MAJOR ARTICLE
An Antimicrobial Role for Zinc in Innate
Immune Defense Against Group A Streptococcus
Cheryl-lynn Y. Ong, Christine M. Gillen, Timothy C. Barnett, Mark J. Walker,a and Alastair G. McEwana
School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, University of Queensland, St. Lucia, Australia
(See the editorial commentary by Jarva on pages 1495–6.)
Background. Zinc plays an important role in human immunity, and it is known that zinc deficiency in the host
is linked to increased susceptibility to bacterial infection. In this study, we investigate the role of zinc efflux in the
pathogenesis of Streptococcus pyogenes (group A Streptococcus [GAS]), a human pathogen responsible for superficial
infections, such as pharyngitis and impetigo, and severe invasive infections.
Methods. The clinically important M1T1 wild-type strain was used in this study, and isogenic mutants were constructed with deletions in the czcD gene (Spy0653; which encodes a putative zinc efflux pump) and adjacent gczA gene
(Spy0654; which encodes a putative zinc-dependent activator of czcD). Wild-type, isogenic mutants and complemented strains were tested for resistance against zinc stress, intracellular zinc accumulation, and virulence.
Results. Both czcD and gczA mutants exhibited increased sensitivity to zinc. Transcriptional analyses indicate that
GczA upregulates czcD in response to zinc. Both mutants displayed increased susceptibility to human neutrophil killing and reduced virulence in a murine infection model. Furthermore, we showed that neutrophils mobilize zinc in
response to GAS.
Conclusions. These data indicate that the innate immune system may use zinc as an antimicrobial agent and that
zinc efflux is an important contributor to GAS pathogenesis.
Keywords. CzcD; zinc efflux; group A Streptococcus; Streptococcus pyogenes; zinc poisoning; innate immunity.
The acquisition of metal ions by bacterial pathogens is
recognized as being critical for their survival, but it has
also been observed that elevated levels of transition metals (especially Fe, Cu, and Zn) in the cytosol may result
in cellular toxicity [1]. Thus, it is essential for bacteria to
maintain metal ion homeostasis, and to achieve this
they have evolved sophisticated systems for the control
of uptake and efflux of metal ions [2]. The need for tight
homeostatic control is particularly true for zinc, an essential transition metal ion that has a key catalytic and/
or structural role in a wide variety of proteins [3]. Intracellular zinc concentration in the bacterial cell is tightly
Received 20 August 2013; accepted 6 December 2013; electronically published
20 January 2014.
a
M. J. W. and A. G. M. contributed equally to this work.
Correspondence: A. G. McEwan, PhD, School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia 4072, Australia ([email protected]).
The Journal of Infectious Diseases 2014;209:1500–8
© The Author 2014. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected].
DOI: 10.1093/infdis/jiu053
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regulated; zinc has a high affinity for S-ligands and
N-ligands, and if zinc ion concentrations were allowed
to rise then this ion would have the potential to inhibit
the action of many enzymes. As a consequence, the concentration of intracellular zinc is such that there is essentially no exchangeable zinc in the cell [3]. These
considerations suggest that excess zinc is highly toxic
toward microorganisms.
It is established that zinc deficiency is linked to increased susceptibility to bacterial infections [4–6].
Altered levels of zinc have been measured during inflammation, and high levels of zinc result in the activation of various immune cells [6, 7]. Zinc has been shown
to prevent the acquisition of manganese by Streptococcus pneumoniae, and this is consistent with the observation that zinc deficiency is linked to increased
susceptibility to pneumococcal infection [8]. Recently,
it has been shown that zinc is trafficked to the phagosome of macrophages infected with Mycobacterium tuberculosis, indicating that elevated zinc levels may play
an important antimicrobial role [9]. However, it is also
the case that the loss of ability to acquire zinc can also lead to
reduced virulence of some pathogens [10], indicating that bacterial pathogens may have to cope with both zinc deficiency and
the toxic effects of excess zinc during infection.
