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Peek Zoo's holding an animal parade to celebrate the birth of its baby panda, but when the day arrives, nothing's ready. As Mr. Peek races around preparing, he leaves a trail of chaos. Thank goodness Mr. Peek's son Jimmy is there to save the day and ensure the crowd gets the best animal parade ever.

Copper is an essential micronutrient for most organisms that is required as a cofactor for crucial copper-dependent enzymes encoded by both prokaryotes and eukaryotes. Evidence accumulated over several decades has shown that copper plays important roles in the function of the mammalian immune system. Copper accumulates at sites of infection, including the gastrointestinal and respiratory tracts and in blood and urine, and its antibacterial toxicity is directly leveraged by phagocytic cells to kill pathogens. Copper-deficient animals are more susceptible to infection, whereas those fed copper-rich diets are more resistant. As a result, copper resistance genes are important virulence factors for bacterial pathogens, enabling them to detoxify the copper insult while maintaining copper supply to their essential cuproenzymes. Here, we describe the accumulated evidence for the varied roles of copper in the mammalian response to infections, demonstrating that this metal has numerous direct and indirect effects on immune function. We further illustrate the multifaceted response of pathogenic bacteria to the elevated copper concentrations that they experience when invading the host, describing both conserved and species-specific adaptations to copper toxicity. Together, these observations demonstrate the roles of copper at the host–pathogen interface and illustrate why bacterial copper detoxification systems can be viable targets for the future development of novel antibiotic drug development programs.

Staphylococcus aureus is a devastating mammalian pathogen for which the development of new therapeutic approaches is urgently needed due to the prevalence of antibiotic resistance. During infection pathogens must overcome the dual threats of host-imposed manganese starvation, termed nutritional immunity, and the oxidative burst of immune cells. These defenses function synergistically, as host-imposed manganese starvation reduces activity of the manganese-dependent enzyme superoxide dismutase (SOD). S. aureus expresses two SODs, denoted SodA and SodM. While all staphylococci possess SodA, SodM is unique to S. aureus, but the advantage that S. aureus gains by expressing two apparently manganese-dependent SODs is unknown. Surprisingly, loss of both SODs renders S. aureus more sensitive to host-imposed manganese starvation, suggesting a role for these proteins in overcoming nutritional immunity. In this study, we have elucidated the respective contributions of SodA and SodM to resisting oxidative stress and nutritional immunity. These analyses revealed that SodA is important for resisting oxidative stress and for disease development when manganese is abundant, while SodM is important under manganese-deplete conditions. In vitro analysis demonstrated that SodA is strictly manganese-dependent whereas SodM is in fact cambialistic, possessing equal enzymatic activity when loaded with manganese or iron. Cumulatively, these studies provide a mechanistic rationale for the acquisition of a second superoxide dismutase by S. aureus and demonstrate an important contribution of cambialistic SODs to bacterial pathogenesis. Furthermore, they also suggest a new mechanism for resisting manganese starvation, namely populating manganese-utilizing enzymes with iron.

Key Points More than 25% of proteins are thought to need metals, such as zinc, iron, copper, cobalt, nickel, manganese, magnesium and calcium. Proteins tend to bind metals such as copper and zinc tightly, but bind metals such as manganese, magnesium and calcium weakly, and for essential divalent cations the order of affinity is defined by the Irving–Williams stability series. Some non-essential metals, such as cadmium and mercury, can also be highly competitive. The cell must supply sufficient atoms of each metal to satisfy the demands of proteins that require the element and must also act to keep the tight-binding metals out of the binding sites of proteins that require weaker-binding metals. Mechanisms by which cells meet this challenge to correctly populate metalloproteins have been proposed, although to date few studies have explicitly set out to test them. By restricting the numbers of metal atoms within the cytoplasm, it is presumed that rather than metals competing with other metals for a limited pool of protein, each protein competes with other proteins for a limited pool of metal. Under these conditions, metal occupancy is determined by the relative metal affinities of the different proteins rather than their absolute affinities. However, this requires precise control over the numbers of atoms of each metal and molecules of the respective metalloproteins. A balance between the actions of importers and exporters for each metal controls how many atoms accumulate in the cell. The catalogue of metal transporters includes ATP-binding cassette-type ATPases, P 1 -type ATPases, RND (resistance and nodulation), CDF (cation diffusion facilitator) and NiCoT (Ni and Co transporter) proteins, CorA (Co resistance), NRAMP (natural resistance associated with macrophage protein) and ZIP (Zrt/Irt-like protein)-family transporters. The number of protein binding sites for each metal can be adjusted to match metal supply; for example, by switching from a protein that requires iron to one that uses copper when it becomes available or iron becomes limiting. The synthesis of storage proteins, such as metallothioneins for zinc or ferritins for ferric iron, sequesters surplus metal atoms to restrain them from other binding sites. Expression of genes that encode metal transporters and storage proteins is generally controlled by metal sensors, including two-component histidine kinases and response regulators plus seven known families of soluble DNA-binding, metal-binding transcriptional regulators (Fur, DtxR, NikR, MerR, ArsR–SmtB, CsoR–RcnR and CopY). The metal affinities of these proteins can determine the boundaries between metal sufficiency and metal excess or deficiency for each element. These affinities become increasingly tight on moving up the Irving–Williams series, such that the metals at the top of the series must be bound and buffered to extremely low concentrations. Some metals are passed to the correct metalloproteins by dedicated delivery pathways that involve metallochaperones, in which case the specificity of a protein–protein interaction can ensure that only the correct proteins acquire the metal. Metal discrimination by the proteins of metal homeostasis is especially important if these proteins influence metal occupancy of other metalloproteins. Analysis of nickel specificity by the DNA-binding repressor NmtR from Mycobacterium tuberculosis revealed that the discernment of metals by metal sensors can be determined by the sensors' allosteric mechanism and/or its access to metal, which is predicted to be a function of the relative metal affinities of the cells complement of metal sensors. Metalloproteins constitute up to one-third of the total cellular cohort of proteins, and cells have evolved elaborate mechanisms for scavenging and storing metal atoms. In this Review, the authors summarize the homeostatic mechanisms by which bacteria and archaea ensure that metalloproteins receive and bind the correct metal. Protein metal-coordination sites are richly varied and exquisitely attuned to their inorganic partners, yet many metalloproteins still select the wrong metals when presented with mixtures of elements. Cells have evolved elaborate mechanisms to scavenge for sufficient metal atoms to meet their needs and to adjust their needs to match supply. Metal sensors, transporters and stores have often been discovered as metal-resistance determinants, but it is emerging that they perform a broader role in microbial physiology: they allow cells to overcome inadequate protein metal affinities to populate large numbers of metalloproteins with the right metals.

