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Coppertoxicityandtheoriginofbacterial
resistance-Newinsightsandapplications
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Copper toxicity and the origin of bacterial resistance—new insights and
applicationsw
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Published on 10 October 2011 on http://pubs.rsc.org | doi:10.1039/C1MT00107H
Christopher L. Dupont,a Gregor Grassb and Christopher Rensing*c
Received 18th July 2011, Accepted 27th September 2011
DOI: 10.1039/c1mt00107h
The bioavailability of different metals has likely changed over the course of Earth’s history. Based
on geochemical models, copper became much more bioavailable with the advent of an oxidizing
atmosphere. This posed both a challenge and an opportunity for the organisms at that time.
Specifically, copper resistance mechanisms were required first and to do this Bacteria appear to
have modified already existing protein structures. Later, Cu-utilizing proteins evolved and
continue to be used sparingly, at least relative to later evolving Eukarya, by Bacteria but with
significant biogeochemical consequences. Copper is a strong soft metal that can attack
intracellular iron–sulfur centers of various proteins under primarily anoxic conditions. In oxic
conditions, copper can catalyze a Fenton-like reaction that may cause lipid peroxidation and
protein damage. The inherent ability of copper to inflict damage upon multiple cellular functions
has been harnessed by macrophages and perhaps amoeba to kill and later digest bacteria and
other microorganisms. Notably, these organisms, unlike Bacteria, most likely evolved after
increases in copper availability, implying that Eukarya utilized their own trafficking and resistance
mechanisms, in addition to the natural toxicity of copper, as leverage in interactions with
Bacteria. In an ‘‘arms race,’’ some pathogenic bacteria have evolved new mechanisms for copper
resistance, which is relevant given renewed interest in the use of copper surfaces due to their
antimicrobial properties.
a
Microbial and Environmental Genomics, J. Craig Venter Institute, San Diego, CA, USA
Bundeswehr Institute of Microbiology, Munich, Germany
c
Department of Soil, Water, and Environmental Science, University of Arizona, Shantz Bld #38 Rm 429, Tucson, AZ 85721, USA.
E-mail: [email protected]; Fax: +1 (520) 621-1647; Tel: +1 (520) 626-8482
w This article is published as part of a themed issue on Metal Toxicity, Guest Edited by Gregor Grass and Christopher Rensing.
b
Christopher
L.
Dupont,
Assistant
Professor
in
Microbial and Environmental
Genomics, J. Craig Venter
Institute. Chris L. Dupont
received a MS in Environmental
Engineering from Cornell University and a PhD in Marine
Biology from the Scripps Institution of Oceanography. Chris
joined the J. Craig Venter
Institute in 2008, where he is
an assistant professor in Microbial and Environmental Genomics. His research focuses on
Christopher L. Dupont
microbial physiology and the
role of environment in shaping molecular adaptations and
diversification.
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The Royal Society of Chemistry 2011
Gregor Grass, Oberregierungsrat, Bundeswehr Institut of
Microbiology. Gregor Grass
works as a tenured senior
research scientist at the Bundeswehr Institut of Microbiology
(Munich, Germany) since 2011.
He is interested in how bacteria
acquire essential transition
metals and in systems involved
in metal detoxification. Currently, his research focuses on
the antimicrobial mode-of-action
of metallic copper and biodefense
issues. Gregor studied Biology at
Gregor Grass
the
Martin-Luther-University
(MLU, Halle, Germany). He completed his Ph.D. in microbiology
in 2000 and then worked as a postdoctoral fellow with Chris Rensing
in Tucson, AZ. In 2002, Gregor become an independent non tenuretrack group leader at MLU before temporarily accepting a position as
assistant professor at the University of Nebraska-Lincoln in 2008.
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Introduction
The transition metal copper (Cu) occurs as Cu(I) and Cu(II) in
nature. Cu(I) is a strong soft metal whereas Cu(II) is borderline
according to the Hard and Soft Acids and Bases (HSAB)
classification.1 Therefore, Cu binding sites are dominated by
amino acids with soft (sulfur donor atoms) and borderline
(nitrogen donor atoms) ligands. These are cysteine and
methionines as soft ligands and histidine as a borderline
ligand. Cu(I) [Ar]3d10 can be found coordinated by 2, 3 or
4 ligands and showing linear, trigonal planar, or tetrahedral
geometries. Cu(II) [Ar]3d9 can be found coordinated by 4, 5, or
6 ligands in geometries such as square planar, square pyramidal,
or distorted octahedral. Proteins using Cu as a cofactor have
positive reduction potentials, as do some iron-containing
proteins that evolved after the rise in oxygen. Since Cu
proteins first appeared under oxidizing conditions, most
evolved functions related to oxygen handling and are involved
in electron transfer, oxygen transport and in redox reactions
of a broad substrate range such as multicopper oxidases
(laccases, Fet3, CueO). Most of these proteins bind Cu tightly
with high coordination binding sites of 4 to 5 to prevent loss of
Cu during redox cycling. Cu trafficking proteins naturally
contain low affinity binding sites or have flexible geometries,
so that metal transfer is possible between two binding sites.
