Mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in Streptococcus pyogenes.

All bacteria possess homeostastic mechanisms that control the availability of micronutrient metals within the cell. 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 Cu and the Zn and Mn homeostasis 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 growth in the presence of supplemental Cu promotes downregulation of genes that contribute to Zn or Mn uptake. This effect is not associated with changes in cellular Zn or Mn levels. Co-supplementation of the culture medium with Zn or, to a lesser extent, Mn alleviates key Cu stress phenotypes, namely bacterial growth and secretion of the fermentation end-product lactate. However, neither co-supplemental Zn nor Mn influences cellular Cu levels or Cu availability in Cu-stressed cells. In addition, we provide evidence that the Zn or Mn uptake transporters in GAS do not promote Cu uptake. Together, the results from this study strengthen and extend our previous proposal that mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in GAS.

Salivary Antimicrobial Peptide Histatin-5 Does Not Display Zn(II)-Dependent or -Independent Activity against Streptococci.

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.

Role of 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. 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.

Copper stress in Staphylococcus aureus leads to adaptive changes in central carbon metabolism.

Copper toxicity has been a long-term selection pressure on bacteria due to its presence in the environment and its use as an antimicrobial agent by grazing protozoa, by phagocytic cells of the immune system, and in man-made medical and commercial products. There is recent evidence that exposure to increased copper stress may have been a key driver in the evolution and spread of methicillin-resistant Staphylococcus aureus, a globally important pathogen that causes significant mortality and morbidity worldwide. Yet it is unclear how S. aureus physiology is affected by copper stress or how it adapts in order to be able to grow in the presence of excess copper. Here, we have determined quantitatively how S. aureus alters its proteome during growth under copper stress conditions, comparing this adaptive response in two different types of growth regime. We found that the adaptive response involves induction of the conserved copper detoxification system as well as induction of enzymes of central carbon metabolism, with only limited induction of proteins involved in the oxidative stress response. Further, we identified a protein that binds copper inside S. aureus cells when stressed by copper excess. This copper-binding enzyme, a glyceraldehyde-3-phosphate dehydrogenase essential for glycolysis, is inhibited by copper in vitro and inside S. aureus cells. Together, our data demonstrate that copper stress leads to the inhibition of glycolysis in S. aureus, and that the bacterium adapts to this stress by altering its central carbon utilisation pathways.

Handling of nutrient copper in the bacterial envelope.

In bacteria, copper (Cu) is often recognised for its potential toxicity and its antibacterial activity is now considered a key component of the mammalian innate immune system. Cu ions bound in weak sites can catalyse harmful redox reactions while Cu ions in strong but adventitious sites can disrupt protein or enzyme function. For these reasons, the outward transport of Cu from bacteria has received significant attention. Yet, Cu is also a bacterial nutrient, required as a cofactor by enzymes that catalyse electron transfer processes, for instance in aerobic and anaerobic respiration. To date, the inward flow of this metal ion as a nutrient and its insertion into target cuproenzymes remain poorly defined. Here we revisit the available evidence related to bacterial nutrient Cu trafficking and identify gaps in knowledge. Particularly intriguing is the evidence that bacterial cuproenzymes do not always require auxiliary metallochaperones to insert nutrient Cu into their active sites. This review outlines our effort to consolidate the available experimental data using an established energy-driven model for metalation.