Molecular responses of Campylobacter jejuni tocadmium stressNadeem O. Kaakoush1, Mark Raftery2 and George L. Mendz3

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 (Cd2+) 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 Zn2+, reduction of Cd2+ 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 mm cadmium chloride, and the metal ions

were lethal at 1 mm concentration. 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 mm cadmium 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 ª 2008 The Authors Journal compilation ª 2008 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

Cd2+ 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

Cd2+ 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 Cd2+ 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) at 0 mm Cd2+ (Fig. 1). Inhibition

of C. jejuni growth increased with Cd2+ concentration,

and the cation was lethal at 1 mm concentration

(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 Cd2+ concentration at which C. jejuni cells could

Campylobacter jejuni and cadmium stress N. O. Kaakoush et al.

5022 FEBS Journal 275 (2008) 5021–5033 ª 2008 The Authors Journal compilation ª 2008 FEBS

be subjected to cadmium stress with only partial inhi-

bition of cell growth. At 0.1 mm Cd2+, 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 mm Cd2+ 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 Cd2+, 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 Cd2+ (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

CdCl2 at various concentrations. Controls were cultures grown

without CdCl2. Bacteria were growth for 18 h in liquid cultures

under microaerobic conditions at 37 �C.

4 pI 7 pI 74

Fig. 2. Two-dimensional pI 4–7 protein pro-

files of C. jejuni NCTC 11168 grown without

CdCl2 (left) and in the presence of 0.1 mM

CdCl2 (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 ª 2008 The Authors Journal compilation ª 2008 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 mM CdCl2 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 a subunit 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 b chain 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 ª 2008 The Authors Journal compilation ª 2008 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 Cd2+ 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 c encoded 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 mM CdCl2 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 c 55

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 ª 2008 The Authors Journal compilation ª 2008 FEBS 5025

to pyruvate and then to succinate; this last step is cata-

lyzed by pyruvate reductase. Expression of this enzyme

was downregulated under cadmium stress; as a result,

the pyruvate produced by pyruvate dehydrogenase

could be directed to the synthesis of aspartate.

Increased production of this amino acid could reduce

intracellular cadmium concentrations by chelating the

metal ions [34,35], and could remove free ammonium

by incorporating it into aspartate. Reduction of the

intracellular ammonium concentration could explain

the downregulation of expression of the urea cycle

enzyme argininosuccinate lyase, as reduced use of the

urea cycle is necessary to maintain homeostasis of

intracellular nitrogen levels.

At the same time, an increase in malate concentra-

tion in the cells could play an important role in the

solubilization of cadmium, which is a function of

malate and other organic acids, as shown in rhizo-

sphere soil [36]. The ability of malate to bind cadmium

[37] and to detoxify metals in other organisms [38,39]

suggests that it could be part of a cadmium detoxifica-

tion process used by C. jejuni.

Effects on amino acid biosynthesis

Effects of cadmium on amino acid biosynthesis have

been reported previously; for example, cadmium

inhibits or blocks the threonine pathway in E. coli

[40]. In C. jejuni, cadmium appeared to enhance the

synthesis of aspartate from pyruvate through upregu-

lation of the expression of aspartate ammonia lyase

(CJ0087). The upregulation of expression of pAM-

P ⁄APP hydrolase encoded by cj1604 under cadmium

stress suggested an increase in purine and ⁄or histidine

biosynthesis. Since expression of the last enzyme of

de novo purine biosynthesis, PurH (CJ0953c), is

downregulated, the results suggest that synthesis of

histidine, an amino acid with very high affinity for

metal ions, was upregulated. The downregulation of

PurH and dihydrodipicolinate synthase (CJ0806) sug-

gest a decrease in the synthesis of arginine and lysine

using aspartate as a precursor. The increased produc-

tion of aspartate and decreased utilization in synthetic

pathways could constitute another mechanism used

by the bacterium for cadmium ion detoxification. The

downregulation of serine hydroxymethyl transferase

(CJ0402) suggests inhibition of glycine synthesis, as

this is the only de novo glycine pathway that has been

identified in C. jejuni.

In summary, cadmium had an inhibitory effect on

central metabolic pathways of C. jejuni, and appeared

to enhance the production of metabolites that may be

utilized for detoxification.