The coordination numbers here would be 2 to 3. Recently
available protein structures confirm this general principle. In
the central transport protein CusA Cu(I) is coordinated in a
trigonal planar geometry to three methionines. CusA is part of
the CusCBA RND-type transport complex common in Gram
negative bacteria.2 In the unrelated Cu(I)-translocating P-type
ATPases CopA from Legionella pneumophila, Cu(I) also
appears to be coordinated in a trigonal planar geometry at
both binding sites along the transport pathway, first by two Cys
and Tyr in transmembrane (TM) binding site I, then by Asn,
Met and Ser in TM binding site II.3 This has also been predicted
in homologous proteins such as CopA from Archaeglobus
fulgidus4 and might be universally applicable for all
Christopher Rensing, Associate Professor of Microbiology, University of Arizona.
Chris Rensing studied and
obtained his PhD at the Freie
Universität Berlin and later
Martin-Luther
Universität
Halle-Wittenberg supervised
by Dietrich Nies in 1996. He
then joined Barry Rosen’s lab
at Wayne State University in
Detroit as a postdoctoral fellow mainly working on the
biochemical characterization
of two metal-transporting
Christopher Rensing
P-type ATPases, ZntA and
CopA. In 1999, he accepted a faculty position at the University
of Arizona and in 2007 was promoted to associate professor. His
research is looking at different aspects of metal–microbe interactions, encompassing both environmental as well as medical
microbiology.
Metallomics
Cu(I)-translocating P-type ATPases. Interestingly, Cu(I) is also
coordinated in trigonal planar geometry by three Mets in the
eukaryotic Cu uptake transporter Ctr15 that seems to be absent
in bacteria.
Evolutionary perspectives on Cu utilization and
resistance in bacteria
The so-called Great Oxidation Event (GOE), which occurred
between 2.4–2.7 billion years ago, refers to an increase in
atmospheric oxygen that potentially changed the bioavailability
of metals including Cu.6 Specifically, according to geochemical
models, in an anoxic or euxinic (anoxic and sulfidic) ocean, a
chalcophilic metal like Cu would be insoluble.6 Thus increasing
global oxygen should augment a source of Cu, specifically,
continental weathering, and also increase the solubility of Cu in
the ocean. Phylogenomic reconstructions have recently been
used to determine the temporal evolution of the Structural
Classification of Proteins (SCOP) fold superfamilies,7 which
are evolutionarily related protein structures. These were
combined with a genome census for these fold superfamilies
and aligned to the modeled geological record using several
transitional arguments.8 These aligned genome and geological
records suggest that Cu-binding enzymes evolved after critical
oxidative defense enzymes like superoxide dismutase and
catalase (Fig. 1).8a These enzymes are essential for oxygen
tolerance in bacteria, thus their evolution must have preceded
or coincided with the invention of oxygenic photosynthesis
and subsequent accumulation of oxygen in the atmosphere.
Just as with oxygen itself, the increased bioavailability of Cu
posed both an opportunity and a challenge for all organisms;
as a cofactor it participates in new biochemical enzymes and
pathways, as a free ion it acted as poison. In the period
following the GOE, new Cu binding proteins with metabolic
roles in aerobic and in facultative aerobic bacteria evolved,
including the Cu binding cupredoxin fold superfamily, which
encompasses the Cu binding proteins plastocyanin, nitrosocyanin,
azurin, and the periplasmic domain of cytochrome c oxidase
subunit II (Fig. 1). Imposed over this timeline of protein
evolution is organismal evolution. Many bacterial lineages,
including cyanobacteria, were already greatly diversified at the
GOE. Given that phylogenetic speciation prior to Cu-protein
evolution would hinder widespread usage, it is not surprising
that none of the new Cu-binding proteins are found in all
extant bacteria. That is, new Cu proteins were likely invented
by specific lineages, with sparing horizontal gene transfer later.
Overall, incorporation of genes of Cu-binding proteins into
bacterial genomes is rare, with less than 0.3% of the annotated
proteome being Cu-binding in Bacteria (Fig. 2). Laterevolving Cu-binding proteins are found in small numbers of
modern bacterial genomes. Consistent with Cu utilization
being tied to aerobic metabolism, obligate anaerobic bacteria
have even fewer Cu binding proteins (Fig. 2). In contrast, the
genomes of modern Eukarya appear to reflect an evolution in
an environment with elevated Cu concentrations; a greater
percentage of Eukaryotic genomes code for Cu binding proteins
(Fig. 2), particularly late-evolving Cu-binding proteins.
