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. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, 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 noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations 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. These results indicate that GSH buffers 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 and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion. IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been 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 has long been 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 physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. 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.

Pathogenic Staphylococcus aureus respond to copper stress by altering central carbon metabolism in response to a specific inhibition of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase.

The insertion of copper into bacterial cuproenzymes in vivo does not always require a copper-binding metallochaperone – why?

27 28 Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To 29 limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally-responsive system 30 for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (Group A 31 Streptococcus, GAS), this system is encoded by the copYAZ operon. In this study, we 32 demonstrate that the site of GAS infection in vivo represents a Cu-rich environment but 33 inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of 34 invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including 35 defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde 3-36 phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as 37 a consequence of mismetalation of non-cognate metal-binding sites. Surprisingly, the onset of 38 these effects is delayed by ~4 h even though expression of copZ is upregulated immediately 39 upon exposure to Cu. We further show that the onset of all phenotypes coincides with 40 depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH 41 replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu 42 treatment. Our results indicate that GSH contributes to buffering of excess intracellular Cu 43 when the transcriptionally-responsive Cu export system is overwhelmed. Thus, while the 44 copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance that 45 allows bacteria to maintain metabolism even in the presence of an excess of this metal ion. 46This study advances fundamental understanding of Cu handling in the bacterial cytoplasm. 47