The present study focused on the obligate human pathogen
Streptococcus pyogenes (group A Streptococcus [GAS]), in which
a number of metal ion uptake/efflux systems have been studied
[10–12]. GAS possesses a number of genes involved in zinc acquisition that are regulated by the zinc-dependent repressor
AdcR [12]. This includes an extracellular solute-binding component protein (Lsp) that has high affinity and selectivity for
zinc. It has been shown that mutations in lsp resulting in hypersensitivity to zinc starvation are attenuated in a murine subcutaneous ulcer model of infection [10]. In addition to zinc uptake
systems, GAS possesses czcD (Spy0653), which encodes a cation
diffusion facilitator that is associated with resistance of cobalt,
zinc, and cadmium [13]. This was first identified in genome
analysis of the clinically important GAS serotype M1T1 clone
[14], but to date there has been no phenotypic characterization
of czcD mutants in GAS. Upstream of czcD is a gene that encodes a putative TetR-family transcriptional regulator that has
been shown in S. pneumoniae to regulate the transcription of
czcD [15].
GAS colonizes the skin and throat asymptomatically or causes mild superficial infections, such as impetigo and pharyngitis.
Occasionally, GAS penetrates into deeper tissues and may cause
severe invasive diseases such as septicemia, streptococcal toxic
shock-like syndrome and necrotizing fasciitis [16, 17]. GAS
must overcome a variety of stresses raised by the host innate immune system, including oxidative stress, in the form of hydrogen peroxide and other reactive oxygen species, and nitrosative
stress, in the form of nitric oxide and nitrite [18, 19]. Additionally, both reactive oxidative and nitrosative species have enhanced deleterious effects in the presence of metals such as
copper, iron, and zinc [20, 21]. Therefore, it is necessary to deploy the appropriate response to fluctuating levels of metal ions
in the various environments that the pathogen encounters within the human host. In this study, we investigated the role of the
CzcD zinc efflux pump and the TetR-family response regulator,
designated group A streptococcal czcD activator (GczA), in zinc
resistance. We demonstrate that CzcD and GczA confer resistance to zinc and not other divalent metal ions and that
GczA positively regulates czcD gene expression in response to
zinc. We also examine the role of the zinc efflux system in the
survival of GAS in human neutrophils and for GAS virulence in
a murine infection model.
MATERIALS AND METHODS
Bacterial Strains and Growth Conditions
The S. pyogenes M1T1 clinical isolate 5448 [22] and derivatives
were routinely grown on either 5% horse blood agar or statically
in liquid cultures at 37°C in Todd Hewitt broth plus 1% yeast
extract (THY). Escherichia coli MC1061 was grown on LuriaBertani medium. Where required, erythromycin was used at
2 μg/mL (for GAS) or 500 μg/mL (for E. coli), and spectinomycin was used at 100 μg/mL (for both GAS and E. coli). GAS isogenic mutants were constructed as previously described [23].
Bacterial strains used in this study are listed in Supplementary
Table 1.
DNA Manipulation and Genetic Techniques
GAS mutants 5448ΔczcD and 5448ΔgczA were constructed by
deletion replacement. The 1-kb regions upstream of czcD and
gczA were amplified using primers czcD1 plus czcD2 and
gczA1 plus gczA2, respectively, whereas the 1-kb downstream
regions were amplified using primers czcD3 plus czcD4 and
gczA3 plus gczA4, respectively. The spectinomycin cassette
was amplified using primers spec-F plus spec-R. The three
PCR fragments generated were joined together with primers
czcD1 plus czcD4 and gczA1 plus gczA4, respectively. The subsequent fragment was cloned into the pHY304 shuttle vector
and transformed into E. coli MC1061. Electrotransformation
and allelic replacement mutagenesis was undertaken using standard protocols [24, 25]. Complementation of 5448ΔczcD and
5448ΔgczA was performed by marker rescue. The entire regions
including the 1-kb regions upstream and downstream of czcD
and gczA were amplified using primers czcD1 plus czcD4 and
gczA1 plus gczA4, respectively. The construct was cloned into
pHY304 via restriction ligation and transformed into E. coli
MC1061. Electrotransformation and allelic replacement mutagenesis was undertaken as described above, and complemented
strains were screened for the loss of spectinomycin resistance.
All strains were confirmed by DNA sequencing (Australian
Equine Genome Research Centre, University of Queensland,
Brisbane, Australia). Plasmids used in this study are listed in
Supplementary Table 1. The primer sequences used in this
study are listed in Supplementary Table 2.