While increasing Cu concentrations provided a redox-facile
ligand for metabolic enzymes, it also posed a toxic threat.
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Fig. 1 The evolution of Cu utilization and modern day genomic incorporation of Cu proteins. Shown are parallel timelines of the evolution of
oxygen in the atmosphere, trace metal chemistry in the ocean, and the evolution of Cu binding protein structures. Several key events, like the Great
Oxidation Event and the first physical Eukaryotic fossils are shown for references. For each noted protein fold superfamily, the histograms show
the percent of Bacterial and Eukaryotic genomes that contain them. Data is compiled and replotted from ref. 8a,71.
What would have been the initial manifestation of Cu toxicity
to early life? One way that Cu can be toxic is through the
generation of free radicals, though this would have been
minimal during the early stages of the planetary oxidation
(See O2 trace on Fig. 1). As discussed in more detail below,
Fe–S cluster -containing proteins are a target of Cu(I) based
toxicity. At the time of the GOE, at least over half of the
known Fe–S cluster binding structures had evolved,8a as had
the structures involved in Fe–S biosynthesis, thus damage
and inhibition of Fe–S metabolism is the more likely initial
manifestation of Cu toxicity.
Within the same phylogenies, it appears that ancient
bacteria co-opted already existing protein families to develop
Cu resistance. In modern bacteria, intracellular Cu concentrations
are carefully controlled by a system of Cu-sensing regulatory
proteins, Cu transporters, and possibly Cu chelators. While
metal homeostasis proteins might appear divergent in terms of
amino acid sequence, from a structural standpoint, many of
the proteins involved in sensing and trafficking numerous
metals are quite similar and belong to one of three fold
superfamilies. One is the Ferric Uptake Regulatory (FUR)
protein family, which includes sensors for Fe, Zn, and Ni. At
the even grander structural scale, the FUR protein family is
related to 83 protein families with a ‘‘winged helix DNAbinding’’ domain (SCOP id 46785), including the arsenic and
cadmium binding ArsR and CadC.9 The ‘‘helical backbone
metal receptor domain’’ (SCOP id 53807) is involved in metal
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transport in modern bacteria (Fig. 1). Finally, the ‘‘heavy
metal associated domain’’ (SCOP id 55008) includes the metal
binding domains of Zn and Cu transporting ZntA and CopA,
the mercury binding protein MerP, and Cu chaperones found
Fig. 2 The average number of Cu binding domains in the genomes of
the three superkingdoms of life (A) and the percent of predicted
proteins in those superkingdoms that are Cu binding (B). In B, the
black line in the Bacterial column shows the percent from obligate
anaerobic bacteria.
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in Eukaryotes like ATX1. On structural protein phylogenies,
these protein families evolved prior to the GOE (Fig. 1),
though the ‘‘parent’’ metal bound at that time is unknown.
They are also nearly ubiquitous and abundant in bacterial
genomes.
In summary, the combination of phylogenies of protein
architectures and surveys of extant bacteria genomes suggests
that the protein architecture required to traffic Cu arose prior
to the GOE, though it might not have been used for Cu at
that time. As changes in global redox chemistry increased
environmental Cu availability, our interpretation is that
bacteria tuned existing metal sensing, transporter, and storage
proteins. Concomitantly, this facilitated new metabolic
utilizations of Cu, but only by select bacterial lineages.
Copper toxicity
A question that is of interest in this review is how Cu becomes
toxic to cells. What are the targets of Cu? Many studies
focused on the ability of Cu ions to redox-cycle between
Cu(I)/Cu(II). This Fenton-like reaction would then generate
reactive oxygen species leading to lipid peroxidation and
protein oxidation and DNA damage. However, as shown
quite impressively in ref. 10, Cu under anaerobic, reducing
conditions was most toxic. Cu(I) is a strong soft metal with high
affinity for thiolates and can destabilize iron–sulfur clusters.
This is often overlooked as toxicologists and others focus on
oxidative damage in many diseases where Cu plays a role.
