Glyoxalase III HchA confers protection against electrophiles and oxidative stress and contributes to the virulence in Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a major human pathogen, which has to cope with reactive electrophilic species (RES), such as the toxic α,β-dicarbonyl compound methylglyoxal during macrophage infection. The DJ-1/ThiJ/PfpI enzyme HchA has been previously structurally and functionally characterized as glyoxalase III and chaperone in vitro . In this work, we have used transcriptional studies, phenotype analyses and biochemical assays to study the expression, redox regulation, and function of HchA under various redox stress conditions and macrophage infection in S. aureus . Transcription of hchA was strongly upregulated by diamide, formaldehyde (FA), methylglyoxal (MG), and allicin stress, and less strongly by hypochlorous acid (HOCl) and H 2 O 2 in S. aureus . Phenotype analyses revealed an increased sensitivity of the Δ hchA mutant towards MG, FA, methylhydroquinone (MHQ), and HOCl stress, indicating that HchA is involved in the defense against electrophiles and oxidants, depending on the active site Cys190 in vivo . Moreover, the Δ hchA mutant was impaired in survival during infection of murine J774A.1 macrophages. Activity assays showed that HchA converts MG directly to lactate, confirming its function as glyoxalase III with similar kinetic parameters as HchA from Escherichia coli . Using mass spectrometry and PEG-maleimide alkylation assays, we further showed that Cys190 is highly sensitive to H 2 O 2 -induced overoxidation in vivo and in vitro . However, citrate synthase aggregation assays did not show any specific chaperone activity of reduced or oxidized HchA protein. Altogether, our data revealed that HchA functions as redox-sensitive glyoxalase III and provides resistance towards electrophiles, as well as during macrophage infections in S. aureus . IMPORTANCE S. aureus is a major human pathogen, which can cause life-threatening infections, such as septic shock, osteomyelitis, or pneumonia, especially in the hospital. During infections, S. aureus is exposed to an antimicrobial cocktail of redox-active compounds, generated by activated macrophages and neutrophils, with the aim to kill invading pathogens. Methylglyoxal was recently shown to accumulate as an antimicrobial factor during aerobic glycolysis in activated macrophages. Thus, we aimed to understand the defense mechanisms of S. aureus against methylglyoxal by investigating the function of the conserved glyoxalase III HchA. Our results revealed that HchA functions as redox-sensitive glyoxalase III, which protects against electrophiles and oxidants and contributes to the virulence of S. aureus . Moreover, the active site Cys190 of HchA was found to be sensitive to H 2 O 2 -induced overoxidation, leading to inactivation of its glyoxalase function. Thus, the discovery of drugs inhibiting HchA activity could be promising strategies to combat S. aureus infections.