Competition for zinc binding in the host-pathogen interaction

Abstract

Due to its favorable chemical properties, zinc is used as a structural or catalytic cofactor in a very large number of proteins. Despite the apparent abundance of this metal in all cell types, the intracellular pool of loosely bound zinc ions available for biological exchanges is in the picomolar range and nearly all zinc is tightly bound to proteins. In addition, to limit bacterial growth, some zinc-sequestering proteins are produced by eukaryotic hosts in response to infections. Therefore, to grow and multiply in the infected host, bacterial pathogens must produce high affinity zinc importers, such as the ZnuABC transporter which is present in most Gram-negative bacteria. Studies carried in different bacterial species have established that disruption of ZnuABC is usually associated with a remarkable loss of pathogenicity. The critical involvement of zinc in a plethora of metabolic and virulence pathways and the presence of very low number of zinc importers in most bacterial species mark zinc homeostasis as a very promising target for the development of novel antimicrobial strategies.

Full text

MINI REVIEW ARTICLE
published: 24 December 2013
doi: 10.3389/fcimb.2013.00108
Competition for zinc binding in the host-pathogen
interaction
Mauro Cerasi1, Serena Ammendola 1and Andrea Battistoni1,2*
1Dipartimento di Biologia, Università di Roma Tor Vergata, Rome, Italy
2Istituto Nazionale Biostrutture e Biosistemi, Consorzio Interuniversitario, Rome, Italy
Edited by:
Frédéric J. Veyrier, Institut Pasteur,
France
Reviewed by:
Klaus Hantke, Universität Tübingen,
Germany
Robert D. Perry, University of
Kentucky, USA
*Correspondence:
Andrea Battistoni, Via della Ricerca
Scientifica, Dipartimento di Biologia,
Room 372, Università di Roma Tor
Vergata, Rome 00133, Italy
e-mail: andrea.battistoni@
uniroma2.it
Due to its favorable chemical properties, zinc is used as a structural or catalytic cofactor
in a very large number of proteins. Despite the apparent abundance of this metal in
all cell types, the intracellular pool of loosely bound zinc ions available for biological
exchanges is in the picomolar range and nearly all zinc is tightly bound to proteins.
In addition, to limit bacterial growth, some zinc-sequestering proteins are produced by
eukaryotic hosts in response to infections. Therefore, to grow and multiply in the infected
host, bacterial pathogens must produce high affinity zinc importers, such as the ZnuABC
transporter which is present in most Gram-negative bacteria. Studies carried in different
bacterial species have established that disruption of ZnuABC is usually associated with a
remarkable loss of pathogenicity. The critical involvement of zinc in a plethora of metabolic
and virulence pathways and the presence of very low number of zinc importers in most
bacterial species mark zinc homeostasis as a very promising target for the development
of novel antimicrobial strategies.
Keywords: zinc uptake, ZnuABC, antibacterial therapies, metal cofactor, host-pathogen interaction, Salmonella
enterica, zinc transporter, nutritional immunity
ZINC: CHEMICAL PROPERTIES AND ROLE IN BACTERIAL
PROTEINS
Among transition metals, zinc is likely the one which is used as
a structural or catalytic cofactor in the wider number of pro-
teins. The widespread use of zinc in proteins can be related to
its peculiar chemical properties (Andreini et al., 2008). Unlike the
other biological relevant transition metals (Fe2+,Mn
2+,Cu
2+,
Ni2+) the zinc ion (Zn2+)hasafilleddorbital and, therefore,
it is redox stable. Zinc mainly participates to catalytic reactions
by acting as a Lewis acid able to accept electron pairs or, as an
alternative, by attracting or stabilizing negative charges of the
substrates. Moreover, zinc binding to proteins is facilitated by its
capability to form stable chemical bonds with nitrogen, oxygen
and sulfur atoms and assume different coordination numbers.
