Molecular responses of Campylobacter jejuni to
cadmium stress
Nadeem O. Kaakoush
1
, Mark Raftery
2
and George L. Mendz
3
1 School of Medical Sciences, University of New South Wales, Sydney, Australia
2 Biological Mass Spectrometry Facility, University of New South Wales, Sydney, Australia
3 School of Medicine Sydney, University of Notre Dame Australia, Sydney, Australia
Cadmium ions (Cd
2+
) are a potent carcinogen in
animals, and cadmium is a toxic metal of significant
environmental and occupational importance for
humans [1–5]. Cadmium ions are very toxic even at
low concentrations, but the basis for their toxicity is
not fully understood. Cadmium is not a redox-active
metal and does not participate in Fenton-type reac-
tions. Moreover, it does not bind to DNA or interact
with DNA in a stable manner [1,2].
Several mechanisms have been proposed to explain
how bacteria and lower eukaryotes protect themselves
against cadmium toxicity. These include accumulation
of intracellular Zn
2+
, reduction of Cd
2+
uptake,
enhanced expression of the low-molecular weight cys-
teine-rich protein metallothionein that sequesters
cadmium, binding of cadmium ions by other heavy
metal-associated proteins, and an increase in intracellu-
lar disulfide content that contributes to effective bind-
ing of cadmium [6].
Disulfide reductases are responsible for the modula-
tion of intracellular disulfide concentrations. They are
essential enzymes in the antioxidant mechanisms of
Keywords
cadmium detoxification;
Campylobacter jejuni; citrate cycle;
glutathione; thioredoxin reductase
Correspondence
G. L. Mendz, School of Medicine Sydney,
University of Notre Dame Australia, Sydney,
NSW 2010, Australia
Fax: +61 293577680
Tel: +61 282044457
E-mail: GMendz@nd.edu.au
(Received 30 May 2008, revised 9 July
2008, accepted 11 August 2008)
doi:10.1111/j.1742-4658.2008.06636.x
Cadmium ions are a potent carcinogen in animals, and cadmium is a toxic
metal of significant environmental importance for humans. Response
curves were used to investigate the effects of cadmium chloride on the
growth of Camplyobacter jejuni.In vitro, the bacterium showed reduced
growth in the presence of 0.1 mmcadmium chloride, and the metal ions
were lethal at 1 mmconcentration. Two-dimensional gel electrophoresis
combined with tandem mass spectrometry analysis enabled identification of
67 proteins differentially expressed in cells grown without and with 0.1 mm
cadmium chloride. Cellular processes and pathways regulated under cad-
mium stress included fatty acid biosynthesis, protein biosynthesis, chemo-
taxis and mobility, the tricarboxylic acid cycle, protein modification, redox
processes and the heat-shock response. Disulfide reductases and their sub-
strates play many roles in cellular processes, including protection against
reactive oxygen species and detoxification of xenobiotics, such as cadmium.
The effects of cadmium on thioredoxin reductase and disulfide reductases
using glutathione as a substrate were studied in bacterial lysates by spectro-
photometry and nuclear magnetic resonance spectroscopy, respectively.
The presence of 0.1 mmcadmium ions modulated the activities of both
enzymes. The interactions of cadmium ions with oxidized glutathione and
reduced glutathione were investigated using nuclear magnetic resonance
spectroscopy. The data suggested that, unlike other organisms, C. jejuni
downregulates thioredoxin reductase and upregulates other disulfide reduc-
tases involved in metal detoxification in the presence of cadmium.
Abbreviations
GSH, reduced glutathione; GSSG, oxidized glutathione; MTA, 5¢-methylthioadenosine; SAH, S-adenosylhomocysteine; TCA, tricarboxylic acid.
FEBS Journal 275 (2008) 5021–5033 ª2008 The Authors Journal compilation ª2008 FEBS 5021
many bacteria, and also play a role in protection
against the toxic effects of heavy metals [7–9]. CXXC
motifs and CXXC-derived motifs are present in the
active sites of disulfide reductases [10], and are capable
of metal coordination and metal detoxification. Clus-
ters of cysteinyls capable of coordinating zinc atoms
are known as ‘zinc knuckles’ or ‘zinc fingers’ [10,11].
