Microbial pathogens and strategies for combating them: science, technology and education (A. Mndez-Vilas, Ed.

)
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Mechanism of action and applications of the antimicrobial properties of

copper
J. Stevenson1, A. Barwinska-Sendra1, E. Tarrant1 and K. J. Waldron1
1
Institute for Cell & Molecular Biosciences, Medical School, Newcastle University, Framlington Place, Newcastle upon
Tyne, NE2 4HH, United Kingdom

Copper is an essential micronutrient for most living organisms, serving as a redox-active cofactor for important enzymes
such as the terminal respiratory oxidases and superoxide dismutase. However, in excess copper can be extremely toxic,
due in part to this same redox-activity by which copper ions can catalyse the production of deleterious reactive oxygen
species (ROS), and also due to its propensity to form extremely stable complexes with cellular components.
In recent decades, much has been learned about how organisms acquire, handle, distribute, store, sense and export copper
ions, collectively termed copper homeostasis. Yet, despite this progress in our understanding of the molecular bases of
copper homeostasis, the molecular mechanisms by which unregulated concentrations of copper ions kill cells remain
largely unknown. This question has gained importance in recent years with the recognition that copper, both as metal salts
and as solid metal surfaces, is an attractive antimicrobial. In a world of decreasing efficacy of traditional antibiotics,
together with the recognition that prevention of infection is better than cure, such antimicrobial substances and 'self-
sanitising' surfaces hold much appeal. As with any new antimicrobial, it is important that its mechanism of action is
defined prior to widespread adoption, primarily to assess the risk of spontaneous resistance emerging amongst the 'wild'
microbial population.
In this review, we first summarise our current understanding of microbial copper homeostasis, then review what is known
of the mechanisms by which copper kills cells. Finally, we look at the current and potential applications of copper toxicity,
in both the medical field and commercial activities.

Keywords copper; copper homeostasis; copper toxicity; antimicrobial copper

1. Introduction
All living organisms require a complement of essential metal ions, which act as co-factors that enable enzymes to
catalyse a wider range of chemical transformations than would be achievable using solely organic catalysts. The precise
metal requirements of organisms vary between species, between environmental niches, between metabolic states and
circadian rhythms, and have varied over evolutionary timescales due to changes in geological, marine and atmospheric
chemical conditions.
Among the most common essential metals (magnesium, calcium, manganese, iron, copper and zinc), copper is one of
the least abundant in biology and yet is of crucial importance, playing a central role in two of the most fundamental
metabolic pathways on Earth; as a co-factor in the electron transfer protein plastocyanin in oxygenic photosynthesis,
and in the terminal aerobic respiratory oxidases such as cytochrome c oxidase. Most organisms require trace quantities
of copper for one or more cellular enzymes (see section 3), and even those that apparently have no enzymatic copper
requirement generally possess homeostatic systems for the detoxification of copper ions (see section 4). This latter
observation is a result of the hazardous nature of copper ions to biological systems. The chemical properties of copper
(see section 2) are such that excess copper ions can cause cytotoxicity by a number of hypothetical mechanisms (see
section 5), and thus all cells go to great lengths in order to limit the number of atoms of free copper (i.e. copper bound
to no ligands other than water) present to avoid such deleterious reactions and interactions.

2. The chemistry of copper

Under physiological conditions two oxidation states of copper are accessible, the cuprous form Cu(I) and the cupric
form Cu(II), and the ability to cycle between these oxidation states is the chemical property behind its recruitment by
life to enable redox reactions. The Cu(II)/Cu(I) redox couple has a standard redox potential (E0) of 0.16 V, but this
potential is readily tunable by the local chemical environment of the copper ion, through specific second coordination
sphere interactions, through conformational restriction to minimise reorganisation energy, and by solvent protection
(dielectric) effects, thereby allowing the redox potential of copper co-factors in proteins to range from -0.2 to +1.0 V
[1,2].
The oxidised cupric ion is a soft metal according to the Pearson definition, which has a d9 outer shell electronic
configuration, with one unpaired electron, making the ion paramagnetic, whereas the reduced cuprous ion is a
borderline hardness ion which has a d10 closed outer shell, and is thus diamagnetic. The redox activity of copper
makes it a powerful catalyst of Fenton and Haber-Weiss-type reactions, which lead to the generation of the extremely

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reactive hydroxyl radical from less dangerous reactive oxygen species (ROS) such as hydrogen peroxide. The hydroxyl
radical, in turn, is able to react with a wide range of biological molecules, including DNA, causing cellular damage [3].
Such oxidative mechanisms may be important in copper toxicity (see section 6).
The stabilities of metal-ligand complexes follow regular trends across the periodic table. For the essential metal ions,
the trend in stabilities is defined by the Irving-Williams series [4] which states that, in general, a given ligand will form
increasingly stable octahedral complexes (i.e. lower dissociation constant, Kd) with the divalent cation of each metal as
we move from left to right across the row of the periodic table. For the essential metal ions, therefore, the trend in
stability is as follows:

Mg(II) < Ca(II) < Mn(II) < Fe(II) < Co(II) < Ni(II) < Cu(II) > Zn(II)

Note that the exception to the linear trend is Cu(II), which actually forms more stable complexes with its ligands than
Zn(II). This is due to the d9 electronic configuration of the Cu(II) cation, which leads to a tetragonal distortion of
octahedral Cu(II) complexes called the Jahn-Teller effect, giving Cu(II) complexes increased stability relative to those
of Zn(II). Further, it should be noted that this series considers only divalent cations; though not originally compared in
the studies of Irving and Williams, the Cu(I) cation also forms exceptionally stable complexes, especially with soft
ligands such as sulphur.
One further chemical property of the Cu(I) ion creates technical difficulties for researchers, but has played an
interesting role in the biological use of copper over evolutionary time. The Cu(I) ion is highly unstable in aqueous,
aerobic conditions, both due to its propensity to undergo disproportionation reactions, yielding Cu(II) and solid copper,
and due to the insoluble nature of Cu(I) oxide and hydroxide salts. This requires anaerobic conditions to be maintained
when working with solutions of Cu(I), and great care must be taken to ensure that the copper present in the solution is
maintained in the cuprous form. As a result of this insolubility, copper would have been relatively biounavailable on the
early Earth when reducing, sulphidic conditions prevailed [5,6], and organisms likely focused on the use of iron,
abundant under these same conditions, for redox activity of metalloenzymes. However, the oxidation of the atmosphere
~2.4 Gyr ago by the evolution of ancestral cyanobacteria using oxygenic photosynthesis [6,7] would have profoundly
altered this availability, with iron in the oceans precipitating as Fe(III) hydroxides concomitant with a liberation of
Cu(II) ions from rocks. It has been suggested that during this period, there must have been a strong evolutionary
advantage to organisms that could reduce their over-reliance on the now difficult to obtain iron by replacing some redox
functions with copper-requiring enzymes [6]. Conversely, the requirement for systems to defend the cell against copper
toxicity would have simultaneously increased.

3. The usage of copper by biological systems

Copper is used almost exclusively by oxidoreductase enzymes [8], exploiting coppers redox activity. Remarkably,
there are only approximately 20 distinct types of known copper-requiring enzymes in biology, and only about half of
these are represented in prokaryotic genomes [9,10]. In addition, at least 14 different protein families are known to be
involved in bacterial copper homeostasis [9,11]. Taken together, a genomes complement of these protein families
constitutes that organisms cuproproteome, which varies between organisms, and can be studied using bioinformatic
analyses of genomic data [9-12].
Among the bacterial genomes studied, the majority (72% users) possess at least one copper-requiring enzyme and are
thus defined as copper users, and this number rises to 94% amongst exclusively aerobic organisms [9]. The most
commonly occurring cuproenzymes among bacteria are cytochrome oxidase (91% of copper users), followed by NADH
dehydrogenase-2 (34%) and Cu,Zn-superoxide dismutase (20%) [9]. The pre-eminence of the cytochrome oxidase
family is perhaps not surprising, given its central role in respiratory energy generation by pumping protons across
biological membranes (coupled to the reduction of oxygen to water) to generate an electrochemical gradient for
subsequent use by ATP synthase. NADH dehydrogenase-2 is also an important enzyme complex in the respiratory
chain, though the role of copper in this enzyme is unclear. Superoxide dismutase (SOD) is an enzyme involved in the
cellular defence against oxidative stress, which decomposes the reactive oxygen intermediate superoxide anion (O2-) to
water and hydrogen peroxide (H2O2). The copper-containing form of this enzyme is not related by sequence to either
the Mn/Fe-dependent or the Ni-dependent SOD superfamilies. Importantly, bacterial homologues of Cu,Zn-superoxide
dismutase are exclusively localised to the periplasmic space of Gram negative bacteria (Figure 1) or are secreted by
Gram positive bacteria [13,14], in contrast to the cytosolic SOD1 of eukaryotes (Figure 2). Other bacterial uses of
copper co-factors are in numerous multicopper oxidases (e.g. laccases), copper-dependent amine oxidases, blue copper
electron transfer proteins (e.g. plastocyanin), particulate methane monooxygenase (pMMO), nitrite reductase and
nitrous oxide reductase, each of which are present in fewer genomes.
It is worthy of note that the majority of organisms, including those that apparently have no requirement for copper
(i.e. their genome does not encode any known copper-dependent enzymes), do possess one or more copper efflux
protein, suggesting that copper toxicity is a near-universal selection pressure, especially among organisms that live an
aerobic lifestyle [9]. In multiple bioinformatics studies fewer than 30% of organisms possessed genomes devoid of any

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of the known copper homeostasis systems, and those that did were predominantly anaerobic or host-associated
organisms, suggesting they may make use of the hosts copper homeostasis machinery [9,11]. By far the most abundant
copper homeostasis system is the P-type ATPase CopA (see section 5.2), which was found to be present in 80-85% of
all bacterial genomes [9,11].

