CodY Regulates Thiol Peroxidase Expression as Part of the Pneumococcal Defense Mechanism against H2O2 Stress

Abstract

<i>Streptococcus pneumoniae</i> is a facultative anaerobic pathogen. Although it maintains fermentative metabolism, during aerobic growth pneumococci produce high levels of H₂O₂, which can have adverse effects on cell viability and DNA, and influence pneumococcal interaction with its host. The pneumococcus is unusual in its dealing with toxic reactive oxygen species (ROS) in that it neither has catalase nor the global regulators of peroxide stress resistance. Previously, we identified pneumococcal thiol peroxidase (TpxD) as the key enzyme for enzymatic removal of H₂O₂, and showed that TpxD synthesis is up-regulated upon exposure to H₂O₂. This study aimed to reveal the mechanism controlling TpxD expression under H₂O₂ stress. We hypothesize that H₂O₂ activates a transcription factor which in turn up-regulates <i>tpxD</i> expression. Microarray analysis revealed a pneumococcal global transcriptional response to H₂O₂. Mutation of <i>tpxD</i> abolished H₂O₂-mediated response to high H₂O₂ levels, signifying the need for an active TpxD under oxidative stress conditions. Bioinformatic tools, applied to search for a transcription factor modulating <i>tpxD</i> expression, pointed toward CodY as a potential candidate. Indeed, a putative 15-bp consensus CodY binding site was found in the proximal region of <i>tpxD-</i>coding sequence. Binding of CodY to this site was confirmed by EMSA, and genetic engineering techniques demonstrated that this site is essential for TpxD up-regulation under H₂O₂ stress. Furthermore, <i>tpxD</i> expression was reduced in a Δ<i>codY</i> mutant. These data indicate that CodY is an activator of <i>tpxD</i> expression, triggering its up-regulation under H₂O₂ stress. In addition we show that H₂O₂ specifically oxidizes the 2 CodY cysteines. This oxidation may trigger a conformational change in CodY, resulting in enhanced binding to DNA. A schematic model illustrating the contribution of TpxD and CodY to pneumococcal global transcriptional response to H₂O₂ is proposed.