Glutathione reductase is an enzyme that is responsi-
ble principally for maintaining intracellular levels of
reduced glutathione (GSH, c-Glu-Cys-Gly) by recy-
cling the oxidized tripeptide (GSSG) to its reduced
form at the expense of oxidizing a molecule of
NAD(P)H. GSH has many roles in cellular processes,
including protection against reactive oxygen species
(ROS) and detoxification of xenobiotic compounds
[12]. GSH is therefore an essential metabolite in the
antioxidant mechanisms of many bacteria, and protects
them from the toxic effects of heavy metals [13,14].
For example, glutathione reductase was found to be
upregulated under cadmium stress in Lemna polyrrhiza
[15].
Cadmium has multiple molecular effects in various
organisms. In Chlamydomonas reinhardtii, exposure to
cadmium resulted in the downregulation of central
metabolism pathways such as fatty acid biosynthesis,
the tricarboxylic acid (TCA) cycle, and amino acid and
protein biosynthesis [16]. In contrast, proteins involved
in glutathione synthesis, ATP metabolism, response to
oxidative stress and protein folding were upregulated
in the presence of cadmium [16]. The effect of cad-
mium on protein expression in Rhodobacter capsulatus
B10 involved upregulation of heat-shock proteins
GroEL and 70 kDa heat shock protein (DnaK),
S-adenosylmethionine synthetase, ribosomal protein
S1, aspartate aminotransferase and phosphoglycerate
kinase [17]. An interesting study in Escherichia coli
found that cadmium-stressed cells recovered more rap-
idly than unexposed cells when subsequently subjected
to other stresses such as ethanol, osmotic, heat shock
or nalidixic acid treatment [18]. In Saccharomyces cere-
visiae, cells exposed to cadmium showed increased syn-
thesis of glutathione and proteins with antioxidant
properties [19]. A proteomic evaluation of cadmium
toxicity on Chironomus riparius Meigen larvae showed
downregulation of energy production, nucleotide bio-
synthesis, cell division, transport and binding of ions,
signal transduction regulating citrate ⁄malate metabo-
lism, and fatty acid and phospholipid metabolism [20].
Campylobacter jejuni belongs to an important group
of gastrointestinal spiral bacteria that have natural res-
ervoirs in many animals and birds that are in contact
with humans [21]; most human diseases caused by
organisms of the genus Campylobacter are due to
Campylobacter jejuni [21]. Little is known about the
detoxification defenses against metals in this micro-
aerophilic bacterium, which lives in habitats that are
subject to continual change. In the human gut, this
pathogen experiences turnover of the proliferative
intestinal epithelium and is exposed to the ever-chang-
ing chemical environment of the gastric tract that
results from the variety and combinations of food
ingested by higher animals. In addition, the bacterium
may encounter environments with diverse chemical
compositions before transmission to the host.
The inhibition of C. jejuni growth by cadmium ions
[22] and the reduction of inhibition by ferrous sulfate
[23] have been reported. Campylobacter isolates from
meat samples were shown to have higher tolerance to
Cd
2+
than clinical isolates [22], providing evidence
that strains with different habitats vary in their physi-
ologies. An important observation is that the genome
of C. jejuni NCTC 11168 does not contain genes
orthologous to those encoding glutathione reductase
or enzymes of the c-glutamyl cycle that are involved in
the synthesis of glutathione in other organisms.
In this study, changes induced in the proteome of
C. jejuni cells subjected to cadmium stress in vitro were
determined using two-dimensional gel electrophoresis
and mass spectrometry. In particular, a better under-
standing of the cellular role of disulfide reduction in
this microaerophilic human pathogen was achieved by
investigating the inhibition of glutathione reduction by
Cd
2+
in situ and in vitro, and the interactions of these
ions with glutathione and glutathione reductase.
