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

The family of human salivary histidine-rich peptides known as histatins bind zinc (Zn) and copper (Cu), but whether they contribute to nutritional immunity by influencing Zn and/or Cu availability has not been examined. We hypothesised that histatin-5 (Hst5) limits Zn availability (and promotes bacterial Zn starvation) and/or raises Cu availability (and promotes bacterial Cu poisoning). To test this hypothesis, Group A Streptococcus (GAS), which colonises the human oropharynx, was used as a model bacterium. Contrary to our hypothesis, Hst5 did not strongly influence Zn availability. This peptide did not induce expression of Zn uptake genes in GAS, nor did it suppress growth of an ΔadcAI mutant strain that is impaired in Zn uptake. Equilibrium competition measurements confirmed that Hst5 binds Zn weakly and does not compete with the high-affinity Zn uptake protein AdcAI for binding Zn. By contrast, Hst5 bound Cu with a high affinity and strongly influenced Cu availability. However, contrary to our hypothesis, Hst5 did not promote Cu toxicity. Instead, this peptide suppressed expression of Cu-inducible genes in GAS, stopped intracellular accumulation of Cu, and rescued growth of a ΔcopA mutant strain that is impaired in Cu efflux in the presence of added Cu. These findings led us to propose a new role for Hst5 and salivary histatins as major Cu buffers in saliva that reduce the potential negative effects of Cu exposure to microbes. We speculate that histatins promote oral and oropharyngeal health by contributing to microbial homeostasis in these host niches.

Histatin-5 (Hst5) is a member of the histatin superfamily of cationic, His-rich, Zn(II)-binding peptides in human saliva. Hst5 displays antimicrobial activity against fungal and bacterial pathogens, often in a Zn(II)-dependent manner. In contrast, here we showed that under in vitro conditions that are characteristic of human saliva, Hst5 does not kill seven streptococcal species that normally colonize the human oral cavity and oropharynx. We further showed that Zn(II) does not influence this outcome. We then hypothesized that Hst5 exerts more subtle effects on streptococci by modulating Zn(II) availability. We initially proposed that Hst5 contributes to nutritional immunity by limiting nutrient Zn(II) availability and promoting bacterial Zn(II) starvation. By examining the interactions between Hst5 and Streptococcus pyogenes as a model Streptococcus species, we showed that Hst5 does not influence the expression of Zn(II) uptake genes. In addition, Hst5 did not suppress growth of a ΔadcAI mutant strain that is impaired in Zn(II) uptake. These observations establish that Hst5 does not promote Zn(II) starvation. Biochemical examination of purified peptides further confirmed that Hst5 binds Zn(II) with high micromolar affinities and does not compete with the AdcAI high-affinity Zn(II) uptake protein for binding nutrient Zn(II). Instead, we showed that Hst5 weakly limits the availability of excess Zn(II) and suppresses Zn(II) toxicity to a ΔczcD mutant strain that is impaired in Zn(II) efflux. Altogether, our findings led us to reconsider the function of Hst5 as a salivary antimicrobial agent and the role of Zn(II) in Hst5 function.

All bacteria possess homeostastic mechanisms that control the availability of micronutrient metals within the cell. Regulatory cross-talks between different metal homeostasis pathways within the same bacterial organism have been reported widely. In addition, there have been previous suggestions that some metal uptake transporters can promote adventitious uptake of the wrong metal. This work describes the cross-talk between the Cu homeostasis pathway and the Zn, Mn, and Fe pathways in Group A Streptococcus (GAS). Using a ∆copA mutant strain that lacks the primary Cu efflux pump and thus traps excess Cu in the cytoplasm, we show that Cu stress leads to strong downregulation of Zn and Mn uptake genes, and mild downregulation of Fe uptake genes. This effect is associated with depletion of cellular Zn levels, but not those of Mn or Fe. Co-supplementation of the culture medium with Zn and, to a lesser extent, Mn, but not Fe alleviates key Cu stress phenotypes, namely bacterial growth and production of the fermentation end-product lactate. However, neither Zn nor Mn treatment influences cellular Cu levels or Cu availability in Cu-stressed cells. In addition, we show that the Zn and Mn uptake transporters in GAS do not promote Cu uptake. Taken together, the results strengthen and extend our previous proposal that mis-repression of Zn uptake genes and cellular Zn depletion are key mechanisms of Cu stress in GAS. By comparison, although Mn homeostasis in GAS is perturbed during Cu stress, the relationship between the two metals is yet to be defined.