4. Microbial copper homeostasis

The majority of bacteria require trace quantities of copper for essential cuproenzymes, but the chemical properties of
copper make it toxic in excess. As a result, cells have evolved complex homeostatic systems to carefully regulate the
intracellular copper concentration to allow sufficient metal supply to cuproenzymes while minimising deleterious
effects. In fact, the copper-binding affinities of copper homeostasis proteins from numerous model organisms suggests
that the cytosolic free copper concentration is several orders of magnitude less than one atom of copper per cell [15],
meaning that every copper ion within the cell must be tightly bound to a cellular macromolecule. In this way, the cell
buffers the free copper concentration to a level which prevents its aberrant association with non-copper
metalloenzymes, from which it will be difficult to remove it due to the extreme stability of copper-complexes, and
prevents its uncontrolled redox cycling in the cytosol.
All known bacterial copper-dependent enzymes are localised outside of the cytosol, suggesting that there may be no
need for copper ions to enter bacterial cells (see section 5.1). Conversely, eukaryotic cells have copper-dependent
enzymes in the cytosol, the mitochondria, and even the nucleus, and therefore require intracellular trafficking systems to
ensure its safe delivery to the targets (Figure 2). This section will review the known components of copper homeostasis
systems, with a focus on bacterial systems; however, some aspects of eukaryotic copper homeostasis will also be
introduced, as these are important with respect to fungal pathogens and to how pathogens interact with their mammalian
hosts.

4.1. Copper import

The mechanisms by which copper ions enter bacterial cells are largely unknown. Indeed the absence of cytosolic
copper-dependent enzymes makes it theoretically possible that all bacterial copper enzymes, localised to the
cytoplasmic membrane or periplasmic space, acquire their co-factor directly from exogenous sources. In eukaryotic
systems, the Ctr family of membrane transporters act to import copper [16], yet there are no Ctr homologues found in
bacterial genomes, suggesting it evolved after the divergence of eukaryotes.
Several bacterial copper import systems have been postulated, though all are controversial and await biochemical
verification. For more than a decade, a sub-class of P-type ATPases were postulated to act to import copper across the
cytoplasmic membrane [17,18], despite the fact that the orientation of these pumps was modelled as being identical to
those members of the same family, often within the same cell, that were proposed to be canonical efflux pumps. Such a
proposition presented biochemical difficulties, but was based on the simplest interpretation of the phenotypes of
deletion mutant strains, where increased copper resistance and decreased cellular accumulation of copper were observed
[17,18]. More recent in vitro biochemical analysis has clarified that this sub-group of P-type ATPases, like all others,
act to efflux copper [19,20] (see section 4.2).
In Pseudomonas the CopCD system has also been implicated in copper import [21]. CopC is a Cu(I)-and Cu(II)-
binding periplasmic protein [22] which is thought to provide copper to the integral membrane protein CopD [21]. Only
expression of both of these proteins enabled copper accumulation in Pseudomonas [21]. A CopCD homologue from
Bacillus subtilis, called YcnJ, has recently been suggested to be involved in copper import [23]. However, neither the
CopCD nor the YcnJ proteins have been characterised biochemically yet, making it difficult to determine their role as
importers at this stage. It is feasible that copper may access the bacterial cytosol solely through hijacking of import
systems intended for metals other than copper, such as zinc transporters.
An unusual copper import system has been identified in one class of bacteria, namely the methanotrophic -
proteobacteria. These organisms synthesise and secrete a low molecular weight copper-binding molecule,
methanobactin, which binds Cu(I) with high affinity and is proposed to act as a chalkophore [24] analogous to
siderophores for iron acquisition. The active internalisation of copper-methanobactin complexes has recently been
observed [25], consistent with the model that high affinity copper binding by this modified peptide aids in copper
acquisition by these bacteria, which have an unusually high demand for copper as a co-factor in particulate methane
monooxygenase (pMMO), the first enzyme in the methane metabolic pathway [26]. This internalised copper is
presumably liberated from the methanobactin complex and is subsequently made available for provision to pMMO [27].
However, methanobactin-like molecules have not been identified from any bacteria other than the methanotrophic -
proteobacteria, suggesting that this unusual system may be unique to these organisms.