Growth Curve Analysis in the Presence and Absence of Zinc
Overnight cultures were diluted at a ratio of 1:2 in fresh THY and
grown in a static environment for 2 hours. The growing cells were
then diluted to an OD600 of 0.01 in fresh THY (in the presence of
0–2 mM ZnSO4•7 H2O). The cells were statically grown in a microaerobic environment (25 mL of medium in a 50-mL tube).
The OD600 of the cells was measured every half hour.
Metal Sensitivity Assay, Using a Drop Test
Overnight cultures were diluted to an OD600 of 0.01 in fresh
THY and grown until the OD600 reached 0.6. Cells were then
serially diluted 10-fold up to a 10−5 dilution, and 5 μL of each
dilution were spotted on THY plates supplemented with varying concentrations of zinc sulfate (0, 0.1, 0.25, 0.5, and 1 mM),
cobalt sulfate (0, 0.1, 0.25, 1, and 2 mM), or cadmium chloride
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(0, 5, 10, 50, and 100 μM). Plates were photographically documented following overnight incubation at 37°C.
Intracellular Zinc Concentration Measurement
Cells from an overnight THY agar plate (grown in the absence
or presence of 0.5 mM ZnSO4•7 H20) were resuspended and
washed 3 times with phosphate buffer/0.25 M EDTA and 3
times with phosphate buffer, resuspended in 80% nitric acid,
and incubated at 80°C for 24 hours. The samples were then diluted to 2% nitric acid and submitted for inductively coupled
plasmid mass spectrometry (ICP-MS) analysis at the School
of Earth Sciences, University of Queensland. The final value
was normalized to the amount of cells present by measuring
the total protein content in accordance with the QuantiPro
BCA assay kit (Sigma) instructions.
Quantitative Gene Expression Studies
RNA was isolated from cells harvested under the desired growth
phase (grown in the absence or presence of different metals in
the following concentrations: ZnSO4•7 H20, 1 mM; CoSO4•7
H2O, 0.5 mM; CdCl2, 10 μM; and NiCl2•6 H2O, 0.5 mM) in accordance with the RNeasy Mini kit (Qiagen) with the additional
mechanical lysis step in lysing matrix B tubes (MP Biomedicals). The isolated RNA was DNase treated using the RNaseFree DNase set (Qiagen) and quantified using a Nanodrop
instrument (Thermo Scientific). One microgram of RNA was
converted to complementary DNA, using the SuperScriptIII
first-strand synthesis system for reverse-transcription polymerase chain reaction (RT-PCR; Invitrogen). Real-time RT-PCR
was performed using the primers described in Supplementary
Table 2. The PCR reaction was performed using SYBR Green
Master Mix (Applied Biosystems) according to manufacturer’s
instructions. All data were analyzed using SDS 2.2.2 software
(Applied Biosystems). Relative gene expression was calculated
using the 2−ΔCT method, with gyrA as the reference gene.
Neutrophil Killing Assay
GAS survival following incubation with human neutrophils
in vitro was assayed as previously described [23]. Experiments were performed in triplicate using mid-logarithmic
phase (A600 = 0.4) GAS at a multiplicity of infection of 10:1
(GAS:neutrophils).
Immunofluorescence Microscopy
Immunofluorescence microscopy was performed using a modification of a previously described method [26]. In brief, human
neutrophils were seeded onto glass coverslips and infected with
GAS strains as described above. Thirty minutes after infection,
coverslips were washed 3 times with Dulbecco’s phosphatebuffered saline (PBS) and fixed in 4% paraformaldehyde. The
fixed cells were blocked overnight in PBS containing 2% bovine
serum albumin and 0.02% sodium azide. Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes and stained
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with DPBS containing AlexaFluor 555–conjugated rabbit antigroup A polysaccharide (1:100 dilution; PAB13831, Abnova),
Alexa 647–conjugated phalloidin (1 U/mL; Life Technologies)
and Zinquin (25 μM; Sigma-Aldrich) for 30 minutes. Coverslips
were then washed with DPBS and mounted onto glass slides,
and images were acquired on a Personal Deltavision inverted
microscope (Applied Precision). Maximum projection of deconvoluted images was performed using the ImageJ Fiji software package (available at: http://fiji.sc/wiki/index.php/Fiji).