Effects on protein repair and oxidoreduction systems

The expression of proteins involved in translation ⁄modification and oxireduction and of chaperones was

upregulated. Cadmium is capable of displacing metal

ions in proteins and affecting their structure and fold-

ing [41]. The upregulation of protein translation and

modification and of expression of chaperones such as

heat-shock proteins in response to Cd2+ stress has been

reported previously [17]. The elongation factors upregu-

lated in C. jejuni exposed to cadmium are required for

extending the polypeptide chain in protein translation,

and the heat-shock proteins are required for proper

protein folding. These findings indicate that the cells

are responding to the negative effects of cadmium on

protein synthesis. Further evidence is provided by the

downregulation of an ATP-dependent protease subunit

encoded by cj0662c that is capable of degrading heat-

shock proteins. The NifU protein homolog encoded by

cj0239c participates in iron–sulfur center assembly [42];

its upregulation may help to counter cadmium-induced

displacement of iron from proteins.

Removal of iron bound to various cellular compo-

nents can cause a cascade of reactions leading to an

increase in oxidative stress in the cells. Upregulation of

proteins involved in oxireduction reactions helps to

combat the toxic effects of oxidative stress. This

response is found in E. coli, in which cadmium upregu-

lated proteins of heat shock and oxidative stress regu-

lons [43]. Similarly, exposure of anterior gills of the

Chinese mitten crab Eriocheir sinensis to cadmium

upregulated the expression of several antioxidant

enzymes and chaperonins [44]. Metaproteomic analyses

of the response of bacterial communities to cadmium

indicated that oxidoreductases were differentially

expressed [45]. Finally, transcriptional analyses of Cau-

lobacter crescentus cells exposed to cadmium showed

that the principal response to this metal was protection

against oxidative stress [46]. These observations

support the view that induction of oxidative stress and

binding of sulfhydryl groups are mechanisms of

cadmium toxicity [44].

An important detoxification mechanism is the trans-

formation of metals into organometallic compounds

by methylation, and the synthesis of several organo-

cadmium compounds has been demonstrated [6,47].

Adenosylmethionine occupies a central metabolic

position in both eukaryotes and prokaryotes, serving

as a major methyl group donor in biological systems

[27]. The upregulation of S-adenosylmethionine

synthetase encoded by cj1096c in bacteria exposed to

cadmium could promote cadmium methylation, and

thus neutralize the toxic effects of the metal.

Campylobacter jejuni and cadmium stress N. O. Kaakoush et al.

5026 FEBS Journal 275 (2008) 5021–5033 ª 2008 The Authors Journal compilation ª 2008 FEBS

Effects on chemotaxis and motility

No chemotaxis or motility genes showed regulated

expression under cadmium stress in E. coli or C. cres-

centus [41,46], but heavy metal ions strongly affect Bor-

relia burgdorferi motility [48]. The downregulation of

five proteins involved in chemotaxis and motility in

C. jejuni exposed to cadmium stress (Table 1) suggested

a decrease in these functions of the bacterium. Bacterial

motion is driven by either a proton motive force or a

sodium motive force [49,50], and the presence of heavy

metal ions may interfere with this function, leading to

downregulation of genes involved in motility.

The N-terminal half of CJ1024c has a signal-receiver

domain (REC) for proteins such as the chemotaxis

protein (CheY), the outer membrane protein (OmpR),

the bacterial enhancer-binding protein (NtrC), and the

activator protein (PhoB), and in its middle segment

there is a r54 interaction domain [51]. Transcription

of the flaB gene encoding flagellin B is regulated by

sigma factor r54 [51]. Thus, downregulation of

CJ1024c in the presence of cadmium may result in

downregulation of signaling by chemotaxis proteins

and transcription of flagellin B.