4.2. Copper export

The most common mechanism by which bacteria detoxify excess copper ions is through efflux, and by far the most
abundant copper efflux systems are members of the family of PIB-type ATPases, usually designated CopA [28]. These

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enzymes use energy derived from ATP to translocate Cu(I) ions against a concentration gradient across the cytoplasmic
membrane [28,29], releasing the cations either into the extracellular milieu, in Gram positive bacteria, into the
periplasmic space, in Gram negative bacteria, or, in a smaller subset of the family, directly to putative receptor
cuproenzymes [14,19,30] (see figure 1). In some organisms, the CopA-exported Cu(I) ions are further detoxified in the
periplasmic space by the action of a multicopper oxidase (CueO in E. coli, CuiD in S. enterica) [31,32], which oxidises
the copper to the less hazardous Cu(II). Deletion of the copA gene commonly gives rise to copper-sensitive phenotypes
and decreased cellular copper accumulation [28,29,33,34]. Usually the copA gene is regulated in a copper-dependent
manner [18,28,29,33] at the transcriptional level by one of several families of copper-sensing transcriptional regulators
(see section 4.4).
CopA enzymes possess eight transmembrane helices and, in common with all P-type ATPases, feature an actuator
(A) domain, a nucleotide-binding (N) domain, and a phosphorylation (P) domain on the cytoplasmic side of the pump
[35]. However, they have additional features that are unique to the members of the family that transport soft metal
substrates, namely a membrane-embedded CPX motif proposed to coordinate the substrate [35], and one or more
soluble, N-terminal metal-binding domains. These N-terminal domains adopt a ferredoxin-like fold,
homologous to the structures of the soluble Atx1-like metallochaperones (see section 4.3), with which they have been
shown to interact in vitro and in vivo [36-38]. The structural similarities and their inherent interacting properties lead to
the conclusion that this interaction must be for copper transfer; an elegant model was developed in which
metallochaperones scavenge and sequester intracellular Cu(I) ions and then transfer them to the CopA domain via
specific protein-protein interaction followed by a series of ligand exchange reactions [36,38]. However, it has
subsequently been shown that the N-terminal domains are not essential; deletion of this domain or modification of the
cysteine residues leads to minimal loss of copper tolerance [39]. Furthermore, the thermodynamic gradient for vectorial
transfer of copper from the metallochaperone to the N-terminal domain of the ATPase is extremely shallow in vitro
[38], and the intermolecular interaction between the two proteins is remarkable stable, long-lived enough in fact for the
structure of the complex to be solved using nuclear magnetic resonance (NMR) spectroscopy [37]. As a result of these
observations, the current model for CopA function suggests a regulatory role of the N-terminal soluble domains
whereby their copper-dependent interaction with the metallochaperone signals the abundance of copper to the pump,
and relieves inhibition of the ATPase effected by the positioning of the N-terminal domain, exposing the throat of the
pump [19]. The location of this domain in a recent crystal structure of Legionella pneumophila CopA is inconclusive,
but this structure did identify a platform region [35] that interacts with the metallochaperone through complementary
electrostatic charges, and it is this interaction that is proposed to be critical for copper supply to the pump [40].
As discussed above, a sub-group of the family of the Cu(I)-transporting ATPases was, for a time, postulated to act as
importers (see section 5.1), a model that has now been rejected based on biochemical data. These studies have,
nonetheless, demonstrated an important functional difference between this sub-group and their canonical colleagues; a
significantly slower kinetic rate [19]. It has been postulated that this slower pump rate is related to the function of
members of this group in supply of copper ions to copper-dependent apo-proteins on the extracellular face of the
membrane [20], leaving the copper detoxification function to the pumps that exhibit a higher rate. Importantly, recent
evidence from a number of organisms have demonstrated a role for copper-exporting P-type ATPases in direct copper-
supply to extracellular bacterial copper-enzymes [14,19,30]. One important consequence of such a model is that, despite
the apparent lack of cytosolic copper-dependent enzymes in bacteria, it would imply that copper must initially enter the
cytoplasm as part of the pathway to supply extracellular enzymes.
Many Gram negative bacteria also possess a second copper efflux system, designated Cus in Escherichia coli in
which it has been best characterised [32,41,42]. This is a multipartite system consisting of a large multi-protein
complex, CusCBA, which spans the entire periplasmic space and integrates into both the cytoplasmic and the outer
membrane. A further component, CusF, is a soluble periplasmic protein which binds Cu(I) through an unusual Cu(I)-
interaction [43], and provides copper to the CusCBA transporter for export [44]. The Cus system drives the efflux of
periplasmic Cu(I) across the outer membrane, and is therefore especially important under anaerobic growth conditions
[32], whereby the activity of periplasmic multicopper oxidases (e.g. CueO/CuiD) is limited by the absence of oxygen,
and therefore the action of CopA would otherwise lead to accumulation of Cu(I) in the periplasm.
It is worth noting that the closest homologue of the E. coli Cus system in Salmonella, designated GesCBA, has not
been experimentally linked to copper resistance, but instead to resistance to Au(I) ions [45]. This was previously also
the case for one of the two Salmonella P-type ATPases, designated GolT [46], despite the seemingly unlikely selection
pressure for gold-specific resistance mechanisms among enterobacteria. Recent studies, crucially using minimal (as
opposed to rich) growth medium with minimal metal-chelating capacity, has demonstrated that the GolT P-type ATPase
is indeed important for copper resistance, and not gold resistance, in Salmonella [33]. For these reasons, we predict that
the GesABC system, studied under these and probably also anaerobic growth conditions, is likely to play a role in
Salmonella copper homeostasis analogous to the Cus system in E. coli (see figure 1).