Virulence of GAS in a Humanized Plasminogen Transgenic
Mouse Model
Transgenic humanized plasminogen AlbPLG1 mice heterozygous for the human transgene were backcrossed >6 generations
with C57BL/J6 mice [23]. GAS strains were prepared to obtain
the target dose of approximately 107 colony-forming units, 10
mice were subcutaneously infected, and virulence was observed
as previously described [23]. Bacterial load was determined by
enumerating colonies from blood and excised lesions 72 hours
after subcutaneous infection.
Statistical Analysis
Differences in growth rate, intracellular metal ion concentrations, and relative gene expression were analyzed using the 2tailed t test, and differences in neutrophil survival and Zinquin
fluorescence were analyzed using 1-way analysis of variance
(GraphPad, Prism 5). Mouse survival curves were compared
using the log-rank (Mantel-Cox) test (GraphPad, Prism 5).
Ethics Approval
All animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals (National
Health and Medical Research Council, Australia) and were approved by the University of Queensland Animal Ethics Committee. Human blood donation and use for the neutrophil
killing experiments were conducted in accordance with the
National Statement on Ethical Conduct in Human Research,
complied with the regulations governing experimentation on
humans, and were approved by the University of Queensland
Medical Research Ethics Committee.
RESULTS
The well-characterized M1T1 GAS isolate 5448 was examined
in this study. To determine the role of czcD and gczA in zinc
tolerance, isogenic deletion mutants and complemented mutants were constructed, and their genotypes were confirmed
using standard procedures (PCR and DNA sequencing). In
the absence of zinc, all deletion mutants and complemented
strains displayed growth characteristics similar to those of the
wild-type strain (Figure 1A). With the addition of increasing
zinc, the lag phase of the czcD and gczA deletion mutants was
significantly increased in THY (Figure 1A). Furthermore, the
Figure 1. Comparison of the growth of wild-type (WT) bacteria, isogenic deletion mutants, and complemented strains. A, Growth curve of 5448 (black
circles), 5448ΔczcD (white triangles), 5448ΔczcD::czcD (black triangles), 5448ΔgczA (white squares), and 5448ΔgczA::gczA (black squares) in Todd Hewitt
broth plus 1% yeast extract supplemented with increasing zinc concentrations (0.2 mM, 0.4 mM, 0.5 mM, 1.0 mM, and 1.5 mM). Graph is a representative
of 3 independent experiments. Drop test analysis of 5448, 5448ΔczcD, 5448ΔczcD::czcD, 5448ΔgczA, and 5448ΔgczA::gczA on THY agar supplemented with:
0, 0.1, 0.5 and 1 mM zinc sulfate (B); 0, 0.1, 0.25, 1, and 2 mM cobalt sulfate (C); 0, 5, 10, 50, and 100 μM cadmium chloride (D). Cells were grown up and
adjusted to OD600 0.6 and serially diluted, and 5-μL drops were spotted onto the plate, starting from the 100 dilution (top) down to the 10−5 dilution (bottom).
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specific growth rate of the deletion mutants in 1 mM zinc was
also significantly reduced, compared with the wild-type strain,
when grown in the presence of zinc (0.02 and 0.012 per hour,
compared with 0.28 per hour, respectively; P < .05). Similarly,
growth on solid medium demonstrated the same zinc inhibitory
effects on the czcD and gczA mutants. Serial dilutions of wildtype, deletion mutants, and isogenic complemented strains onto
THY plates supplemented with varying concentrations of zinc
demonstrated that both mutants displayed a distinct growth defect in the presence of increasing zinc concentration (Figure 1B).
Furthermore, complementation of both deletion mutants restored the ability to grow in the presence of zinc (Figure 1A
and 1B) to the same level as wild-type cells, confirming that
the phenotype of the mutants is attributable to the mutation
in czcD and gczA. Although previous studies have shown that
CzcD in Cupriavidus metallidurans, Bacillus subtilis, and
S. pneumoniae is responsible for resistance to zinc, cobalt,
and cadmium [13, 15, 27], no difference in inhibition of 5448
wild-type strain, deletion mutants, and complemented mutants
was observed in the presence of cobalt or cadmium (Figure 1C
and 1D). Thus, in GAS, CzcD appears to be specific for zinc
resistance.
