining intracellular redox homeostasis. The ypdA mutant showed increased levels of BSSB and a lower bacillithiol redox ratio vs. the isogenic parent, indicating a higher level of oxidative stress within the bacterial cytosol. We showed that YpdA consumed NAD(P)H; and YpdA protein levels were augmented in response to stress. Wild type strains overexpressing YpdA showed increased tolerance to oxidants and electrophilic agents. Importantly, YpdA overexpression in the parental strain caused an increase in BSH levels accompanied by a decrease in BSSB concentration in the presence of stress, resulting in an increase in bacillithiol redox ratio vs. the vector control. Additionally, the ypdA mutant exhibited decreased survival in human neutrophils (PMNs) as compared with the parent, while YpdA overexpression protected the resulting strain from oxidative stress in vitro and from killing by human neutrophils ex vivo. Taken together, these data present a new role for YpdA in S. aureus physiology and virulence through the bacillithiol system.

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

“…Phylogenomic profiling of protein interaction networks using EMBL STRING search has suggested the flavoenzyme YpdA (SACOL1520) as putative NADPH-dependent BSSB reductase (Supplementary Figure S1), since YpdA co-occurs together with BrxA/B and the BSH biosynthesis enzymes (BshA/B/C) only in BSH-producing bacteria, such as B. subtilis and S. aureus (Supplementary Figure S2; Gaballa et al, 2010). While our work was in progress, a recent study provides first evidence for the function of YpdA as putative BSSB reductase in S. aureus in vivo since an increased BSSB level and a decreased BSH/BSSB ratio was measured in the Δ ypdA mutant under control and H 2 O 2 stress conditions (Mikheyeva et al, 2019). YpdA overproduction was shown to increase the BSH level and contributes to oxidative stress resistance, fitness, and virulence of S. aureus (Mikheyeva et al, 2019).…”

“…While our work was in progress, a recent study provides first evidence for the function of YpdA as putative BSSB reductase in S. aureus in vivo since an increased BSSB level and a decreased BSH/BSSB ratio was measured in the Δ ypdA mutant under control and H 2 O 2 stress conditions (Mikheyeva et al, 2019). YpdA overproduction was shown to increase the BSH level and contributes to oxidative stress resistance, fitness, and virulence of S. aureus (Mikheyeva et al, 2019). However, biochemical evidence for the function of YpdA as BSSB reductase and the association of YpdA to the BrxA/B enzymes have not been demonstrated in B. subtilis or S. aureus .…”

“…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 .…”

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

“…The BSSB reductase activity of YpdA was shown to be dependent on the conserved Cys14, which is located in the glycine-rich Rossmann-fold NAD(P)H binding domain (GGGPC14G) (Bragg et al, 1997;Mikheyeva et al, 2019). Cys14 might be S-bacillithiolated by BSSB and reduced by electron transfer from NADPH via the FAD co-factor.…”

The Disulfide Stress Response and Protein S-thioallylation Caused by Allicin and Diallyl Polysulfanes in Bacillus subtilis as Revealed by Transcriptomics and Proteomics

“…Similarly, as shown for HOCl and diamide stress, allicin and DAS4 resulted in up-regulation of regulons controlled by the redox-sensitive regulators HypR YodB, CatR and HxlR in B. subtilis (Figure 2A,B, Tables S1 and S2). The MarR/DUF24-family regulators HypR, YodB, and CatR were shown to sense HOCl, quinones, and diamide via conserved N-terminal Cys residues, while HxlR is more specific to control aldehydes detoxification [45,47,[53][54][55][56]. Among the genes of the HypR, YodB, and CatR regulons, hypR, hypO, azoR1, yodC, and the catDE operon were most highly induced under allicin and DAS4 with log2-fold changes of 3.2-7.9 (Tables S1 and S2).…”