As a consequence zinc can be found in a large variety of dis-
tinct chemical environments, which may significantly modulate
its reactivity. However, a potential problem of zinc is that it binds
to proteins stronger than the other divalent metals (Irving and
Williams, 1948) and, therefore, cells maintain the intracellular
pool of “free” metal at very low levels to prevent its unspecific
binding to proteins (Colvin et al., 2010).
Different studies have attempted to measure the amount of
zinc in bacteria. It has been shown that microorganisms have a
remarkable capability to modify their intracellular zinc content
in response to variations in the environmental availability of the
metal (Outten and O’Halloran, 2001; Garmory and Titball, 2004)
and that the total cellular zinc in bacteria growing in rich media is
in the submillimolar range (10−4M), i.e., a concentration com-
parable to that usually observed in most eukaryotic cells (Eide,
2006). More complex is to obtain a careful evaluation of the intra-
cellular pool of metal ions not tightly bound to proteins. In vitro
studies carried out with purified zinc-responding transcriptional
regulators have initially suggested that cellular “free” zinc levels
are in the femtomolar range, i.e., around 10−15 M(Outten and
O’Halloran, 2001). However, recent studies involving protein-
based ratiometric biosensors have established that in vivo the
concentration of intracellular exchangeable zinc is around 20pM,
i.e., 2 ×10−11M(Wang et al., 2011). Picomolar values of “free”
zinc have been reported also in several eukaryotic systems (Colvin
et al., 2010).
Interestingly, although the zinc concentration in bacterial cells
is close to that of iron, a significant fraction of iron may be found
in association to proteins such as ferritins, bacterioferritins or
DPS (Andrews et al., 2003), whereas bona fide zinc-storage pro-
teins are present only in a few bacteria (Blindauer et al., 2002).
An experimental attempt to explore the complexity of the bacte-
rial zinc proteome has shown that more than 3% of the proteins
expressed in Escherichia coli contain zinc (Katayama et al., 2002),
whereas bioinformatics investigations have revealed that about
5% of all bacterial proteins contain recognizable zinc-binding
sites (Andreini et al., 2006). This means that an E. coli cell with
about 4300 protein-encoding genes contains more than 200 zinc-
binding proteins. These figures, however, are not sufficient to have
an accurate idea of the actual importance of this metal in the
physiology of a bacterial cell. In fact, in addition to being an essen-
tial cofactor in a large number of enzymes involved in central
metabolic pathways, zinc is bound to several proteins involved
in the management of gene expression, including some riboso-
mal proteins (Hensley et al., 2011), RNA polymerases (Scrutton
et al., 1971), tRNA synthetases (Miller et al., 1991), sigma factor
interacting proteins (Campbell et al., 2007) and zinc responding
transcriptional factors (Chivers, 2007). Moreover, zinc is involved
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Cerasi et al. Zinc and bacterial virulence
in other crucial processes, including DNA repair (Kropachev
et al., 2006), response to oxidative stress (Ortiz De Orue Lucana
et al., 2012), antibiotic resistance (Meini et al., 2013)andpro-
duction of virulence-related proteins (Ammendola et al., 2008).
It follows that changes in the intracellular concentrations of zinc
can have pleiotropic effects on the composition of the bacte-
rial proteome, involving changes in the expression and activity
of zinc-containing proteins as well as of proteins which do not
employ this cofactor.
BACTERIAL ZINC UPTAKE SYSTEMS AND RESPONSE TO
ZINC SHORTAGE
Although in bacteria exposed to high levels of zinc the metal
may enter through a large number of unspecific channels, only
a few metal transporters are known to mediate the specific uptake
of zinc (Hantke, 2005)(Figure 1). Some recent studies on the
pneumococcal PsaA protein involved in manganese uptake have
provided interesting hints to understand the mechanisms ensur-
ing specificity in transition metal import (McDevitt et al., 2011;
Counago et al., 2013). PsaA may bind either manganese or other
first-row transition metals, but, due to the propensity of zinc to
form stable complexes with proteins (Irving and Williams, 1948)
it competitively affects Mn2+binding and locks the protein in a
conformation which prevents the entry either of zinc or of man-
ganese. This observation provides an explanation for the ability
of zinc to inhibit pneumococcal growth (McDevitt et al., 2011)
and an elegant example of the strategies used by living cells to
guarantee the correct uptake of specific metal ions (Wal dro n and
Robinson, 2009).