To further understand the biochemical basis of the effect of
zinc on the growth of the czcD and gczA mutants, we determined
the cellular content of zinc, using ICP-MS analysis. Figure 2A
shows that the intracellular zinc content remained unchanged
(P > .05) for each sample, compared with the wild-type strain,
in all the strains when no additional zinc was added to the
growth medium. However, the addition of zinc resulted in a significant accumulation of intracellular zinc in both the czcD and
gczA deletion mutants (P < .001, Figure 2A). This result is consistent with a role for CzcD and GczA in zinc efflux.
In S. pneumoniae, czcD expression is regulated by SczA, a
TetR-family regulator [15]. In accordance with these findings,
czcD was upregulated >25-fold more in the wild-type 5448
strain grown in the presence of zinc (P = .0003, Figure 2B).
Moreover, no czcD gene upregulation in response to zinc was
observed in the gczA mutant (2-fold vs 25-fold; Figure 2B). Additionally, complementation of both czcD and gczA resulted in
the restoration of the czcD response to zinc (Figure 2B). In line
with findings observed in S. pneumoniae [15], these results also
suggest that czcD is upregulated by zinc via the action of GczA.
To determine whether GAS czcD is induced by transition
metals, 5448 wild-type cells were grown in the presence of
zinc, cobalt, cadmium, and nickel at sublethal concentrations,
samples were collected, and the relative gene expression of
czcD was measured using real-time RT-PCR. The results indicate that GAS czcD is upregulated primarily by zinc (Figure 2C).
Also, the effect of Co and Ni on czcD regulation was
insignificant.
Neutrophils are one of the first lines of defense of the innate
immune system against an invading microbial pathogen, and
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Figure 2. Comparison of the zinc accumulation and relative gene expression of wild-type bacteria, isogenic deletion mutants, and complemented strains. A, Intracellular zinc analysis of 5448, 5448ΔczcD,
5448ΔczcD::czcD, 5448ΔgczA, and 5448ΔgczA::gczA grown in Todd Hewitt
broth plus 1% yeast extract in the absence (black bars) or presence (white
bars) of 1 mM zinc. Unpaired, 2-tailed t tests were performed for 5448 versus 5448ΔczcD and for 5448 versus 5448ΔgczA grown in the presence of
zinc. ***P < .001. B, Relative expression of czcD in 5448 wild-type bacteria,
5448ΔczcD, 5448ΔczcD::czcD, 5448ΔgczA, and 5448ΔgczA::gczA grown in
the absence (black bars) or presence (white bars) of 0.5 mM zinc. Samples
were collected during the exponential phase of growth, and error bars are
indicative of the SDs of 3 independent experiments. The relative expression
of each gene was calculated using the 2−ΔCT method, with gyrA as the
reference gene. C, Relative expression of czcD in 5448 wild-type cells
grown in the presence of various transition metals.
they play a key role in the clearance of GAS infections [17].
To determine whether czcD and gczA might play a role in the
defense against neutrophil killing of GAS, we initially examined
the relative gene expression of czcD and gczA in the wild-type
Figure 3. Interaction of wild-type (WT) bacteria, isogenic deletion mutants, and complemented strains with human neutrophils. A, Relative gene expression of czcD and gczA in 5448 wild-type bacteria in contact with neutrophils. Black bars indicate 5448 wild-type control, and white bars indicate 5448
wild-type 0.5 hours after infection with neutrophils. The graph is the relative gene expression of each gene calculated using the 2−ΔCT method, with gyrA as
the reference gene. Samples were collected 30 minutes after combination with neutrophils or Roswell Park Memorial Institute medium (control), and the
error bars are indicative of the SDs of 3 independent experiments. B, Neutrophil killing assay. Percentage survival following coculture with human neutrophils in vitro. One-way analysis of variance (ANOVA) was performed for 5448 versus 5448ΔczcD and for 5448 versus 5448ΔgczA. **P < .005, ***P < .001.