Almost half of all enzymes must associate with a particular metal to function. An ambition is to understand why each metal–protein partnership arose and how it is maintained. Metal availability provides part of the explanation, and has changed over geological time and varies between habitats but is held within vital limits in cells. Such homeostasis needs metal sensors, and there is an ongoing search to discover the metal-sensing mechanisms. For metalloproteins to acquire the right metals, metal sensors must correctly distinguish between the inorganic elements.

ABSTRACT One of the current aims of synthetic biology is the development of novel microorganisms that can mine economically important elements from the environment or remediate toxic waste compounds. Copper, in particular, is a high-priority target for bioremediation owing to its extensive use in the food, metal and electronic industries and its resulting common presence as an environmental pollutant. Even though microbe-aided copper biomining is a mature technology, its application to waste treatment and remediation of contaminated sites still requires further research and development. Crucially, any engineered copper-remediating chassis must survive in copper-rich environments and adapt to copper toxicity; they also require bespoke adaptations to specifically extract copper and safely accumulate it as a human-recoverable deposit to enable biorecycling. Here, we review current strategies in copper bioremediation, biomining and biorecycling, as well as strategies that extant bacteria use to enhance copper tolerance, accumulation and mineralization in the native environment. By describing the existing toolbox of copper homeostasis proteins from naturally occurring bacteria, we show how these modular systems can be exploited through synthetic biology to enhance the properties of engineered microbes for biotechnological copper recovery applications. A review of current technologies in bacterial bioremediation, biorecycling and bioleaching, of copper homeostasis strategies used by bacteria, and how these could be exploited through synthetic biology for bioremediation.

Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis . These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1–2) × 10 17  M −1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria.

The increasing worldwide trend of antibiotic-resistant Neisseria gonorrhoeae strains highlights the urgent need for new therapeutic strategies against this sexually transmitted pathogen, including a gonococcal vaccine. We previously designed a bioinformatics-based candidate selection pipeline (CASS) and identified potential novel gonococcal vaccine targets among hypothetical proteins expressed during natural human infection. One of these candidates, NGO1701, is a predicted periplasmic four-helix bundle protein with amino acid sequence homology to the copper storage protein 1 (Csp1) from Methylosinus trichosporium OB3b. In this study, we confirmed that purified NGO1701 binds 15 Cu(I) ions per monomer in vitro , supporting its function as Csp in N. gonorrhoeae . Using a ngo1701 deletion mutant generated in N. gonorrhoeae F62, we investigated its role in bacteria physiology. We showed that ablation of Csp was not limiting for bacterial growth and fitness in vitro , but the Δ csp strain became significantly more susceptible to copper mediated toxicity. This phenotype was rescued by csp gene complementation, indicating a role in protection against copper toxicity. Our results indicate that Csp participates in periplasmic copper homeostasis in N. gonorrhoeae, buffering excess copper to reduce toxicity and playing a putative role in copper delivery to important copper-enzymes. Csp does not appear to be involved in bacterial host cell interaction and activation in vitro , since no difference in the ability of N. gonorrhoeae to adhere/invade epithelial cells or induce IL-8 secretion was reported among wild type, csp deletion mutant and complemented strains. Furthermore, sera from mice immunized with NGO1701 failed to recognize Δ csp by dot blot and ELISA, and the sera’s ability to kill N. gonorrhoeae was abrogated against Δ csp . However, both functions were restored after gene complementation, supporting the relevance of Csp as a potential vaccine target. Allelic analysis of Neisseria species revealed that this gene is absent in N. meningitidis , thus making it a gonococcal-specific target.

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 (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.

Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal’s redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity. Many metalloenzymes are highly specific for their cognate metal ion but the molecular principles underlying this specificity often remain unclear. Here, the authors characterize the structural and biochemical basis for the different metal specificity of two evolutionarily related superoxide dismutases.