Excess Cu led to an increase in iron acquisition and sulfur
assimilation both in E. coli and in B. subtilis.10,11 This is due to
decreased iron–sulfur cluster stability both during their
biogenesis and when bound to their target proteins. Genes
encoding enzymes containing iron sulfur clusters that are
induced during Cu stress in B. subtilis include biotin (BioB),
molybdopterin (MoaA), pyrimidin and branched-chain amino
acid synthesis. In E. coli dihydroxy-acid dehydratase (IlvD) in
the common branched-chain amino acid synthesis pathway,
isopropylmalate dehydratase (LeuC) in the leucine-specific
branch, fumarase A (FumA) in the tricarboxylic acid cycle
and 6-phosphogluconate dehydratase in the pentose phosphate pathway (Edd) were found to be inactivated by Cu ions;
thus, these proteins constitute targets for lethal damage.10
Since these enzymes fulfill vital cell functions, it is essential
to keep Cu at very low concentrations in the cytoplasm. In
fact, the P-type ATPase regulated by CueR in E. coli allows
only extremely low concentrations of free Cu in the cell.12 In
effect, there is almost no free Cu in cells. In addition to
degradation of iron–sulfur clusters of vital enzymes, excess
Cu would effectively lead to an intracellular sink for iron
making increased iron uptake necessary.11 This would put cells at
a serious disadvantage in an environment with increasing
concentrations of bioavailable Cu and diminishing concentrations
of soluble iron. Most bacteria solved this situation by having Cu
containing enzymes only in the periplasm or facing out in the
cytoplasmic membrane. In the few bacteria that need Cu delivered
to intracellular compartments (e.g. photosynthetic bacteria),
chaperones guide Cu through the cytoplasm as in eukaryotes.
Aerobically, Cu readily catalyzes reactions that result in the
production of hydroxyl radicals through the Fenton and
Metallomics
Haber–Weiss reactions (Cu+ + H2O2 - HO + HO +
Cu2+, O2 + Cu2+ - Cu+ + O2).13 That highly reactive
oxygen intermediates are responsible for lipid peroxidation,
oxidation of proteins and damage to nucleic acids was
proposed as well.13b,14
The effect of Cu(I) on generation of reactive oxygen
species can also be indirect. Free Cu ions are able to oxidize
sulfhydryl-groups, such as cysteine in proteins or the cellular
redox-buffer glutathione.15 Degradation of Fe–S clusters
would lead to the release of iron. An increase in unbound free
iron can then lead to oxidative damage through iron-based
Fenton chemistry.16 All of these events can cause oxidative
damage but it appears only indirectly through Cu. Moreover,
it was believed previously that Cu ion toxicity in bacteria was
mediated by oxidative DNA damage. However, under
anaerobic conditions Cu ions suppressed the growth rate of
E. coli even more strongly than under aerobic conditions.17
More importantly, addition of Cu ions to E. coli cells
decreased oxidative DNA damage when cells were challenged
with hydrogen peroxide.18 Table 1 summarizes the consequences of contact of microbes to different forms of Cu focusing
on aerobic conditions.
Cu homeostasis in bacteria
Little is known about how most bacteria acquire Cu. Much
of what is known about Cu homeostasis in prokaryotes
originated from the bacterial model organism Escherichia coli.
This enterobacterium probably does not require cytoplasmic
Cu, and it is believed that Cu-requiring proteins are exclusively
located in the periplasm or within the cytoplasmic membrane
(reviewed in ref. 19) with the Cu center facing the periplasm and
finally some in intracellular membranes (e.g. cyanobacterial
photosynthesizers and methanotrophs).
In contrast, defense systems against Cu-overload are
conserved in most Gram-negative and even Gram-positive
organisms.