In several Gram-negative bacteria growing in metal replete
conditions, zinc uptake is thought to be primarily mediated by
ZupT, a constitutively expressed low affinity transporter belong-
ing to the ZIP (ZRT-, IRT-like Protein) protein family (Grass
et al., 2002). This metal permease has a broad metal specificity,
but it displays a clear preference for zinc over other divalent met-
als (Grass et al., 2005). ZupT depends on the proton motive force
to energize zinc import (Karlinsey et al., 2010; Taudte and Grass,
2010).
The response to zinc paucity is controlled through the coordi-
nated expression of a set of genes regulated by the transcriptional
factor Zur, which may bind two or more zinc ions, depending on
the species (Outten et al., 2001; Lucarelli et al., 2007; Shin et al.,
2011). One atom of zinc serves a structural role, whereas the other
atom(s) favors the folding of a DNA-binding domain enabling
the protein to tightly bind to a consensus sequence located in
the promoter of said genes. In contrast, when the intracellular
zinc content decreases, zinc-devoid Zur is no longer able to sta-
bly interact with DNA and to repress transcription. The number
of known Zur–regulated genes changes in different bacteria, but
in all species they include a small operon encoding for the com-
ponents of ZnuABC, a high affinity zinc importer of the ABC
family, and one or more genes encoding for paralogs of zinc-
containing ribosomal proteins (Panina, 2003; Graham et al., 2009;
Li et al., 2009; Lim et al., 2013). The ZnuABC uptake system is
composed of three proteins: the ZnuB channel, the ZnuC ATPase
component which provides the energy necessary for ion trans-
port through the inner membrane, and ZnuA, a soluble protein
which captures Zn(II) in the periplasm with high efficiency and
delivers it to ZnuB (Patzer and Hantke, 1998). In some bacteria
there is also an accessory component of the ZnuABC transporter,
ZinT, which is known to form a complex with ZnuA in pres-
ence of zinc and is thought to enhance ZnuA ability to recruit
zinc (Petrarca et al., 2010; Ilari et al., 2013). A similar zinc uptake
system (AdcABC) can be found in pneumococci and some other
Gram-positive bacteria, where the lipoprotein AdcA is charac-
terized by two structural domains that show clear sequence and
structural homology with ZnuA and ZinT, respectively (Dintilhac
et al., 1997; Panina et al., 2003).
An interesting facet of the Zur-mediated response to zinc
shortage is the substitution of ribosomal proteins containing zinc
with homologous proteins lacking the zinc-binding motif. This
change in ribosomal structure reduces the metal requirements
of bacterial cells, as the majority of intracellular zinc is thought
to be associated to ribosomes (Hensley et al., 2011). Moreover,
the production of zinc-independent ribosomal proteins may be
useful to mobilize a relevant amount of metal from pre-existing
ribosomes and facilitates the adaptation to zinc-limiting condi-
tions (Gabriel and Helmann, 2009). From this point of view, the
ribosome may be described as a zinc storage protein complex.
Additional paralogs of zinc-containing proteins have been identi-
fied in several bacteria (Haas et al., 2009) and include a homolog
of the transcriptional factor DksA which is involved in the con-
trol of the bacterial response to stress and starvation (Blaby-Haas
et al., 2011).