C, Neutrophil killing assay in the presence of 1 μM N,N,N’,N’-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Percentage survival following coculture
with human neutrophils in vitro. D, Quantitation of Zinquin (zinc fluorescent probe) fluorescence, demonstrating the release of free zinc by the neutrophils
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Figure 4. Reduced virulence of the czcD and gczA deletion mutants in the murine model of group A streptococci (GAS) infection. A, Survival duration
after subcutaneous challenge of transgenic humanized plasminogen AlbPLG1 C57BL/J6 mice with 5448 wild-type (WT) bacteria, compared with challenge
with the 5448ΔczcD deletion mutant and the isogenic complemented strain 5448ΔczcD::czcD. Infecting dose, 2 × 107 colony-forming units (CFUs). B, Survival duration after subcutaneous challenge of transgenic humanized plasminogen AlbPLG1 C57BL/J6 mice with 5448 wild-type bacteria, compared with
challenge with the 5448ΔgczA deletion mutant and the isogenic complemented strain 5448ΔgczA::gczA. Infecting dose, 1 × 107 CFUs.
strain following a 30-minute exposure to human neutrophils.
The relative gene expression of czcD was increased 6-fold
in the wild-type strain after neutrophil exposure (P = .0001;
Figure 3A). Similarly, the relative gene expression of gczA was
increased 4-fold in the wild-type strain after incubation with
neutrophils (P = .0001; Figure 3A), indicating that expression
of these genes are upregulated in response to neutrophil
exposure.
The survival of the czcD and gczA deletion mutants in the
presence of neutrophils was compared with that of wild-type
strain 5448 and the isogenic complemented strains. The czcD
and gczA deletion mutants had a significantly reduced capacity
to survive neutrophil-killing mechanisms, compared with wildtype and complemented mutant strains (P = .0035 and .0004,
respectively; Figure 3B). These results demonstrate that zinc efflux may be an important component in the ability of GAS to
resist neutrophil killing. Furthermore, addition of a zinc chelator, tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), rescued the deletion mutants to the wild-type resistance levels
against neutrophil killing (Figure 3C). These results suggest
that neutrophils use zinc poisoning as a means of eliminating
microbial pathogens. Changes in exchangeable zinc in the cell
can be measured with the use of a zinc fluorescent probe, Zinquin. We observed that there was a significant increase in fluorescence when neutrophils were infected with GAS, compared
with resting neutrophils (P = .001; Figure 3D). Additionally,
this fluorescence was quenched by the addition of TPEN
(P < .0001, Figure 3D), indicating that it is a zinc-specific signal.
In addition, immunofluorescence microscopy demonstrated the
movement of zinc within the neutrophils during GAS infection
with the use of Zinquin (Figure 3E). Infected neutrophils with
internalized bacteria possessed higher fluorescence when
stained with Zinquin (cyan; Figure 3E), compared with resting
neutrophils.
To assess the virulence of czcD and gczA deletion mutants, we
used a transgenic humanized plasminogen mouse model of invasive infection [28]. Following subcutaneous infection, mouse
survival was significantly higher when infected with the czcD
deletion mutant, compared with the wild-type and complemented strains, at a dose of 2 × 107 colony-forming units
(P = .021; Figure 4A). Similarly, mouse survival was also higher
when infected with the gczA deletion mutant, compared with
the wild-type and complemented strains, at a dose of 1 × 107
colony-forming units (P = .05; Figure 4B). Taken together,
these results indicate that the czcD/gczA regulon plays an
important role in GAS virulence.
DISCUSSION
Protection against zinc toxicity was originally considered in the
context of bacteria isolated from environments contaminated
with heavy metal cations [2]. In Gram-negative bacteria,
CzcCBA has been identified as a major zinc efflux pump that
transports zinc across the outer membrane, leading to the
Figure 3 continued. during an infection with group A streptococci (GAS). The fluorescence was quenched with the addition of an equal concentration of
TPEN. Zinquin fluorescence was measured using the excitation/emission of 370/490 nm, and results are represented by an arbitrary unit (ie, fluorescence
units). Fluorescence was measured 30 minutes after combination of neutrophils with and without GAS, and the error bars are indicative of the SDs of 3
independent experiments. One-way ANOVA was performed for neutrophils with and without GAS and for neutrophils with GAS, with and without TPEN.