Detoxification of superfluous cytoplasmic Cu is accomplished
by P-type ATPases that efficiently pump out Cu(I).20 Periplasmic
Cu is effluxed by huge multi-component protein complexes
such as CusCBA of E. coli.17,21 CusA is a resistance/nodulation/
cell division (RND) protein, driven by the proton motive force,
that together with CusB and CusC transport Cu to the
extracellular space. Mechanistically, CusCBA functions
similar to a peristaltic pump with three alternating enzyme
conformations, as was shown for a related system.22 Three
conserved methionines in the periplasmic cleft of CusA are
responsible for specifically binding and releasing Cu(I).2 The
CusCBA complex can sense periplasmic Cu concentrations
through three conserved N-terminal residues and adjust
transport efficiency accordingly.23 The third component of Cu
homeostasis is a multicopper oxidase (MCO). In E. coli the
periplasmic MCO CueO oxidizes Cu(I) to Cu(II), thus protecting
periplasmic proteins from Cu(I)-mediated toxicity.24 CueO might
also oxidize the siderophore enterobactin when Cu levels
are high, providing a Cu-binding oxidation product diminishing
Cu-uptake into the cytoplasm.25
Surprisingly, a close relative of E. coli, Salmonella enterica
features a variation of this prototypical three-legged Cu
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Table 1
Consequences of diverse kinds of aerobic Cu stress
Stress exposure
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Cu speciation
Cell growth under
stress
Mechanism of cell
damage
Planktonic culture = Biofilms culture =
growth with Cu salts growth on wet Cu surface
Cells exposed to moist
Cu surfaces
Cells exposed to dry
Cu surfaces
Chronic (cells grow
under stress)
Cu(I), Cu(II)
Yes (up to mM Cu)
Acute (hybrid of planktonic
culture and dry surfaces)
Cu(0), Cu(I), Cu(II)
No significant growth
Acute
Fe–S cluster in
hydratases and other
factors (ROS)
Inactivation of sensitive Slow, bacteriostatic,
cells
concentration
dependent
Specific Cu resistance Common, well
mechanisms
studied mechanism
Chronic (cells adapt to
stress and grow)
Cu(0), Cu(I), Cu(II)
Yes (live cells on top of
dead cell barrier)
Probably Fe–S cluster in
hydratases and other factors
(ROS)
Initially fast, negligible
after biofilm formation
Overlap with planktonic
culture but probably
additional unknown
specialized mechanisms
homeostasis system. While CopA and CueO (aka CuiD) are
present, the Cus system is not. In contrast, another periplasmic
Cu defense protein, termed CueP has been identified.26 Similar
CueP proteins are not only restricted to Salmonella but can be
found in other, often pathogenic bacteria as well. The highly
abundant CueP in the periplasm of Salmonella is no transport
protein but the function of CueP is thought to be related to Cu
sequestration particularly under anaerobiosis. Here, CueP
constitutes a major cellular Cu pool.27 Analogous to the Cus
system, CueP is also most important under anaerobic growth
conditions. Thus, CueP might replace the Cus system by a
functionally distinct mechanism.
Though the RND-type Cus system is lacking in Salmonella,
there is an additional unrelated tripartite RND efflux pump
encoded on the Salmonella genome. This Ges system was
initially identified and characterized as a gold-responsive
operon with a MerR-type regulator, GolS, encoded by the
neighboring Gol system.28 Ges does apparently not transport
Cu but the adjacently encoded Gol system does. In addition to
the regulator GolS, the small metallochaperone GolB and the
CopA-like P-type ATPase GolT are also present.29 Though
Gol was identified by its ability to confer gold-resistance, in
the absence of CopA Gol also confers resistance to Cu.
Interestingly, while the secondary function of Gol for
Cu homeostasis was recently corroborated, gold-resistance
conferred by the system could not be confirmed.27
Salmonella does not only exhibit an interesting variation of
the prototype Cu homeostasis of E. coli, it also offered the
opportunity to study Cu homeostasis in an intracellular
pathogen and the role proper Cu-handling plays during
infection. For this it was first established that the expression
of copA was only dependent on Cu ions and no other relevant
factors potentially encountered in macrophages altered copAlevels. Next macrophages were infected with a Salmonella
strain expressing a reporter comprising the copA promoter
and the gene for beta-galactosidase (copAp-lacZ). It was
known from previous studies that such reporters sensitively
and dose-dependently respond to environmental Cu ions.
Expression of copAp-lacZ also occurred during the course
of macrophage infection with increasing reporter activities27
indicating that within host cells the bacteria encountered
increasing Cu ion concentrations maybe as a defense response
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Unknown, probably Cu ion and
ROS related, targets unknown
Fast (min–h)
Overlap with planktonic culture
but probably also overlap with
dry surface resistance mechanisms
Cu(0), Cu(I), Cu(II)
No
Unknown, probably Cu
ion and ROS related,
targets unknown
Very fast (min)
Unknown—resistance
mechanisms different
from that of planktonic
cells
to infection. Further, deletion of both copA and golT resulted in
significantly fewer cells than the wild-type control in infected
macrophages, clearly demonstrating a necessity of Cu resistance
for survival of infecting Salmonella in a macrophage model.
This observation is in good agreement with a previous study
showing that Cu-transporting P-type ATPases from both host
and pathogen play a role during infection.30 When the host
Golgi Cu exporter ATP7A was expressionally silenced, killing
of invading E. coli was attenuated. Conversely, if these E. coli
cells lacked CopA they were hypersensitive to macrophagemediated killing.