Effects on metal uptake and storage

A putative ABC transport system ATP-binding protein

encoded by cj1663 and a hypothetical protein encoded

by cj0172c were downregulated. The STRING tool

[52] predicted that the gene cj0172c is in a network

with cj0173c, cj0174c and cj0175c, which encode an

iron uptake ABC transport system, and with cj0271,

which encodes a bacterioferritin conjugatory protein

homolog. The bacterioferritin CJ1534c, which contains

heme and is involved in iron uptake, was also down-

regulated. In contrast, the heme-free ferritin encoded

by cj0612c involved in intracellular iron storage was

upregulated. Ferritin is involved in the primary detoxi-

fication response to heavy metals including Cd2+ in

Xenopus laevis cells [53]. The principal function of

ferritins is to store iron inside cells in the ferric form; a

secondary function could be detoxification of iron or

protection against O2 and its reactive products. A

C. jejuni CJ0612c-deficient mutant was more suscepti-

ble to killing by oxidant agents than the parent strain,

thus demonstrating that this ferritin makes a signifi-

cant contribution to protection of the bacterium

against oxidative stress [54]. It has been hypothesized

that C. jejuni CJ0612c plays a role mainly in regulating

cellular iron homeostasis by storing and releasing iron

under iron-restricted conditions, whereas C. jejuni

CJ1534c contributes mainly to protection against

oxidative stress by sequestering cellular free iron to

prevent the generation of hydroxyl radicals [55]. This

bacterioferritin may have a greater involvement than

ferritin CJ0612c in protection against oxidative stress,

but it contains heme, whose synthesis might be affected

by cadmium ions. For instance, in Bradyrhizobi-

um japonicum, an engineered d-aminolevulinic acid

dehydratase that uses Zn2+ for activity is inhibited by

Cd2+ ions [56]. d-aminolevulinic acid dehydratase is an

enzyme of the heme synthesis pathway that exists in

C. jejuni. This may explain why expression of the heme-

free ferritin was upregulated and expression of the

heme-containing bacterioferritin was downregulated.

CJ0355c has 58% similarity with CzcR of Strepto-

coccus agalactiae, and was upregulated under cadmium

stress. Czc systems have been studied in detail in Alca-

ligenes eutrophus and Pseudomonas aeruginosa [57,58].

Induced mechanisms of bacterial resistance to heavy

metals increase the expression of the heavy metal efflux

pump CzcCBA and its cognate two-component regula-

tor CzcR–CzcS in A. eutrophus [57] and P. aeruginosa

[58]. Furthermore, the cadmium stress response of

C. crescentus also involved reduction of the intracellu-

lar cadmium concentration using multiple efflux pumps

[46].

Finally, the rubredoxin-like protein encoded by

cj0012c was upregulated. This type of protein is

sensitive to oxidative stress and capable of forming

complexes such as [Cd(CysS)4]2 with metals [59]. The

upregulation of CJ0012c may be another mecha-

nism used by C. jejuni to protect itself against Cd2+

toxicity.

In summary, these observations suggested that, in

the presence of cadmium, C. jejuni downregulates pro-

teins involved in metal uptake and upregulates proteins

that are capable of binding, storing and exporting met-

als. In addition, the upregulation of proteins involved

in iron storage is in agreement with the ability of

cadmium to displace iron from proteins.

Effects on other cellular processes

Expression of the proteins CJ0355c and CJ0448c,

which participate in signal transduction, and CJ0440c

and CJ1071, which are involved in transcription, was

upregulated. Signal transduction cascades are essential

for metal-inducible protein transcription [60]. The

upregulation of these four proteins suggests that

C. jejuni may contain a metal-responsive signal

transduction pathway.

The upregulated ATP synthase subunit B encoded

by cj0107 forms part of the oxidative phosphorylation

pathway responsible for the production of ATP; this

N. O. Kaakoush et al. Campylobacter jejuni and cadmium stress

FEBS Journal 275 (2008) 5021–5033 ª 2008 The Authors Journal compilation ª 2008 FEBS 5027

pathway is also upregulated in other organisms sub-

jected to metal stress [26]. Oxidative phosphorylation

generates high-energy ATP, and upregulation of the

expression of this synthase may serve to offset the

downregulation of expression of TCA cycle enzymes

under cadmium stress.

Finally, a TypA homolog encoded by cj0039c and a

rod shape-determining protein encoded by cj0276 were

upregulated. Homologs of both these proteins have

been associated with virulence in other organisms

[61,62]. Cadmium-stressed E. coli were found to

recover more rapidly during subsequent stress condi-

tions than unexposed cells [18]. Cadmium is capable of

upregulating proteins involved in the virulence pheno-

type of C. jejuni that possibly make the bacterium

more tolerant to stresses such as the oxidative

bursts by the host’s immune system, hence exposure of

C. jejuni to cadmium ions may enhance its virulence,

with significant consequences for the hosts.