Results and Discussion
Effects of cadmium on the survival of
Campylobacter jejuni
The effects of cadmium ions on the growth of C. jejuni
were measured at Cd
2+
concentrations of 0.05, 0.1,
0.3, 0.5 and 1 mm. Two colony-forming unit
(cfuÆmL
)1
) counts were taken at 0 and 24 h from each
culture (n= 3). The bacteria grew approximately
1.5 log (cfuÆmL
)1
)at0mmCd
2+
(Fig. 1). Inhibition
of C. jejuni growth increased with Cd
2+
concentration,
and the cation was lethal at 1 mmconcentration
(Fig. 1); changes in C. jejuni growth were observed at
micromolar concentrations of cadmium (Fig. 1). These
effects were comparable to those observed in other
bacteria and yeast [16,17,19]. The results indicated that
cadmium is highly toxic to C. jejuni, as is the case for
other microorganisms.
The growth-inhibition data enabled determination of
the Cd
2+
concentration at which C. jejuni cells could
Campylobacter jejuni and cadmium stress N. O. Kaakoush et al.
5022 FEBS Journal 275 (2008) 5021–5033 ª2008 The Authors Journal compilation ª2008 FEBS
be subjected to cadmium stress with only partial inhi-
bition of cell growth. At 0.1 mmCd
2+
,C. jejuni
growth was significantly decreased but the bacteria
remained viable.
Proteomic analyses of Campylobacter jejuni
under cadmium stress
The response of C. jejuni to 0.1 mmCd
2+
in the
growth medium was analyzed using two-dimensional
gel electrophoresis to determine the changes in the pro-
teome of the bacterium (Fig. 2). Two-dimensional gel
electrophoresis was performed using proteins extracted
from pairs of bacterial cultures grown with and with-
out Cd
2+
, and included three independent biological
repeats and one technical repeat. The four pairs of gels
obtained from cultures under both conditions were
analyzed to identify spots corresponding to proteins
whose expression was regulated under cadmium stress;
these proteins were identified using tandem mass spec-
trometry analyses. Sixty-seven proteins were differen-
tially expressed, of which 38 were downregulated and
29 were upregulated in the presence of Cd
2+
(Tables 1
and 2).
Bioinformatics analyses on regulated proteins
Effects on central metabolic pathways
Applying the functional classifications available in the
Kyoto Encyclopedia of Genes and Genomes (KEGG)
to the downregulated proteins in Table 1, it was con-
cluded that fatty acid biosynthesis and the TCA cycle
were downregulated. The former pathway is downreg-
ulated by metal ions in both prokaryotes and eukary-
otes [24–26]. Previous studies have suggested that the
effect of metals on fatty acid biosynthesis is indirect,
arising from changes induced in other metabolic path-
ways such as carbohydrate metabolism [25,26]. None-
theless, the modulation of fatty acid biosynthesis in
C. jejuni subjected to cadmium stress was notable. The
enzymes CJ1290c responsible for conversion of
acetyl CoA to malonyl CoA, and CJ0116 and
CJ0442 responsible for conversion of acetyl CoA to
acetyl ACP and malonyl CoA to malonyl ACP, respec-
tively, were all downregulated. In addition, the
enzymes CJ0442 and CJ1400c responsible for produc-
ing hexadecanoyl ACP from acetyl ACP or malo-
nyl ACP were also downregulated, indicating extensive
downregulation of fatty acid biosynthesis.
Fatty acid biosynthesis is the first step in membrane
lipid biogenesis. The downregulation of CJ0858c,
which catalyses the first step of lipopolysaccharide
synthesis, indicates that the pathway is disrupted from
its beginning. Similarly, CJ1054c, which catalyzes the
Fig. 1. Growth of C. jejuni NCTC 11168 in medium containing
CdCl
2
at various concentrations. Controls were cultures grown
without CdCl
2
. Bacteria were growth for 18 h in liquid cultures
under microaerobic conditions at 37 C.
4
p
I7
p
I74
Fig. 2. Two-dimensional pI 4–7 protein pro-
files of C. jejuni NCTC 11168 grown without
CdCl
2
(left) and in the presence of 0.1 mM
CdCl
2
(right). Proteins differentially
expressed between the two growth
conditions are listed in Tables 1 and 2.
N. O. Kaakoush et al. Campylobacter jejuni and cadmium stress
FEBS Journal 275 (2008) 5021–5033 ª2008 The Authors Journal compilation ª2008 FEBS 5023
first step of peptidoglycan biosynthesis, was also down-
regulated, indicating disruption of this pathway also.