However, important parts of the Cu-resistance puzzle
during host–pathogen interaction are still missing. In a murine
model of infection no significant differences in colonization
between wild-type Salmonella and the copA/golT double
mutant were observed.27 This might be a consequence of the
animal model selected or caused by more subtle differences
between the two strains during the course of infection not yet
elucidated. Also, in these mutant cells the CueO protein was
still present. It would be interesting to include CueO in such
studies as well and test a triple mutant for survival in different
models of infection. Recently, a study indicated a role for
CueO in a murine model of systemic infection by Salmonella.31
Thus, new surprises regarding Cu homeostasis can certainly be
expected. For example, cells of E. coli or Salmonella with a
defect in CueO exhibit a mucoid phenotype and aggregative
behavior when Cu-stressed.32 It appears that under these
conditions cells produce the extracellular polysaccharide
colonic acid, and lack of this compound increased Cu toxicity.
It is an open question if colonic acid production has a practical
relevance during infection such as production of a bacterial
capsule for host-defense evasion.
Clearly, more research is needed in addition to these few
piloting studies concerned with this subject in the past including
these in Legionella pneumophila,33 Pseudomonas aeruginosa34 or
Mycobacterium tuberculosis.35 The latter example offers yet
another new variation on an old theme. M. tuberculosis harbors
eleven metal-translocating PIB-type ATPases. An earlier study
has revealed that only one, CtpV, can be directly associated
with Cu homeostasis in this organism. Further, absence of
CtpV resulted in altered cellular transcription as a response to
increased Cu stress. Similar to the case of Salmonella, animal
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models of tuberculosis infection including mouse and guinea pig
did not reveal an obvious decline of fitness of the mutant.
However, in contrast to infection with wild-type M. tuberculosis,
infection with ctpV mutant cells resulted in less lung damage,
attenuated immune response and increased survival times of
the host.
Surprisingly, M. tuberculosis possesses a novel unexpected Cu
homeostasis component that seems to be essential for virulence
in this organism. This MctB (mycobacterial Cu transport
protein B) is located in the outer membrane equivalent of the
mycobacterial cell wall.36 There were no homologs of MctB
with known roles but MctB likely functions analogously to
porins as outer membrane channel for Cu and silver ions.
However, MctB seems to be specifically responsible for Cu
efflux but not for uptake or facilitated diffusion across the
membrane, because a deletion of mctB rendered cells more
susceptible to Cu toxicity and cells accumulated approximately
100-times more Cu than wild-type cells when challenged even
with low mM external Cu ion concentrations. It was suggested
that MctB and the Cu ATPase CtpV interact constituting a
novel dipartite Cu efflux system.36 It remains to be seen if this
interaction is physical or rather functional in nature. Notwithstanding, it appears that lack of MctB in M. tuberculosis elicits
the most severe Cu-dependent phenotype both in growth
experiments and animal models when compared with strains
lacking one of the other two major Cu homeostasis factors,
CtpV and the small metallothionein MymT that may bind
up to six Cu ions.37 A deletion of mctB resulted in a
100-fold reduction in infection with M. tuberculosis in guinea
pig lymph nodes and even 1000-fold reduction in lungs.36 This
result argues against a strict interdependence of CtpV and
MctB but it would be interesting to study M. tuberculosis
mutants only harboring each one of its three Cu homeostasis
systems.
Overall, findings from recent years clearly point to a more
prominent role of the biometal Cu for host–pathogen interaction
than previously thought. Two bacterial model systems are most
advanced, S. enterica and M. tuberculosis each featuring unique
Cu homeostasis systems. In case of M. tuberculosis, a model of
the events occurring during infection was suggested.36 Herein,
host and invader Cu homeostasis systems battle for the upper
hand with the host also employing the adventitious properties of
redox-cycling between Cu’s different oxidation states. The
generated toxic reactive oxygen species would thus additionally
support killing of the bacterial pathogen. It appears that Cu
offers a weak point of defense in M. tuberculosis. Even though
this bacterium expresses three potentially efficient Cu homeostasis factors, its tolerance for Cu ions is rather low, about a
factor of ten lower than in most bacteria including E. coli or
Salmonella.32,36 It is tempting to speculate that inhibition of a
factor such as MctB, which might be reached from the outside
of the cell, could lead to new strategies for anti-tuberculosis
chemotherapy. Recently, however, a potential exit strategy for
M. tuberculosis from such approaches was offered. A deletion
of ricR, a paralog of the long known Cu regulator CsoR,
caused overproduction of MymT with a concomitant highly
increased resistance of M. tuberculosis against Cu ions.38
However, it remains to be tested if such mutant cells can still
efficiently invade and infect host cells.