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Fig. 1 Schematic illustration of the copper homeostasis systems of a model Gram negative organism (Salmonella enterica sv.
Typhimurium, left) and a model Gram positive organism (Staphylococcus aureus ATCC 12600, right). Note, in both Salmonella and
S. aureus two copper transporting P-type ATPases are present (CopA and GolT/CopB) in addition to their cognate metallochaperone
(GolB/CopZ). In addition, Salmonella possesses dual copper sensors (CueR and GolS) and the GesCBA system which is analogous
to the E. coli CusCBA system.

4.3. Intracellular copper trafficking and sequestration

Once inside cells, copper must be bound to macromolecules to prevent aberrant reactions, but must be supplied safely to
copper-requiring enzymes. In most eukaryotes, copper sequestration is primarily achieved by metallothioneins, small,
soluble, cysteine-rich proteins that bind multiple Cu(I) ions (though in most cases, Zn(II) or Cd(II) ions) with high
affinity [47,48]. But copper-sequestering metallothioneins are rare amongst bacteria [49]. More generally, intracellular
copper sequestration and trafficking is achieved by metallochaperones. These are small, soluble proteins that bind Cu(I)
with high affinity and generally participate in specific protein-protein interactions with their target partner protein. In
eukaryotes, two such metallochaperones are known; yeast Atx1 (human Atox1) shuttles copper to the trans Golgi
network (TGN), where it interacts with the yeast Ccc2 (human ATP7A or ATP7B) P-type ATPase for provision of
copper to the secretory pathway [38], and Ccs1 supplies copper to the Cu,Zn-superoxide dismutase (Sod1) localised
primarily in the cytosol [50] (see figure 2). Common to both Atx1 and Ccs1 is small domain containing a Cu(I)-binding
Cys-X-X-Cys motif within a ferredoxin-like fold, structurally related to the N-terminal metal-binding domains of the
P1B-type ATPases with which they interact.
Atx1-like metallochaperones are also encoded in many bacterial genomes, where they are usually named CopZ. The
copZ gene is often encoded within a copper-regulated operon, adjacent to the copA gene [29], indicative of linked
functions. As with eukaryotic Atx1, CopZ proteins are generally small (usually ~60 amino acids), soluble proteins with
a Cu(I)-binding Cys-X-X-Cys motif within a ferredoxin-like fold [37], and they have been shown to interact with their
respective CopA P-type ATPases in vivo and in vitro [37,38]. Indeed, in the absence of bacterial cytosolic copper-
dependent enzymes, almost all known bacterial copper metallochaperones in the cytosol interact solely with the
exporter, suggesting that their primary role in bacteria is to sequester excess cytosolic copper ions and to safely provide
them to the pump for efflux. Yet deletion of copZ genes rarely give rise to severe copper-sensitive phenotypes
[34,51,52,53] as might be expected if their copper sequestration provides a survival advantage by preventing dangerous
side-reactions. It is possible that in the absence of metallochaperones, excess low molecular weight thiols such as
glutathione, which have a significant affinity for copper ions in vitro [54], can play this protective role.
Recently, a new class of copper metallochaperones has been described that are distinct from those of the Atx1/CopZ
family. The Streptococcus pneumoniae protein CupA is a member of the cupredoxin fold family, binds Cu(I) with high
affinity [55] and plays an important role in copper resistance in this bacterium [55,56]. CupA was shown to form a
specific protein-protein interaction with the soluble, N-terminal domain of its cognate partner P-type ATPase, CopA, a
domain which also adopts a cupredoxin fold, and is able to transfer copper to this target in vitro consistent with a
metallochaperone function [55]. Crucially CupA is an integral membrane protein, and this membrane association is
essential to its role in copper detoxification [55]. Bioinformatics suggests that this CupA-like cupredoxin domain
chaperone and CopA N-terminal domain are only present in organisms that lack a traditional Atx1/CopZ-like system.
Although the Cup-type system appears to be widely distributed, it is possible that this membrane-based chaperone

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system is particularly crucial for organisms, like streptococci, that produce high levels of cytosolic ROS as part of their
metabolism [55].
Two periplasmic proteins have also been assigned roles as metallochaperones, in that they sequester periplasmic
copper and transfer it to a cognate partner. CueP from Salmonella (see figure 1) plays a critical role in loading of the
periplasmic SodCII with its copper cofactor, using copper supplied by the efflux P-type ATPases [14]. In addition, the
CusF protein sequesters copper in the periplasm of E. coli and transfers it to the multipartite Cus efflux system for
export [44].