These effects, together with downregulation of the cell
division protein FtsA (CJ0695), could explain the
decreased cell growth observed in bacteria subjected to
cadmium stress.
An interesting finding was the downregulation of
CJ0117, which catalyzes the hydrolysis of 5¢-methyl-
thioadenosine (MTA) to 5¢-methylthioribose or S-ade-
nosylhomocysteine (SAH) to S-ribosylhomocysteine
and adenine in prokaryotes but not mammalian cells;
both MTA and SAH are potent inhibitors of impor-
tant cellular processes in prokaryotes, such as trans-
methylation [27,28]. The accumulation of these
intermediates in the bacterium could induce metabolic
changes responsible for inhibition of central metabolic
pathways in C. jejuni, such as the TCA cycle (Table 1).
It has been proposed that adenylated compounds alert
cells to the onset of stress, thus accumulation of the
adenylated compounds MTA and SAH could simply
be the result of onset of cadmium stress. This response
has been shown in Salmonella typhimurium and
Synechococcus spp. [29,30]. Moreover, phenylalanyl
and seryl tRNA synthetases are the only two synthetases
Table 1. C. jejuni NCTC 11168 proteins identified as downregulated in the presence of 0.1 mMCdCl
2
in three independent cultures (n= 3).
Proteins in spots were identified by LC-MS tandem mass spectrometry analyses. The ORF numbers correspond to those of the annotated
genome of C. jejuni strain NCTC 11168.
Functional category Protein Protein name Spot no.
Amino acid metabolism CJ0117 Probable MTA ⁄SAH nucleosidase 1
CJ0402 Serine hydroxymethyl transferase 2
CJ0665c Argininosuccinate synthase 3
CJ0806 Dihydrodipicolinate synthase 4
CJ0858c UDP-N-acetyl glucosamine carboxyl transferase 5
CJ0897c Phenyl alanyl tRNA synthetase asubunit 6
CJ1054c UDP-N-acetylmuramate-L-alanine ligase 7
CJ1681c CysQ protein homolog 8
Cell division CJ0695 Cell division protein ftsA 9
Chemotaxis and mobility CJ0144 Methyl-accepting chemotaxis protein 10
CJ0262c Putative methyl-accepting chemotaxis protein 11
CJ1338c Flagellin B 12
CJ1339c Flagellin A 13
CJ1462 Flagellar P-ring protein precursor 14
Fatty acid biosynthesis CJ0116 Acyl carrier protein S-malonyltransferase 15
CJ0442 3-oxoacyl acyl carrier protein synthase II 16
CJ1290c Acetyl CoA carboxylase 17
CJ1400c Enoyl acyl carrier protein reductase 18
Glycolysis CJ0597 Fructose bis-phosphate aldolase 19
Nucleic acid metabolism CJ0146c Thioredoxin reductase 20
CJ0953c Bifunctional formyltransferase ⁄IMP cyclohydrolase 21
Redox CJ0779 Probable thiol peroxidase 22
TCA cycle CJ0409 Fumarate reductase 23
CJ0531 Isocitrate dehydrogenase 24
CJ0533 Succinyl CoA synthetase bchain 25
CJ0835c Aconitase 26
CJ0933c Putative pyruvate carboxylase B subunit 27
CJ1287c Malate oxidoreductase 28
CJ1682c Citrate synthase 29
Transport ⁄binding proteins CJ1443c KpsF protein 30
CJ1534c Possible bacterioferritin 31
CJ1663 Putative ABC transport system ATP-binding protein 32
Metabolism of vitamins CJ1046c Thiamine biosynthesis protein ThiF 33
Unknown CJ0172c Hypothetical protein 34
CJ0662c ATP-dependent protease ATP-binding subunit 35
CJ1024c Signal transduction regulatory protein 36
CJ1214c Hypothetical protein 37
CJ1725 Putative periplasmic protein 38
Campylobacter jejuni and cadmium stress N. O. Kaakoush et al.
5024 FEBS Journal 275 (2008) 5021–5033 ª2008 The Authors Journal compilation ª2008 FEBS
involved in the production of adenylated nucleotides
[31], and these two enzymes were found to be regu-
lated under cadmium stress.