Metallomics
Copper and innate immunity
One of the hallmarks of multicellular organisms is the presence
of an immune system. Multicellular eukaryotes evolved
entirely under conditions of relatively high concentrations of
Cu (Fig. 1) and zinc.8a It is therefore not surprising that these
metals were highly utilized for many functions including
development, the nervous system and immunity.39 The role
of Cu in innate immunity is becoming better understood as of
late. Cu is taken up through Ctr1 and delivered to the Golgi
network by the Cu chaperone Atox1. From here the P-type
ATPase ATP7A delivers it to the phagosome where Cu(I) is
actively pumped across the membrane (Fig. 3). Macrophages
use Cu to kill off infectious agents such as microbes in
combination with production of reactive oxygen and nitric oxide
species.27,30 Therefore, inflammation is often characterized by an
increase in Cu concentration in the affected tissue.40 Since Cu is
used to actively kill bacterial cells, pathogens had to find
effective Cu resistance strategies in order to insure survival.
Consequently, copper resistance determinants are important
for virulence in Mycobacterium tuberculosis, Salmonella enterica
and Streptococcus pneumoniae27,36,41 as described above.
However, the mechanism used by macrophages to
kill infectious agents appears to be even more widespread.
Phagocytosis first evolved in primitive unicellular eukaryotes
such as amoeba and ciliates.42 Amoeba are known to occur
in aquatic environments,43 but also appear to use simple
agricultural methods to maintain bacterial food populations
in soil.44 Essentially, in addition to the actual environmental
concentrations of Cu, bacteria in diverse environments are
likely to encounter predators that utilize the inherent toxicity
of Cu. For example, most Vibrio species and the Deep Sea
bacterium Photobacterium profundum contain an operon
encoding CusCBA but also a blue Cu protein in an operon
regulated by a two component system. It is conceivable, that
the presence of these operons is in part protection against
predatory amoeba since these organisms have also been found
in many marine environments including the Deep Sea.45
Moreover, there is strong evidence Acanthamoeba polyphaga
is host to Vibrio cholera.46 Other operons prevalent in many
bacteria including various rhizobia encode an outer membrane
protein, a multicopper oxidase, an azurin-like blue Cu protein
and a CusF-like Cu chaperone.47 Interestingly, many of these
bacteria such as Mesorhizobium amorphae and Bradyrhizobium
japonicum are resistant to predation by amoeba.48
Since many of the processes involved in killing bacteria in
macrophages and amoeba are similar, the defense mechanisms
that developed are manifold but there probably was extensive
pressure to possess an effective copper resistance system to
prolong survival and possibly overcome killing by amoeba and
other predatory protozoa. In contrast to macrophages, it is at
present not known whether amoeba use copper in their arsenal
to kill microorganisms, however, in our opinion it is likely
that at least some predatory protozoa will also use copper,
since well studied Dictyostellium discoideum contains both
P80, a Ctr-type copper transporter localized in endocytic
compartments involved in maturation of the phagosome,
and a Cu(I)-translocating P-type ATPase with a function in
copper resistance.49
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Fig. 3 Role of Cu in the respiratory burst reaction within phagosomes. The respiratory burst reaction in activated macrophages and neutrophils
is a reactive oxygen species (ROS)-mediated antibacterial response. ROS production is metal-catalysed, with Fe(II) being considered the important
metal ion. Compelling new evidence indicates that Cu(I) also has an important role in this reaction. Cu(I) is trafficked in the cytoplasm of
eukaryotic cells by metallochaperone proteins to various intracellular sites of utilization. One such metallochaperone, Atox1, shuttles Cu(I) to a
Cu(I) transporter, ATP7A, which resides within the trans-Golgi network, where it provides Cu(I) for the maturation of secreted cuproenzymes. An
elevation in Cu levels within macrophages is achieved by enhanced activity of the high-affinity plasma membrane Cu(I) permease Ctr1. This results
in the relocation of ATP7A to both perinuclear vesicles and the phagosome. Delivery of Cu(I) to the phagosome might, in turn, enhance
bactericidal activity during the phagocytosis of bacteria by catalysing the formation of ROS (with permission from ref. 72).
Since Cu is such an effective means to kill bacteria not only
in macrophages but perhaps also in amoeba and in plants,50
it is perhaps not surprising that Cu has surfaced as new
antibacterial material as described in the following chapter.