4.4. Copper sensing

In most bacteria the genes encoding copper detoxification systems are transcriptionally regulated in a copper-dependent
manner, ensuring that efflux systems are synthesised only when copper is in excess [14,29]. The sensor that an
organism utilises to sense copper levels varies between organisms. In E. coli and Salmonella, as with most Gram
negative bacteria, a Cu(I)-sensing member of the MerR family, CueR, acts as a copper-dependent activator of gene
expression [15,33]. CueR associates with the sub-optimally spaced operator/promoter sequence that controls expression
of the cop operon, thereby regulating copA [15,33]. Binding of Cu(I) induces a CueR conformational change which
underwinds the bound DNA, enabling transcription from the now optimally-spaced operator/promoter [15]. In Gram
positive bacteria, the sensor is usually a copper-dependent de-repressor, either CsoR or CopY, which dissociates from
its operator sequence when Cu(I) binds [57,58]. Additionally, a two-component system, CusRS, is also present in some
Gram negative bacteria where it is usually a regulator of the expression of the Cus system in response to periplasmic
copper levels [41]. Most organisms possess at least one of these sensors, and many organisms that possess multiple
copper efflux systems also possess multiple copper sensors (Figure 1) [33].
The fact that these sensors detect and respond to the cytosolic copper concentration, and respond by activating
expression of the copper efflux systems, places them at the apex of the copper detoxification hierarchy. As such, the
measured affinities of these sensors for binding of Cu(I), determined to be in the 1018 1021 M-1 range [15,58], has
significance for copper homeostasis. This concentration of free copper equates to several orders of magnitude less
than one atom per bacterial cell, implying that there is no free copper in the cytosol and that all copper must be tightly
bound and buffered [15]. It is also worthy of note that many copper sensors are autoregulatory, suggesting that their
own ability to tightly chelate and sequester copper ions may play a role in detoxification of excess copper ions [33].

5. Copper toxicity mechanisms

Despite the requirement of nearly all organisms for trace quantities of copper, above certain concentrations it causes
toxicity. The preceding section illustrates the lengths that cells go to in order to minimise copper accumulation beyond
their copper requirements to avoid deleterious effects. Toxicity for the purposes of this discussion is defined as a
concentration of exogenous copper that results in lower rates of cellular proliferation than occur in its absence.
Presumably this represents a phase of accumulating intracellular copper when the rate of influx exceeds the rate of
efflux, even with high-level expression of the efflux systems described above. High levels of toxicity may cause cell
death and therefore result in a null-growth phenotype, but this is not the only possible outcome of toxicity as cells are
often able to divide at lower rates in relatively concentrated copper solutions, and to recover upon its removal.
Metal toxicity has been traditionally under-studied, merely representing a confounding factor in studies of
homeostasis through its pleiotropic effects on metabolism. These pleiotropic effects make the study of these
mechanisms technically challenging. Moreover, copper can be toxic both in solution and as a solid surface, and the way
in which these forms of toxicity work may vary. Both of these are relevant for antimicrobial purposes and will be
explored here. Three distinct molecular mechanisms of copper ion toxicity have been proposed, and it is likely that all
play a role in the physiology of copper toxicity, though perhaps to different extents. A goal of current research aims to
determine which cellular molecules associate with excess copper, in which order these associations occur, and to
understand how these associations give rise to their effects on replication, metabolism, and ultimately on cell viability.

5.1. Redox activity generates reactive oxygen species

As we have seen, the redox activity of the copper ion enables redox activity of copper-dependent enzymes. While this is
the reason why copper ions have been recruited by evolution, this activity can also give rise to deleterious effects.
Copper ions are highly efficient catalysts of Haber-Weiss and Fenton chemistry [59], generating the extremely toxic
hydroxyl radical that can damage a range of cellular macromolecules, including causing mutations in DNA [3]. This has
been proposed to be a key mechanism of copper toxicity, yet the evidence for such copper-catalysed ROS generation in
vivo is scarce [60]. In E. coli the excess copper ions accumulated under toxicity conditions are almost exclusively
localised to the periplasmic space, potentially protecting DNA from ROS-induced damage, and making periplasmic
antioxidant systems especially important [60]. In fact copper toxicity is often greater under anaerobic conditions
[60,61], suggesting that any copper-dependent ROS-generating mechanism is less important than has been previously
assumed in toxicity.

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Under normal conditions, it is thought that there is no free copper in the cytosol of any organism, with all copper ions
tightly bound to macromolecules. This presumably limits the ability of the copper ion to undergo redox cycling and thus
to generate ROS. Only under extreme copper conditions, in which the rate of copper influx exceeds the rate of efflux,
will cytosolic copper accumulate. However, even under these conditions it seems unlikely that the cytosols massive
over-capacity to chelate copper will ever be overcome to lead to accumulation of free cytosolic copper ions. For
example, most organisms synthesise millimolar concentrations of low molecular weight thiol-containing compounds
(most commonly glutathione, but also mycothiol and bacillithiol) that maintain the reducing conditions of the cytosol
[62]. Glutathione has a high affinity for Cu(I) which it stabilises relative to Cu(II) thus suppressing redox activity [54],
and so it seems likely that other toxic effects would be observed long before the cellular reduced glutathione pool
became saturated with copper ions, though toxic effects mediated by depletion of the reduced glutathione pool through
copper chelation, thereby preventing normal redox balance and ROS scavenging, cannot be ruled out [62].
Alternatively, ROS generation may not necessarily involve direct redox activity of free copper ions, but instead through
adventitious association of copper ions with enzymes of the respiratory chain, leading to unproductive side-reactions of
those enzymes.