Inhibitory effects of cadmium on the TCA cycle of
other organisms have been reported [26]. The presence
of Cd
2+
modulated expression of all the enzymes of
the TCA cycle in C. jejuni: seven were downregulated
and two (2-oxoglutarate oxidoreductase and fumarate
dehydratase) were upregulated. These data suggest that
operation of the TCA cycle was downregulated, and
that the upregulation of expression of 2-oxoglutarate
oxidoreductase and fumarate dehydratase was a
response to their other metabolic roles. Some bacteria
have developed metal detoxification pathways in which
the metal ion is first reduced by various c-type cyto-
chromes, hydrogenases and reduced ferredoxins, and
subsequently transported outside the cell [6,32]. 2-oxo-
glutarate oxidoreductase can reduce the low-redox-
potential protein ferredoxin, and its activity can lead
to higher intracellular concentrations of reduced ferre-
doxin than normal basal conditions. In the presence of
cadmium, the increased expression by C. jejuni of
2-oxoglutarate oxidoreductase, leading to elevated con-
centrations of reduced ferredoxin, and the upregulation
of a putative cytochrome cencoded by cj0037c are
important responses to cadmium ions that may act as
detoxification pathways in C. jejuni.
Downregulation of the expression of malate oxidore-
ductase and pyruvate decarboxylase decreases the
entry of pyruvate into the TCA cycle via malate or
oxaloacetate, respectively, and avoids futile cycling of
pyruvate driven by these two enzymes. Malate can still
be produced at normal concentrations from phospho-
enol pyruvate via oxaloacetate, and is converted to
aspartate through the activities of pyruvate dehydroge-
nase and aspartate lyase whose expression was upregu-
lated in the presence of cadmium ions. Similarly to
Helicobacter pylori [33], the dicarboxylic acid branch
of the TCA cycle of C. jejuni functions in the reductive
direction in the presence of excess malate converting it
Table 2. C. jejuni NCTC 11168 proteins identified as upregulated in the presence of 0.1 mMCdCl
2
in three independent cultures (n= 3).
Proteins in spots were identified by LC-MS tandem mass spectrometry analyses. The ORF numbers correspond to those of the annotated
genome of C. jejuni strain NCTC 11168.
Functional category Protein Protein name Spot no.
Amino acid metabolism CJ0087 Aspartate ammonia lyase 39
CJ0389 Seryl tRNA synthetase 40
CJ1096c S-adenosylmethionine synthetase 41
CJ1197c Aspartyl ⁄glutamyl tRNA amidotransferase subunit B 42
CJ1604 pAMP ⁄APP hydrolase 43
Cell division CJ0276 Homolog of E. coli rod shape-determining protein 44
Chaperones, heat shock CJ0759 Molecular chaperone DnaK 45
CJ1221 Heat-shock protein GroEL 46
Metabolism of vitamins CJ1045c Thiazole synthase 47
Oxidative phosphorylation CJ0107 ATP synthase subunit B 48
Protein translation and modification CJ0115 Peptidyl prolyl cis–trans isomerase 49
CJ0193c Trigger factor 50
CJ0239c NifU protein homolog 51
CJ0470 Elongation factor Tu 52
CJ0493 Elongation factor EF-G 53
Redox CJ0012c Rbo ⁄Rbr-like protein 54
CJ0037c Putative cytochrome c55
CJ0169 Superoxide dismutase 56
CJ0414 Putative oxidoreductase subunit 57
Signal transduction CJ0355c Two-component regulator 58
CJ0448c Putative MCP-type signal transduction protein 59
TCA cycle CJ0536 2-oxoglutarate ferredoxin oxidoreductase 60
CJ1364c Fumarate dehydratase 61
Transcription ⁄replication CJ0440c Putative transcriptional regulator 62
CJ1071 Single-stranded DNA-binding protein 63
Transport ⁄binding proteins CJ0612c Ferritin 64
CJ0734c Histidine-binding protein precursor 65
Unknown CJ1136 Putative galactosyl transferase 66
Virulence CJ0039c GTP-binding protein TypA homolog 67
N. O. Kaakoush et al. Campylobacter jejuni and cadmium stress
FEBS Journal 275 (2008) 5021–5033 ª2008 The Authors Journal compilation ª2008 FEBS 5025