The outer membrane of Gram-negative bacteria allows the
passive diffusion of low molecular weight molecules. However, a
mechanism of nutrient uptake solely based on diffusion may be
hardly able to ensure the adequate absorption of elements which
are poorly available in the environment. In recent years, an outer
membrane TonB-dependent receptor involved in zinc uptake has
been identified in Neisseria meningitidis and some other Gram-
negative bacteria (Stork et al., 2010). This protein, denominated
ZnuD, mediates either zinc or heme uptake and is regulated either
by Zur or by Fur (Kumar et al., 2012; Pawlik et al., 2012). The
pneumococcal surface protein PhtD has been proposed to play a
functionally similar role in favoring zinc uptake through AdcAII
(Loisel et al., 2011).Nooutermembranezincreceptorshave
been so far identified in Enterobacteria or in other Gram-negative
bacteria. However, it has been observed that apo-ZinT can be
extruded outside the cell, suggesting that it could have some role
in the acquisition of zinc from the environment (Ho et al., 2008;
Gabbianelli et al., 2011).
It should also be noted that a few bacterial species have been
shown to express more than one high affinity zinc uptake sys-
tems. This is the case of Listeria monocytogenes which expresses
two ABC-type zinc importers (ZnuABC and ZurAM), both con-
tributing to full virulence (Corbett et al., 2012) and of non-
typeable Haemophilus influenzae, where the zinc binding system
ZevAB facilitates growth in zinc-limiting conditions and lung
colonization in infected mice (Rosadini et al., 2011). Similarly,
disruption of znuA in Pseudomonas aeruginosa results in a very
limited growth defect under zinc-limiting conditions (Ellison
et al., 2013), possibly due to the expression of a zinc-importing
P-t yp e ATPase (Lewinson et al., 2009).
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Cerasi et al. Zinc and bacterial virulence
FIGURE 1 | Schematic diagram of transporters involved in zinc uptake.
The bacterial outer membrane is thought to be permeable to hydrophilic
solutes of <600 dalton (Nikaido and Vaara, 1985) and, therefore, zinc
concentration in the periplasmic space is largely dependent on zinc availability
in the environment. Under zinc replete conditions (left), the metal is imported
through low affinity import systems, such as ZupT, and Zur inhibits the
expression of the importer ZnuABC. Under conditions of zinc shortage (right),
apoZur is unable to bind DNA and the high affinity zinc importer ZnuABC is
expressed. Neisseria meningitidis expresses a Zur-regulated TonB-dependent
outer membrane protein, ZnuD, involved in zinc uptake.
ZINC IN THE HOST-PATHOGEN INTERACTION
Whereas the competition for iron acquisition has been recognized
as a key element of the host pathogen interaction for a long time,
only in recent years the efficient uptake of other divalent metals
has emerged to play a comparable role (Kehl-Fie and Skaar, 2010).
In particular the importance of zinc has become clear through
a series of investigations which have established that deletion of
the znuABC genes not only decrease bacterial ability to growth
in in vitro environments poor of this metal, but also dramati-
cally affects their pathogenicity. Bacterial pathogens which have
been shown to critically depend on ZnuABC to infect their hosts
include Acinetobacter baumannii (Hood et al., 2012), Brucella
abortus (Kim et al., 2004; Yang et al., 2006), Campylobacter
jejuni (Davis et al., 2009), pathogenic E. coli strains (Sabri et al.,
2009; Gabbianelli et al., 2011), H. ducreyi (Lewis et al., 1999),
Moraxella catarrhalis (Murphy et al., 2013), Pasteurella multo-
cida (Garrido et al., 2003), Salmonella enterica (Campoy et al.,
2002; Ammendola et al., 2007)andYersinia ruckeri (Dahiya
and Stevenson, 2010). In contrast, while being required for zinc
uptake in vitro,ZnuABCdoesnotcontributetoY. pes t is virulence
(Desrosiers et al., 2010) and provides only a limited advantage
to Proteus mirabilis during urinary tract infections (Nielubowicz
et al., 2010). It is not yet clear whether these bacteria possess addi-
tional zinc importers or if they show limited zinc requirements
during infections.