Confirmation of changes in the proteome

Changes in the proteome of C. jejuni exposed to cad-

mium stress were confirmed by measuring enzyme

activities that reflect changes in protein levels. Many

studies use quantitative real-time PCR to verify the

results of proteomic analyses, but this method detects

regulation at the transcription level and is more suit-

able for confirmation of transcriptome data. The activ-

ities of several enzymes of the TCA cycle were

measured because they are involved in the central

metabolism of the cell, and previous studies have

shown that this pathway is commonly regulated under

cadmium stress. Thioredoxin reductase activity was

determined because this enzyme is involved in the

response of other organisms to cadmium; thus, the

downregulation of its expression by C. jejuni required

verification.

Upregulation of fumarate dehydratase and 2-oxo-

glutarate ferredoxin oxidoreductase activities was veri-

fied using proton nuclear magnetic resonance

spectroscopy (1H-NMR) spectroscopy. Fumarate dehy-

dratase activity was 1.4-fold higher in whole-cell

lysates of cells grown with 0.1 mm cadmium than in

lysates of cells grown without cadmium. The activity

of 2-oxoglutarate ferredoxin oxidoreductase was two-

fold higher in cell-free extracts of cells grown with cad-

mium than in extracts of cells grown without

cadmium. Downregulation of fumarate reductase and

thioredoxin reductase activities were confirmed using1H-NMR spectroscopy and spectrophotometry, respec-

tively. Their activities were 1.3-fold lower in lysates

and 1.5-fold lower in cell-free extracts of cells grown

with cadmium than in cells grown without cadmium,

respectively. The changes in the reduction rates of the

four enzymes were in agreement with the regulation of

protein expression observed in the proteomic analyses.

Disulfide reductases in cadmium detoxification

The involvement of disulfide reductases, including

thioredoxin reductase, in cadmium detoxification has

been demonstrated in several microorganisms. For

example, S. cerevisiae strains lacking thioredoxin and

thioredoxin reductase are hypersensitive to cadmium

[19,35]. The genomes of many species of Campylobac-

terales bacteria do not contain genes orthologous to

those in other organisms that encode glutathione

reductases or enzymes of the c-glutamyl cycle for syn-

thesis of glutathione [63], and the thioredoxin system is

the only disulfide redox system that is present in these

bacteria. The activity of the metalloenzyme thioredoxin

reductase is also required to supply reduced thior-

edoxin for the reduction of pyrimidine nucleotides by

ribonucleotide reductase. The downregulation of thior-

edoxin reductase in C. jejuni exposed to cadmium was

unexpected because of its unique roles in cellular

metabolism, but this result was confirmed by the

measurement of enzyme activity by spectrophotometric

analyses.

The absence of glutathione-specific metabolic

pathways in C. jejuni allowed use of GSSG as a non-

specific disulfide substrate. The presence of glutathione

reduction activities in C. jejuni was established by

observing the reduction of GSSG to GSH with con-

comitant oxidation of NADH using 1H-NMR spec-

troscopy. This measurement of disulfide reduction was

validated using several controls described in Experi-

mental procedures. An increase of approximately

1.6-fold in the rate of GSSG reduction was observed

in cells grown with 0.1 mm Cd2+. This result indicates

that disulfide reductases capable of reducing GSSG are

involved in the response of C. jejuni to cadmium ions.

Glutathione reduction was investigated further by

determining the kinetic parameters of this activity in

lysate suspensions; the values calculated for the Micha-

elis constants and maximal velocities were 4.7 ±

0.4 mm and 43 ± 2 nmolÆmg)1Æmin)1 for GSSG, and

2.7 ± 0.1 mm and 42 ± 2 nmolÆmg)1Æmin)1 for

NADH. The presence of Cd2+ inhibited GSSG

reduction activity. The inhibition constant of the

cadmium ions was determined by measuring enzyme

activities in the presence of various concentrations of

the metal, and the Ki value was 6.2 ± 0.6 lm. Addi-

tion of GSH to the assay mixtures relaxed the inhibi-

tion imposed by CdCl2 on glutathione reduction.

