A role for glutathione in buffering excess intracellular copper in Streptococcus pyogenes

Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally-responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (Group A Streptococcus, GAS), this system is encoded by the copYAZ operon. In this study, we demonstrate that the site of GAS infection in vivo represents a Cu-rich environment but inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde 3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of non-cognate metal-binding sites. Surprisingly, the onset of these effects is delayed by [~]4 h even though expression of copZ is upregulated immediately upon exposure to Cu. We further show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. Our results indicate that GSH contributes to buffering of excess intracellular Cu when the transcriptionally-responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance that allows bacteria to maintain metabolism even in the presence of an excess of this metal ion. This study advances fundamental understanding of Cu handling in the bacterial cytoplasm. IMPORTANCEThe control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it is established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and is long assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this role of glutathione beyond measurement of the effect of Cu on growth of glutathione-deficient mutants. Our work with Group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.