Probably, the previous underestimation of the importance of
zinc in the interaction between bacteria and their hosts can be
largely attributed to the apparent abundance of this element in
all tissues. In fact, high levels of zinc are present either within
cells or in the plasma, where most of the metal is loosely associ-
ated to proteins (Zalewski et al., 2006) and, therefore, potentially
available for invading microorganisms. However, it should be
noted that a typical feature of the early response to the infec-
tion is the rapid fall of plasma zinc concentration, accompanied
by zinc accumulation in the liver. Redistribution of zinc among
the various tissues is regulated by a lipopolysaccharide-induced
cytokines cascade (with IL-6 playing a central role) which stim-
ulates increased synthesis of acute phase proteins, such as met-
allothionein, and the hepatic uptake of the metal through the
induction of the solute carrier 39 (SLC39) protein ZIP14 (Liuzzi,
2005). In view of the importance of the ZnuABC transporter in
bacterial zinc uptake during infections, this feature of the acute
phase response appears as an adaptive mechanism intended to
deprive pathogens of zinc.
The role of the ZnuABC transporter has been investigated in
details in S. enterica serovar Typhimurium (S. Typhimurium).
Expression of znuABC is repressed in S. Typhimurium culti-
vated in synthetic media containing zinc concentrations as low
as 1 µM. In contrast, the znuABC operon is strongly induced in
bacteria recovered from the spleens of infected mice or from cul-
tured epithelial or macrophagic cells (Ammendola et al., 2007).
These observations suggest that zinc bound to proteins is not eas-
ily available for invading bacteria and that ZnuABC is required
to have rapid access to the pool of “free” zinc. More recently,
it has been shown that during gut infections ZnuABC confers
resistance to the antimicrobial protein calprotectin (Liu et al.,
2012). Calprotectin is a neutrophilic protein of the S100 fam-
ily of calcium binding proteins, that is abundantly released at
sites of infection to control the multiplication of pathogens by
the sequestration of zinc and manganese (Kehl-Fie and Skaar,
2010). In support to the experimental evidence that calprotectin
starves bacteria for metal ions, structural studies have confirmed
that calprotectin possesses two distinct binding sites for transition
metals, one of which is specific for zinc and the other one may
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Cerasi et al. Zinc and bacterial virulence
accommodate either zinc or manganese (Brunjes Brophy et al.,
2013; Damo et al., 2013). Bacteria expressing ZnuABC are able to
resist to such antimicrobial strategy and this favor their growth
over competing microbes in the inflamed gut (Gielda and Dirita,
2012; Liu et al., 2012). Taken together, these studies suggest that
zinc acquisition through the ZnuABC transporter is essential for
the colonization of Salmonella in mice, and provide a parallelism
between the mechanisms of iron and zinc sequestration in the
host-pathogen relationships. It is worth noting that calprotectin
is not the unique S100 protein involved in zinc sequestration. In
fact, a comparable function has been proposed for the antibacte-
rial protein psoriasin (S100A7), which protects human skin from
E. coli infections (Glaser et al., 2005).
Although, the above mentioned studies have suggested that
zinc sequestration is a strategy widely used by vertebrates to con-
trol microbial infections, a few recent observations have revealed
an alternative way to use zinc in host defense. In fact, it has been
shown that human macrophages control mycobacteria by elevat-
ing zinc levels in the bacteria-containing phagosomes (Botella
et al., 2011). This process is dependent on reactive oxygen species
generated by the phagocytic NADPH oxidase and involves the
mobilization of zinc from intracellular stores. Mycobacterial resis-
tance to zinc intoxication in macrophages relies on their ability to
induce the expression of heavy metal efflux P-type ATPases, which
prevent the intracellular accumulation of zinc at toxic levels.