**P = .001, ****P < .0001. E, Immunofluorescence micrograph demonstrating the release of free zinc within the neutrophils during an infection with GAS.
F-actin of neutrophils is stained with AlexaFluor 647 phalloidin (red). GAS cells are stained with anti-group A carbohydrate (green), and zinc is visualized
with the use of a zinc fluorescent probe, Zinquin (cyan). Arrows indicate neutrophils with internalized GAS chain. Scale bar represents 5 μm.
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term “transenvelope transport” [2]. The inner membrane component of this transport system, CzcD, is also commonly found
in isolation from other Czc components. The energy of the proton-motive force is sufficient to export zinc across the cytoplasmic membrane; in the case of gram-positive bacteria, this means
that zinc ions are excluded from the cell. Our observations that a
czcD mutant of GAS was more susceptible to zinc killing and
increased intracellular zinc accumulation (Figures 1 and 2A)
are consistent with the recognized biological role for this transporter [29]. However, CzcD in other bacteria has been shown to
be involved in the efflux of zinc and a range of other cations,
including Co, Cd, Ni, and Cu [2, 15, 27]. The results of this
study show that CzcD confers only zinc resistance in GAS, suggesting that this particular transporter has more-restricted
metal ion specificity and may have evolved specifically to protect GAS cells against zinc toxicity.
We also observed that czcD expression was increased when
GAS came into contact with neutrophils, indicating that
under these conditions the GAS cells are in an environment
where the zinc level is high. Our observation that the czcD
and gczA mutants deficient in zinc efflux are less able to survive
killing by neutrophils, compared with wild-type GAS, is consistent with this view and leads to the possibility that release of exchangeable zinc is used by neutrophils as an antimicrobial
agent. We also observed that the czcD and gczA mutants exhibited reduced virulence in an invasive murine model of GAS
infection (Figure 4). Furthermore, only mice infected with
wild-type and czcD complemented strains and not the czcD mutant disseminated into the blood (Supplementary Figure 1). It is
established that zinc deficiency is linked to increased susceptibility to bacterial infections [4, 6]. High levels of zinc have been
measured during inflammation, and high levels of zinc in the
body result in the activation of various immune cells [6, 7].
Zinc levels in blood serum of infected mice can reach up to
900 μM [8], a biologically relevant concentration that was
used in our in vitro studies. Recent studies that have demonstrated that zinc poisoning through the release of free zinc
ions was used by macrophages to kill the invading pathogen
[9]. Our results suggest that this innate immune method of
eliminating microbial pathogens may also be used by neutrophils. Typically, mobilizable zinc is stored within vacuoles and
in storage proteins, such as metallothionein [3, 30]. And, although calprotectin represents about 40% of the cytosolic proteins in neutrophils [31, 32], it may not be a site of zinc binding
within neutrophils, since this protein only has a high affinity for
zinc when it encounters elevated calcium concentrations in the
extracellular environment [33]. Although the source of intracellular zinc will be the focus of future research, our results show
that the level of this metal ion is elevated within the neutrophils
following GAS internalization.
So far, no definitive studies have quantified zinc levels in the
human nasopharynx (a common niche for GAS), but research
has been undertaken showing that zinc is relatively higher in the
lungs, kidneys, liver, and skeletal muscle [34, 35]. Zinc levels are
significantly increased in the nasopharynx and blood serum in
the murine model during an infection [8]. Hospital trials have
shown that zinc supplementation resulted in reduced severity to
acute respiratory tract infections and pneumonia in children
[36, 37]. In contrast to the above findings, it is established that
GAS mutants deficient in zinc acquisition have reduced ability
to colonize skin [10]. Thus, it appears that both zinc acquisition
and zinc efflux is required for GAS pathogenesis, but the relative
importance of these 2 processes may be dependent upon the tissue or niche in which the pathogen is located.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org/). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Notes
Acknowledgments. C. Y. O., M. J. W., and A. G. M. designed
experiments. C. Y. O. performed and analyzed the experiments. C. M. G.
provided assistance with mouse virulence experiments, and T. C. B. provided assistance with immunofluorescence microscopy. C. Y. O., A. G. M., and
M. J. W. wrote the manuscript.
Financial support. This work was supported by the National Health
and Medical Research Council of Australia (grant 565526).
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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