5.2. Copper binding to proteins

The chemical properties of copper enable it to form exceptionally stable complexes with ligands. This gives rise to the
possibility that toxicity may be caused by non-specific binding of excess copper ions to non-copper proteins, which may
in turn inactivate essential cellular functions. This may involve binding to metalloproteins that require metals other than
copper, with the stability of the copper complex making it difficult for cellular quality control systems to replace the
bound copper with the correct metal ion. The presence of any cytosolic copper ions is likely to create difficulties for the
cell in correctly populating, for example, zinc-proteins with zinc. Alternatively it may involve binding of copper to non-
metalloproteins at surface-exposed sites.
One such example has recently been demonstrated in E. coli, where excess copper ions associate with a family of
dehydratase enzymes that are involved in amino acid biosynthesis [61]. The dehydratases contain an exposed iron
sulphur (Fe-S) cluster that is essential for function, and in vivo and in vitro studies demonstrated that exposure to excess
copper leads to a disintegration of these clusters, inactivating amino acid biosynthesis and creating copper-dependent
auxotrophies [61]. The Cu(I) affinity of the dehydratase enzymes was not determined [61], and is therefore an important
question for future study in order to shed light on what copper affinity is necessary for an adventitious binding site to
compete with intracellular copper chelators such as glutathione, metallochaperones and native copper-proteins.
Alternatively a transitory interaction of copper with the Fe-S cluster may be sufficient to lead to degradation of the
cluster, or cluster formation may be prevented through interaction of copper with the cluster biosynthetic machinery. It
is noteworthy that amino acid supplementation did partially, but did not completely, restore growth of E. coli in the
presence of copper, suggesting that additional toxicity mechanisms were in operation [61]. Nonetheless, this discovery
shows that copper toxicity can be exerted through adventitious association of copper with non-copper proteins in vivo,
leading to a loss of function.
Though further examples of such adventitious copper binding are lacking in the literature, it is possible to make
predictions. The likely oxidation state of copper in the reducing cytosol is Cu(I), which forms especially stable
complexes with the amino acids cysteine, methionine and histidine, usually in coordination numbers of two or three [8].
Thus, any protein that has two or more such residues on an exposed surface, in close proximity, may be a potential
candidate, especially if these residues participate in a catalytic mechanism. Obvious candidates, therefore, are enzymes
that utilise catalytic cysteine residues, especially pairs of cysteines during catalysis, such as the thioredoxins. It is hoped
that future studies may enable the identification of such adventitious copper binding proteins from a range of organisms,
which may or may not be conserved. It is anticipated that within any one organism there are hierarchies of copper-
binding sites, each progressively weaker than the last, which become sequentially occupied as the copper concentration
increases. Individual identified targets may present opportunities for exploitation to maximise toxicity for specific
applications, perhaps by combinatorial approaches that aim to target an essential enzyme by combining copper
compounds with specific enzyme inhibitors.

5.3. Effects of excess copper ions on metabolism

The possible effects of excess copper on metabolism have been largely overlooked in previous studies. The only role of
small molecules in copper homeostasis previously studied is the interaction of glutathione with copper ions [54], which
may deplete the functional pool of reduced glutathione with consequences for redox homeostasis of the cell. However,
Cu(I) is likely to also form stable complexes with numerous other cellular metabolites, especially those with thiol
groups such as lipoamide and coenzyme A and its derivatives, as well as numerous other small molecules. If such
complexes do form in vivo under conditions of copper excess, they are likely to inhibit cellular metabolic processes,
making determination of the Cu(I)-binding properties of such molecules of interest. Importantly, the rapidly developing
field of metabolomics is beginning to make it feasible to address such questions in a way that has never previously been
possible [63]

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Alternatively, the effects of copper toxicity on metabolism may be through copper-binding to metabolic enzymes. As
we have seen, copper toxicity inactivates a family of dehydratase enzymes in E. coli [61], which prevents the bacterium
from biosynthesising specific amino acids. It is likely that further cellular protein targets for adventitious copper binding
will also be metabolic enzymes. Copper binding to such enzymes is predicted to inhibit enzymatic function, leading to
alterations in metabolic processes. For example, oxidative stress has been shown to lead to a switch in central carbon
metabolism from glycolysis to the pentose phosphate pathway [64], an evolved response that ensures adequate supply
of reducing equivalents (in the form of NADPH) to maintain the antioxidant systems under stress. It is anticipated that
this type of re-direction of metabolism will also occur under metal stress, either as an evolved response to maintain
critical cellular systems, or as an accident of toxicity where copper ions interfere with normal metabolism.

