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 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 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 )at0mm Cd 2+ (Fig. 1). Inhibition of C. jejuni growth increased with Cd 2+ 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 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 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 Cd 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 mm Cd 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 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 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. 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 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 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 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. 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 Cd 2+ 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 Cd 2+ 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 O 2 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 Zn 2+ for activity is inhibited by Cd 2+ 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 Cd 2+ 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 ( 1 H-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 1 H-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 1 H-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 Cd 2+ . 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 )1 Æmin )1 for GSSG, and 2.7 ± 0.1 mm and 42 ± 2 nmolÆmg )1 Æmin )1 for NADH. The presence of Cd 2+ 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 K i value was 6.2 ± 0.6 lm. Addi- tion of GSH to the assay mixtures relaxed the inhibi- tion imposed by CdCl 2 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 Cd 2+ 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 Cd 2+ ions with GSSG, GSH and glutathione reductase were investigated using 1 H-, 13 C- and 113 Cd-NMR spectros- copy. The 1 H-NMR spectrum of 2 mm GSSG was not affected by the presence of 2 mm CdCl 2 ; under the same experimental conditions, the b-CH 2 cysteinyl pro- ton resonances of GSH were strongly broadened in the presence of cadmium. The 13 C-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 CdCl 2 . 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 C a and C b of the cysteine residues. Moderate broadening and a small change in chemical shift were observed for the 113 Cd-NMR resonance of 50 mm CdCl 2 solutions by adding 5 mm GSSG. How- ever, strong broadening and a marked upfield shift occurred for the 113 Cd-NMR resonance of CdCl 2 in the presence of 5 mm GSH; a binding constant K b =7±1lm was determined from these data (Fig. 3). The NMR spectroscopy data suggest that Cd 2+ ions interact weakly with the residues of oxidized glutathione, but show strong and specific interactions with the cysteinyl of reduced glutathione. The interactions of Cd 2+ ions with bovine glutathi- one reductase were studied by 113 Cd-NMR spectros- copy by titrating 50 mm CdCl 2 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 CdCl 2 solutions produced downfield shifts in the 113 Cd-NMR resonance that were a linear function of the protein concentration. Thus, the NMR spectroscopy data showed significant binding of Cd 2+ ions to glutathione reductase and GSH, but not to GSSG. These results could be explained by a simplified model that considers three populations of Cd 2+ 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 Cd 2+ 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 Cd 2+ 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 Cd 2+ 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 Cd 2+ 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 mM CdCl 2 in aqueous NaCl (75 m M), 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 Ò ), 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 °ConCampylobacter selective agar plates [64] under microaerobic conditions (6% O 2 , 10% CO 2 ). 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 ( 1 H-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 1 H-NMR spec- troscopy. At substrate concentrations well below the K m , the inhibition constant can be calculated from the expression m 0 =m ¼ 1 þ I=K i where v o 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 [...]... 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