A role for glutathione in buffering excess intracellular copper inStreptococcus pyogenes

Abstract

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

The role of metal binding in the function of the human salivary antimicrobial peptide histatin-5

“…Two mechanisms are again plausible (Figure 4): (i) Hst5 binds extracellular Cu and suppresses entry of Cu into the GAS cytoplasm, leading to less Cu toxicity, or (ii) Hst5 binds intracellular Cu, allowing intracellular Cu levels to rise without significant toxicity. The latter would resemble the model described for GSH in binding (buffering) excess intracellular Cu 20 . Since Cu(II) is not thought to exist within the reducing cytoplasm, the first model is more likely.…”

“…The effects of Hst5 on growth of GAS were examined in a metal-deplete, chemically defined medium (CDM) 20 . In this medium, up to 50 µM Hst5 ( ca .…”

“…Further examination using a Cu-sensitive Δ copA mutant strain that lacks the Cu-effluxing P-type ATPase 20 (Figure 4) revealed no difference between the survival phenotype of this mutant strain and that of the wild-type in the presence of Hst5 (Figure 7C). There was, however, a clear difference in their growth phenotypes.…”

“…By contrast, it did not require the predicted Cu(I) binding sites 17 , since the effects of the ∆H7,8 and ∆H15,18,19 variants lacking either of the bis-His motifs (Table 1) were indistinguishable to that of the wild-type peptide (Figure 7B). Further examination using a Cu-sensitive ∆copA mutant strain that lacks the Cu-effluxing P-type ATPase 20 (Figure 4) revealed no difference between the survival phenotype of this mutant strain and that of the wild-type in the presence of Hst5 (Figure 7C). There was, however, a clear difference in their growth phenotypes.…”

Cu Homeostasis in Bacteria: The Ins and Outs

“…In contrast, a GSH-deficient E. coli strain shows no increase in Cu-sensitivity unless CopA is also deleted [ 158 ]. Studies using a S. pyogenes Δ copA strain show that mis-metallation in the presence of Cu is reduced when cells grow under GSH supplementation and that GSH competes with CopY for Cu when Cu concentration is high [ 213 ]. Thus, GSH that is present at millimolar concentrations inside the cell likely binds Cu primarily when the maximal capacity of Cu chaperones and exporters to bind Cu is consumed.…”