Campylobacter jejuni and cadmium stress N. O. Kaakoush et al.

5028 FEBS Journal 275 (2008) 5021–5033 ª 2008 The Authors Journal compilation ª 2008 FEBS

The results suggest several possible Cd2+ detoxifica-

tion mechanisms in which the metal is bound by: (a)

GSSG, (b) the enzyme, and ⁄or (c) GSH. To differenti-

ate between these alternatives, the interactions of Cd2+

ions with GSSG, GSH and glutathione reductase were

investigated using 1H-, 13C- and 113Cd-NMR spectros-

copy. The 1H-NMR spectrum of 2 mm GSSG was not

affected by the presence of 2 mm CdCl2; under the

same experimental conditions, the b-CH2 cysteinyl pro-

ton resonances of GSH were strongly broadened in the

presence of cadmium. The 13C-NMR resonances of the

c-glutamate, cysteine and glycine residues of 50 mm

GSSG suspensions were slightly broadened by the addi-

tion of 10 mm CdCl2. At similar concentrations of the

cadmium salt, the resonances arising from the c-gluta-mate and glycine residues of 50 mm GSH were slightly

broadened, but strong broadenings and upfield shifts

were observed in the Ca and Cb of the cysteine residues.

Moderate broadening and a small change in chemical

shift were observed for the 113Cd-NMR resonance of

50 mm CdCl2 solutions by adding 5 mm GSSG. How-

ever, strong broadening and a marked upfield shift

occurred for the 113Cd-NMR resonance of CdCl2 in the

presence of 5 mm GSH; a binding constant

Kb = 7 ± 1 lm was determined from these data

(Fig. 3). The NMR spectroscopy data suggest that

Cd2+ ions interact weakly with the residues of oxidized

glutathione, but show strong and specific interactions

with the cysteinyl of reduced glutathione.

The interactions of Cd2+ ions with bovine glutathi-

one reductase were studied by 113Cd-NMR spectros-

copy by titrating 50 mm CdCl2 solutions with the

purified protein. Bovine glutathione reductase was

utilized because it is commercially available and is able

to reduce GSSG. Addition of the enzyme to the CdCl2solutions produced downfield shifts in the 113Cd-NMR

resonance that were a linear function of the protein

concentration. Thus, the NMR spectroscopy data

showed significant binding of Cd2+ ions to glutathione

reductase and GSH, but not to GSSG.

These results could be explained by a simplified model

that considers three populations of Cd2+ ions: (a) bound

to the enzyme, (b) bound to the reduced thiol, and (c) a

heterogenous ensemble of ions that are free in solution,

bound to cellular components, etc. The proportion of

Cd2+ ions bound by the reduced thiol will increase with

time as more thiol is produced by the reaction. This will

induce redistribution of ions in the other two popula-

tions. In particular, removal of Cd2+ cations that are

available to interact with the protein will decrease inhibi-

tion of the enzyme activity. The redistribution of ions

between the three populations will continue until it

reaches a new equilibrium, which depends on factors

such as total Cd2+ ion concentration, substrate concen-

tration, maximal rates of enzyme activity, etc.

Conclusion

This study identified features in the response of C. jejuni

to cadmium stress that are unique to it as well as

others that are common with the responses of other

bacteria. The modulation of expression of enzymes of

fatty acid biosynthesis and the TCA cycle by C. jejuni

is similar to that reported previously for other organ-

isms [24,26]. On the other hand, the downregulation

by C. jejuni of thioredoxin reductase expression and

the upregulation of expression of a disulfide-reducing

system capable of reducing GSSG are demonstrated

here for the first time. Cadmium affected the central

metabolism of C. jejuni, and the bacterium responded

by downregulating proteins involved in metal uptake,

and upregulating proteins involved in metal storage

and xenobiotic detoxification. Further studies will

characterize the glutathione-reducing system of C. jeju-

ni that is modulated by the presence of Cd2+ ions; 35

putative redox proteins have been identified in this

bacterium [63] that are potentially responsible for this

activity. Finally, similar GSSG reduction activities

have been observed in four genera belonging to two

families of the order Campylobacterales [63], suggest-

ing that these bacteria may have in common a novel

system that is capable of detoxification of metal ions.