5.4. Solid copper surfaces

In recent years, there has been a surge of research aiming to understand the toxicity of solid copper surfaces, often with
contradictory results [65-73]. Such contradictions are probably the result of the difficulty in the standardisation of
methods for such studies, particularly between wet and dry application to such surfaces [74]. The consensus,
nonetheless, is that dry surfaces made from copper or a copper alloy are able to kill a wide range of pathogenic
organisms in a matter of minutes, whereas killing tends to take a longer duration on wet surfaces. This might argue that
copper ions, expected to be more of an influence under wet conditions, play only a minor role, and several alternative
mechanisms have been proposed for such contact killing including membrane depolarisation, membrane rupture and
even DNA damage, although the order in which these events occur remains in dispute [73,75]. Nonetheless, several
studies have shown that copper ion does play some role in contact killing, as demonstrated by phenotypes of strains that
lack one or more of the copper detoxification systems described [72,76,77].

6. Applications of copper toxicity

The antimicrobial properties of copper have been exploited by man for hundreds of years. Early civilisations such as the
Egyptians, Greeks, Chinese, Romans and Aztecs used copper to sterilise wounds and water and to treat a number of
minor ailments [78]. During cholera epidemics in the 19th century it was observed that copper workers showed a
reduced susceptibility to the disease [78]. From the 1880s copper found use as an anti-fungal agent, as part of Bordeaux
mixture and Burgundy mixture, a purpose for which it remains important today [79]. As well as an anti-fungal for
plants, copper has also been used to minimise the growth of algae in water and on timber, including for anti-fouling of
ships hulls, and in the preservation of fabrics [79], and current studies are investigating the use of copper compounds as
antiproliferative, anticancer drugs [80].

Fig. 2 Schematic diagram of the copper homeostasis system in a mammalian macrophage, illustrating the copper trafficking
pathways to SOD1 and the ATP7A TGN/phagolysosomal transporter.

Although its use by man has a long history, evolution has been making use of copper toxicity for a great deal longer
as a weapon in the arsenal of the mammalian immune system. When phagocytic cells engulf pathogens, they exploit the

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redox activity of copper, possibly to catalyse the production of reactive oxygen and reactive nitrogen species within the
phagolysosomal compartment [81]. This compartment of the activated macrophage has been shown to be enriched in
copper [82], supplied through the action of the mammalian P-type ATPase ATP7A (see figure 2) whose activity is
crucial for bacterial killing [83]. Pathogens that are unable to produce their copper detoxification systems due to genetic
modification are more susceptible to macrophage/immune killing in vitro and in vivo [33,56,83-87], and bacterial cells
display increased expression of copper-dependent genes inside phagolysosomes [33,88]. Some bacterial pathogens use
siderophores to chelate the exogenous copper, to minimise toxicity [89]. Furthermore, increased dietary copper has been
shown to improve the immune defence against pathogens in mammals [86,90]. It is this final observation that probably
underpins one common application of copper as a growth promoter in the meat production industry [91].
In an age of decline for the traditional antibiotics, and a dearth of promising new classes of such compound, there is a
need for new strategies to fight pathogens. For these reasons, there is an increasing interest in the application of metals
such as copper as antimicrobial materials, in medical and veterinary settings, in food preparation and in commercial
products. Many bacteria, including spores, are killed by copper surfaces in a matter of minutes under laboratory
conditions [66,69,70,72,75], whereas copper shows little toxicity to humans exemplified by the long, safe history of the
use of copper-based intrauterine contraceptive devices [92]. A number of small scale clinical trials have been performed
to test whether copper touch surfaces in hospitals can reduce nosocomial transmission of pathogens, with promising
initial results in both reducing the microbial burden on such surfaces and in reducing hospital-acquired infections [93-
96]. It is worth noting that copper has become the first-line defence against bacterial contamination of water storage and
transport systems, being particularly important in controlling Legionella [97].

7. Conclusions and prospective

The utility of copper toxicity, both as metal salts and as solid metal surfaces, has gained much interest of late. This
interest is predicted to increase in the future, due largely to the decreasing effectiveness of traditional antibiotics, and
the recognition that prevention of infection is better than cure. Laboratory testing and small-scale initial trials in
healthcare settings show great promise for copper-containing materials for their antimicrobial properties. However,
despite the excellent progress made over the last two decades in understanding how cells, from bacteria to humans,
acquire, store, transport and detoxify this essential yet toxic metal, we are still far from understanding the molecular
mechanisms by which copper kills pathogens. An understanding of these mechanisms is key to developing new, more
effective copper-based materials, optimising the killing effect against the cost and potential for human side-effects. Just
as importantly, this research field can provide information about the likelihood of spontaneous copper resistance
developing among pathogens, something which coppers long history of use by man perhaps argues against, but which
must be assessed before wide adoption of antimicrobial copper. The ongoing research of many groups around the world
aims to understand these toxicity mechanisms in order to aid their exploitation.

Acknowledgements K.J.W. and E.T. are supported by a Wellcome Trust/Royal Society funded Sir Henry Dale fellowship
(098375/Z/12/Z). Support by a BBSRC studentship (to J.S.) is also gratefully acknowledged.

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