Metallic copper as an antimicrobial agent
Many historic cultures around the globe explored the utility of
Cu compounds for hygiene and health problems such as ulcer
treatment, wound dressings or water preservation, taking
advantage of the antimicrobial properties of Cu without
knowing the basis of these effects. This empirical knowledge
of certain transition metal cations, including Cu when in
surplus, exerting antibacterial properties was later described
as the ‘‘Oligodynamic Effect’’.51 In general, single-celled
organisms are especially prone to Cu ion toxicity, enabling
use of Cu as a selective agent against microbes without
harming animals or man.52 Even today, our understanding
of the molecular mode of action of Cu ions against bacteria is
still limited and best understood in Escherichia coli.10,19,53
The antimicrobial efficacy of metallic Cu surfaces has been
established for a variety of bacteria.54 In these studies, cells in
buffer were applied to Cu surfaces, incubated under ambient
conditions and cells were killed within hours. Recently, a
method was established that aimed to mimic contact of
microbes to dry Cu touch surfaces. Under these conditions
most microbes are killed within minutes.55 Surfaces can be an
important reservoir of bacterial contamination; especially the
hospital environment provides multiple transmission and
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cross-infection opportunities for pathogenic bacteria. Thus,
contaminated surfaces contribute to an increase in healthcareassociated infections and costs.56 Furthermore, some pathogenic micro-organisms, such as the bacteria Staphylococcus
aureus or Acinetobacter ssp. and the yeast Candida albicans,
can contaminate and persist on surfaces in the hospital
environment for months.57 Frequent and efficient removal of
such organisms from surfaces combined with hand hygiene
practice reduces such transmission opportunities.58 Problems
arise when these active procedures are not properly followed.59
Self-sterilizing surfaces can be envisioned to help counter such
shortcomings. In 2008 the Environmental Protection Agency
(EPA) registered Cu surfaces as antimicrobial (http://www.
epa.gov/pesticides/factsheets/copper-alloy-products.htm).
Thus, the use of anti-microbial metallic Cu surfaces is likely to
provide protection from infectious microbes by reducing
surface contamination as was recently shown in successful
hospital trials.60 Importantly, bacteria in contact with dry Cu
surfaces, in contrast to those exposed to Cu ions, do not
proliferate and most are killed within minutes.55
Unsurprisingly, there are similarities between the antimicrobial
properties of ionic and metallic Cu: recent studies have
demonstrated that Cu ions are released from metallic Cu upon
contact with bacteria.53,55b,61 Further, extracellular supplementation with protective substances against oxidative stress
such as catalase, superoxide dismutase or mannitol, a hydroxyl
radical quencher, increased the time needed to kill Cu surfaceexposed E. coli cells.55b Similarly, Cu surfaces protected by
thermal oxide layers or the application of corrosion inhibitors
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prolonged the time needed for complete inactivation of
Cu surface-exposed bacteria.62 We are only beginning to
comprehend the molecular mode-of-action exerted by Cu ions
on microbes.10 However, the specific kinds of stress exerted by
metallic Cu surfaces and the identity of sensitive cellular
targets are less understood and still under investigation. Recent
studies suggest that cells are not killed via accumulation of
lethal mutations but rather that initially membranes are
damaged.53,63 This, in turn, also leads to DNA damage in
killed cells giving the impression of genotoxicity as the
underlying mechanism of kill. Conversely, it is more likely
that lipid peroxidation or irreversible inactivation of essential
membrane proteins compromises membranes of Cu surfaceexposed cells beyond the point of recovery.
Cu nanoparticles are another type of antimicrobial metallic
Cu surface that can, however, enter cells. Different species
of bacteria64 have been subjected to nano Cu previously.
Concentrations of Cu nanoparticles (40–100 nm, Ø) required
for 90% antimicrobial efficiency were in the low, microgramper-milliliter range for both E. coli and Bacillus subtilis,65 but the
mechanism of action of nano Cu remains unknown.
Use of ionic Cu as a feed-supplement in piggeries66 has
resulted in the emergence of high-level Cu ion resistance
systems.67 Similarly, application of Cu-containing antifungal
and antibacterial sprays in agriculture has selected for similar
Cu ion resistance systems in plant-pathogenic bacteria.68
Importantly, cells exposed to dry Cu surfaces are not in an
environment in which they can proliferate, in contrast, for
example, to a Cu ion-treated animal gut,67a Cu catheters,69 Cu
implants70 and Cu in other environments where biofilms may
form. Comparable studies on the long-term consequences of
exposure to metallic Cu on bacterial communities, however,
are lacking.
Most bacteria54 are metallic Cu surface sensitive and only a
few of the bacteria isolated from Cu coins survived prolonged
exposure to pure Cu.55a Remarkably, some coin-isolates
survived from 16 times (Micrococcus luteus) up to 5760 times
(Pseudomonas oleovorans) longer on pure Cu than their type
strains or controls but were not especially resistant to ionic Cu.
It is thus unlikely that Cu ion and Cu surface resistance are
governed by the same mechanisms because metallic Cu resistant
isolates from coins were sensitive to dissolved Cu ions.55a
Further research is also required to investigate and address
the likelihood of the possible emergence and spread of Cu
surface resistant bacteria.
Acknowledgements
CLD was supported by a NASA Astrobiology Institute DDF.
Research in the laboratories of CR and GG was supported by
grants from the International Copper Association.
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