Báo cáo khoa học: Molecular responses of Campylobacter jejuni to cadmium stress ppt

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

cadmium stress

Nadeem O Kaakoush1, Mark Raftery2and 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 concentramodula-tions 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.

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 jejunicells 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 situand 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

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 jejunisubjected 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.

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 m M

CdCl2(right) Proteins differentially

expressed between the two growth

conditions are listed in Tables 1 and 2.

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 m M CdCl 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.

CJ0953c Bifunctional formyltransferase ⁄ IMP cyclohydrolase 21

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 m M CdCl 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.

CJ1197c Aspartyl ⁄ glutamyl tRNA amidotransferase subunit B 42

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 utilizaproduc-tion 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 cj0239cparticipates 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 uplated 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

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 jejuniexposed 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 japonicBradyrhizobi-um, 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 propro-teins 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

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 using

1H-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 CdCl2on glutathione reduction

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 using1H-, 13C- and113Cd-NMR

spectros-copy The1H-NMR spectrum of 2 mm GSSG was not

affected by the presence of 2 mm CdCl2; under the

same experimental conditions, the b-CH2cysteinyl

pro-ton resonances of GSH were strongly broadened in the

presence of cadmium The13C-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 Caand Cbof 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 the113Cd-NMR resonance of CdCl2in 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 CdCl2 solutions 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 redistribuinhibi-tion 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 3 113 Cd-NMR resonances of 50 m M CdCl2 in aqueous NaCl

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

to the solution (top) Instrument parameters are described in

Exper-imental procedures.

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 jejunistrain NCTC 11168 isolated from humans [51] was

grown at 37C 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⁄ v

methanol, 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 80C 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 using1H-NMR spec-troscopy At substrate concentrations well below the Km, the inhibition constant can be calculated from the expression

m0=m¼ 1 þ I=Ki where vo and v are the uninhibited and inhibited rates

of reduction, respectively, and I is the concentration of inhibitor [67]