Structurally homologous zinc efflux pumps have been identi-
fied in a large number of bacteria, including E. coli,wherethe
P-type zinc exporter ZntA has been proved to be critical for
zinc tolerance (Beard et al., 1997; Rensing et al., 1997)andfor
the maintaining of appropriate levels of intracellular zinc (Wang
et al., 2012). Whereas mycobacteria lacking the efflux pump CtpC
or E. coli cells devoid of ZntA display a reduced ability to survive
in human macrophages, disruption of CtpC does not affect the
ability of M. tuberculosis to infect mice (Botella et al., 2011).
To add to the complexity, a mobilization of zinc in the oppo-
site direction to that found in response to mycobacteria has
been observed in murine macrophages infected with the fungus
Histoplasma capsulatum (Subramanian Vignesh et al., 2013). In
this case, phagosomes are deprived of zinc and the metal accu-
mulates in the Golgi or in the cytoplasm, in association to met-
allothioneins. Further studies are needed to understand whether
these different responses depend on the cross-talk between each
specific microorganism and phagocytic cells, or whether the abil-
ity to poison bacteria through an excess of zinc is a prerogative of
human macrophages.
ZINC HOMEOSTASIS AS A TARGET FOR ANTIBACTERIAL
THERAPIES
Different studies have proposed that ABC transporters could be
effective targets for the development of novel antibacterial drugs
or vaccines (Garmory and Titball, 2004; Counago et al., 2012). In
this view, ZnuABC appears as a particularly promising candidate.
Whereas a large number of pathogens produce multiple metal
import systems that enable the uptake of different iron forms
(reduced or oxidized, “free” or bound to proteins or to heme),
in most bacteria there is only one high affinity zinc importer.
Moreover, it has been shown that Salmonella strains lacking the
whole znuABC operon display the same dramatic loss of virulence
of strains producing ZnuB and ZnuC, but lacking ZnuA (Petrarca
et al., 2010). This finding indicates that the ability of ZnuA to
effectively compete for zinc binding with other periplasmic pro-
teins is critical to ensure zinc import in the cytoplasm (Berducci
et al., 2004) and suggests that drugs targeting the soluble com-
ponent of the transporter could block zinc import. This is a very
interesting possibility because ZnuA is a suitable substrate for bio-
chemical and structural studies (Banerjee et al., 2003; Chandra
et al., 2007; Wei et al., 2007; Yatsunyk et al., 2008; Ilari et al., 2011;
Castelli et al., 2013). It is worth noting that several molecules
able to interfere with zinc uptake in Candida albicans have been
recently identified through the screening of a small-molecule
library of 2000 compounds (Simm et al., 2011), thus providing
a proof of concept that it is possible to pharmacologically target
zinc homeostasis in pathogenic microorganisms.
Bacterial mutant strains unable to produce the ZnuABC trans-
porter are also putative candidate for the development of live–
attenuated vaccines. It has been shown that a S.Typhimurium
znuABC strain is able to induce short lasting infections in mice
which induce a solid and durable immune-based protection
against virulent strains of S. Ty phi mu r ium ( Pasquali et al., 2008;
Pesciaroli et al., 2011). The same strain proved to be attenuated,
immunogenic and protective also in pigs (Gradassi et al., 2013;
Pesciaroli et al., 2013). Similarly, it has been shown that vacci-
nation with Brucella znuA mutants protects mice from Brucella
infections (Yang et al., 2006; Clapp et al., 2011). In addition, it has
been shown that ZnuD is able to elicit the production of antibod-
ies triggering complement-mediated killing of several Neisseria
meningitidis serogroup B strains, suggesting that it is a promising
candidate for the generation of an effective vaccine (Hubert et al.,
2013). Taken together, these studies suggest that zinc homeosta-
sis offers interesting options to generate vaccines against different
pathogenic bacteria.
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Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Received: 01 October 2013; accepted: 11 December 2013; published online: 24
December 2013.
Citation: Cerasi M, Ammendola S and Battistoni A (2013) Competition for zinc bind-
ing in the host-pathogen interaction. Front. Cell. Infect. Microbiol. 3:108. doi: 10.3389/
fcimb.2013.00108
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