Fig. 3. 113Cd-NMR resonances of 50 mM CdCl2 in aqueous NaCl

(75 mM), KCl (75 mM) buffer (bottom), and with 5 mM GSH added

to the solution (top). Instrument parameters are described in Exper-

imental procedures.

N. O. Kaakoush et al. Campylobacter jejuni and cadmium stress

FEBS Journal 275 (2008) 5021–5033 ª 2008 The Authors Journal compilation ª 2008 FEBS 5029

Experimental procedures

Materials

Blood agar base no. 2, brain heart infusion, defibrinated

horse blood and horse serum were obtained from Oxoid

(Heidelberg West, Australia). Amphotericin (Fungizone�),

bicinchoninic acid, BSA, chloramphenicol, copper II sul-

fate, dithiobis-2-nitrobenzoic acid, GSSG, GSH, NADH,

NADPH, polymixin B, trimethoprim, and bovine gluta-

thione reductase were obtained from Sigma (Castle Hill,

Australia). Vancomycin was obtained from Eli Lilly

(North Ryde, Australia), and Tris base was obtained from

Amersham Biosciences (Melbourne, Australia). All other

reagents were of analytical grade.

Bacterial strain and growth conditions

C. jejuni strain NCTC 11168 isolated from humans [51] was

grown at 37 �C on Campylobacter selective agar plates [64]

under microaerobic conditions (6% O2, 10% CO2). Liquid

cultures were grown in vented flasks using 50 mL brain

heart infusion supplemented with cadmium chloride (Sigma)

at concentrations of 0, 0.05, 0.1, 0.3, 0.5 and 1 mm. Cells

were tested for purity using phase-contrast microscopy.

Two-dimensional PAGE and mass spectrometry

identification of proteins

Preparation of cell-free protein extracts was performed as

described previously [65]. For the first dimension of two-

dimensional gel electrophoresis, samples were loaded onto

an 18 cm Immobiline DryStrip pH 4-7 (Amersham

Biosciences), and left to incubate sealed for 20 h at room

temperature. Isoelectric focusing was performed using a

flatbed Multiphor II unit (Amersham Biosciences). For the

second dimension, SDS–PAGE was performed on 11.5%

acrylamide gels using the Protean II system (Bio-Rad,

Sydney, Australia). The experimental conditions for two-

dimensional PAGE were as described previously [65]. Gels

were fixed individually in 0.2 L of fixing solution (50% v ⁄ vmethanol, 10% v ⁄ v acetic acid) for a minimum of 1 h, and

were subsequently stained using a sensitive ammoniacal

silver method. For comparative image analysis, statistical

data were acquired and analyzed using z3 compugen soft-

ware (Sunnyvale, CA, USA). Proteins were considered to

be regulated if the intensities of the corresponding spots on

test and control gels differed at least twofold.

The protocol to excise proteins from gels and digest

them, as well as the preparation of peptides for sequencing

by mass spectrometry, has been described previously [65].

Peptide identifications were performed using an API

QStar Pulsar I tandem MS instrument with the instrument

parameters used previously [65]. Protein searches were

performed on the National Center for Biotechnology Infor-

mation non-redundant database.

Bioinformatics

blastp searches were performed using the complete protein

sequences available at the NCBI database (http://

www.ncbi.nlm.nih.gov/). The Kyoto Encyclopedia of Genes

and Genomes (KEGG) available at http://www.genome.jp/

kegg was used to determine the biochemical pathways to

which the proteins were assigned. The Search Tool for the

Retrieval of Interacting Proteins (STRING), available at

http://string.embl.de/, which comprises known and pre-

dicted protein–protein interactions, was used to examine

predicted interactions between proteins.

Enzyme assays

Preparation of lysate fractions and cell-free protein extracts

for enzyme assays was carried out as previously described

[66]. Proton nuclear magnetic resonance (1H-NMR) spec-

troscopy was used to measure disulfide reduction. Free

induction decays were collected using a Bruker DMX-600

NMR spectrometer (Karlsruhe, Germany) operating in the

pulsed Fourier transform mode with quadrature detection

and the instrumental parameters used previously [66]. Disul-

fide reduction activities were measured in C. jejuni cell-free

extracts using GSSG and NADH as substrates. Chemical

reduction of GSSG in this system was ruled out because no

reduction was observed in the absence of cell-free extracts.

