GpoA Glutathione Peroxidase Links Oxidative Stress Response, Antibiotic Persistence, and Virulence in Streptococcus pneumoniae

Abstract

Glutathione (GSH) peroxidases are conserved enzymes found in prokaryotic and eukaryotic organisms that reduce H2O2 to protect cells from oxidative stress damage. In this study, we identified and characterized the GpoA glutathione peroxidase of S. pneumoniae, one of the most important human bacterial pathogens. This bacterium is unable to synthesize endogenous GSH as a cofactor for this enzyme but acquires GSH from host tissues via the GshT transporter. We demonstrated that recombinant GpoA exhibits GSH peroxidase activity and that the Cys36 residue is essential for this function. The gpoA transcripts, as well as tpxD and ahpD, which encode the TpxD thiol peroxidase and the AhpD alkylhydroperoxidase, respectively, were upregulated when pneumococci were exposed to H2O2. The{Delta} gpoA, {Delta}tpxD, {Delta}ahpD, and{Delta} gshT mutants exhibited increased susceptibility to H2O2, and also impaired intracellular survival in pneumocytes, macrophages, and neutrophils compared to the wild-type strain. These findings indicate that GpoA, TpxD, and AhpD constitute a robust H2O2 detoxification system that depends on extracellular GSH uptake. These three peroxidases also contribute to the fluoroquinolone persistence mechanism, which is closely associated with the oxidative stress response in S. pneumoniae. Additionally, we investigated the effect of GpoA on virulence in a murine model. The {Delta}gpoA mutant exhibited diminished survival across multiple organs relative to the wild-type strain, suggesting that GpoA contributes to pneumococcal pathogenesis. The molecular, biochemical, and functional analysis of GpoA elucidates an effective bacterial mechanism that incorporates the TpxD and AhpD peroxidases alongside the GshT transporter, promoting extracellular and intracellular survival under oxidative stress conditions.