Negative controls showed that reduction of GSSG did not

take place if NADH was not present. The enzymatic origin

of the reactions was established by determining that no

activity was present in suspensions of cell-free extracts that

had been denatured by heating at 80 �C for 2 h.

Assays of fumarate reductase, fumarate dehydratase and

2-oxoglutarate ferredoxin oxidoreductase activities were

performed in whole-cell lysates as described previously [33].

Thioredoxin reductase activity was measured by dithiobis-

2-nitrobenzoic acid reduction in the presence of NADPH

using a Varian Cary-100 UV-visible spectrophotometer

(North Ryde, Australia) as described previously [63].

Effects of cadmium ions on enzyme activities

The effect of cadmium ions on glutathione reduction was

determined by measuring glutathione rates of reduction in

suspensions of whole bacterial lysates using 1H-NMR spec-

troscopy. At substrate concentrations well below the Km, the

inhibition constant can be calculated from the expression

m0=m ¼ 1þ I=Ki

where vo and v are the uninhibited and inhibited rates

of reduction, respectively, and I is the concentration of

inhibitor [67].

Campylobacter jejuni and cadmium stress N. O. Kaakoush et al.

5030 FEBS Journal 275 (2008) 5021–5033 ª 2008 The Authors Journal compilation ª 2008 FEBS

Interactions of cadmium ions with glutathione

and glutathione reductase

The interactions of oxidized and reduced glutathione with

Cd2+ were studied using 1H- and 13C-NMR. Solutions of

GSSG or GSH were placed in 5 or 10 mm tubes at con-

centrations between 2 and 50 mm. 1H-NMR free induction

decays were collected using a Bruker DMX-500 NMR

spectrometer, operating in the pulsed Fourier transform

mode with quadrature detection. Proton spectra were

acquired with presaturation of the water resonance. The

instrumental parameters were: operating frequency

500.13 MHz, spectral width 5000 Hz, memory size 16 K,

acquisition time 1.64 s, number of transients 64, pulse

angle 50� (3 ls), and relaxation delay with solvent presatu-

ration 1.4 s. Spectral resolution was enhanced by Gaussian

multiplication with line broadening of )0.6 Hz and Gauss-

ian broadening factor of 0.19. Chemical shifts are quoted

relative to sodium 4,4-dimethyl-4-silapentane-1-sulfonate

at 0 p.p.m.

One-dimensional natural-abundance 13C-NMR spectra

were acquired with composite pulse proton decoupling in

an ACP-300 Bruker NMR spectrometer. The instrumental

parameters were: operating frequency 75.5 MHz, spectral

width 16129 Hz, memory size 16 K, acquisition time

0.508 s, number of transients 1600, and pulse angle 66�(9 ls). Exponential filtering of 1 Hz was applied prior to

Fourier transformation. Chemical shifts are quoted with

respect to HCO3) at 160 p.p.m.

The interactions of Cd2+ with reduced or oxidized gluta-

thione and with bovine mucus glutathione reductase were

studied using 113Cd-NMR spectroscopy. Solutions of CdCl2(50 mm) in buffer were placed in 10 mm tubes, and titrated

with either metabolite, the enzyme or both. The changes in

the spectral position and linewidth of the 113Cd resonance

were measured from spectra of mixtures at various concen-

trations of GSSG, GSH or glutathione reductase.113Cd-NMR spectra were acquired using a Bruker DMX-

500 NMR spectrometer with composite pulse decoupling of

protons. The instrumental parameters for observing 113Cd

were: operating frequency 110.9 MHz, spectral width

8865 Hz, memory size 16 K, acquisition time 0.92 s, relaxa-

tion delay 30 s, and pulse angle 90� (12 ls). The number of

transients was 128. Exponential filtering of 3 Hz was

applied prior to Fourier transformation. Chemical shifts

are quoted with respect to 0.1 m aqueous Cd(ClO4)2 at

0 p.p.m.

Protein determination

Protein concentrations were determined using the bicinchoni-

nic acid method and a microtitre protocol (Pierce, Rockford,

IL, USA). Absorbances were measured using a Beckman

Du 7500 spectrophotometer (Gladesville, Australia).

Acknowledgement

This work was supported by the Australian Research

Council.

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