Abstract

Copper, though toxic in excess, is an essential trace element that serves as a cofactor in many critical biological processes such as respiration, iron transport, and oxidative stress protection. To maintain this balance between requirement and toxicity, biological systems have developed intricate systems allowing the preservation of homeostasis while ensuring delivery of copper to the appropriate cellular component. The nematode Caenorhabditis elegans was exploited to assess the effects of copper toxicity at the population level to identify key changes in life cycle traits including, lethality, brood size, generation time, growth, and life span. To enhance our understanding of the complexities of copper homeostasis at the genetic level, the expression profile and functional significance of a putative copper cytoplasmic metallochaperone cutc-1 were analyzed. Using quantitative PCR technology, cutc-1 was found to be downregulated with increasing CuSO4 concentrations. However, although total (whole body) copper levels increased in nematodes exposed to elevated levels of copper, wild-type and knock down of cutc-1 by RNA-mediated interference (RNAi) were statistically indistinguishable. Nevertheless, RNAi of cutc-1 affected brood size, growth and induced a marked increase in protruding vulva and bagging phenotypes at higher copper exposures. This indicates that cutc-1 plays a crucial role in the protection from excess copper.

Copper ions can adopt distinct redox states, oxidized Cu2+ or reduced Cu+, allowing the metal to play a pivotal role in cell physiology as a catalytic cofactor in the redox chemistry of enzymes and proteins (Tapiero et al., 2003). These enzymes and proteins carry out fundamental biological functions that are required for growth and development (Linder, 1991). Copper proteins are involved in vital processes such as respiration (mitochondrial oxidative phosphorylation), iron transport, oxidative stress protection (free radical scavenging), blood clotting, hormone production, neurotransmitter synthesis and maturation, elastin crosslinking, and pigmentation (Petris et al., 2003; Puig et al., 2002; Tapiero et al., 2003). As copper-requiring proteins are involved in such a wide variety of biological processes, deficiency can cause an alteration in enzyme activity, often causing disease states or pathophysiological conditions (Mak and Lam, 2008).

Excess copper ions can cause damage to cellular components because of an imbalance between the uptake and efflux of copper ions (Tapiero et al., 2003). Copper is toxic to cells because of its ability to reversibly donate and receive electrons and is responsible for the intracellular generation of superoxide and other reactive oxygen species (Kimura and Nishioka, 1997). In addition, under anaerobic conditions, copper appears to shift from Cu++ to the Cu+ oxidation state thereby exacerbating the Cu+ concentration gradient and increasing its toxicity by facilitating passive or active uptake (Outten et al., 2001). If allowed to engage in uncontrolled redox chemistry in a cell, copper can cause devastating and irreparable damage to proteins, lipids, and DNA (Rees and Thiele, 2004). It is therefore important that organisms have appropriate mechanisms for uptake and detoxification, as well as possessing cellular sensors to ensure that copper is present in the cell to drive the essential biochemical processes while preventing its accumulation to toxic levels (Mufti et al., 2007; Pena et al., 1998).

Several families of proteins have been shown to control the activity and distribution of intracellular metal ions (Rensing and Grass, 2003), including integral transmembrane transporters, metalloregulatory sensors, and diffusible cytoplasmic metallochaperone proteins that protect and guide metal ions to targets (Finney and O'Halloran, 2003). On the basis of the preliminary characterization of Cu-sensitive mutants in Escherichia coli, it was proposed that six genes (cutA, cutB, cutC, cutD, cutE, and cutF) are involved in the uptake, intracellular storage, and delivery and efflux of copper. A mutation in one or more of these genes results in increased copper sensitivity (Gupta et al., 1995). A human ortholog of E. coli cutC was identified and found to be distributed throughout the cytoplasm, which is consensus with E. coli cutC, and suggests that cutC may be a cytoplasmic Cu-binding protein which plays a role in copper homeostasis and acts as a shuttle protein in intracellular copper trafficking (Li et al., 2005).

The aim of this study was to assess the whole organism response of C. elegans exposed to copper, identify functional and expression characteristics of cutc-1, a putative copper transporter, and utilize RNA-mediated interference (RNAi) technology to knock down cutc-1 to determine its involvement in copper toxicology.

MATERIALS AND METHODS

Caenorhabditis elegans strains and culture conditions

Wild-type (N2, Bristol) C. elegans was used in all experiments and maintained on either nematode growth medium (NGM) agar in 55 mm Petri dishes at 20°C in a constant temperature incubator or in NGM liquid culture grown in 50 ml conical flasks within an orbital shaker at 150 rpm maintained at 20°C, according to standard procedures (Brenner, 1974; Lewis and Fleming, 1995; Sulston and Hodgkin, 1988). The bacterial food source was E. coli OP50 (an uracil-requiring E. coli strain to prevent overgrowth of the bacterial culture).

Metal content of exposure media and nematodes

Exposures were performed by supplementing the NGM agar–based support media and the fresh overnight culture of E. coli OP50 (immediately prior to seeding on agar plates) and with equimolar amounts of CuSO4. The copper concentration of fully oxidized nitric acid–digested samples was quantified either by Atomic Absorption Spectrophotometry (for NGM agar analysis) using an air-acetylene flame on a Varian SpecrAA-100 (Varian Instruments, Walton-on-Thames, Surrey, UK) with automatic background correction or, for nematodes exposed to copper-containing NGM plates, a Graphite Furnace (41102L, PerkinElmer, Waltham, MA). For the purpose of this study, all copper concentrations stated refer to nominal, not nitric acid extractable, amounts.

Quantitative PCR

Nematodes were cultured in 750-ml S-basal media (5.85 g NaCl, 1 g K2HPO4, 6 g KH2PO4, made up to 999 ml with ddH20, and following autoclave sterilization and cooling to below 55°C, 1 ml of cholesterol [5 mg/ml] was added) inoculated with 20 ml OP50 and LB broth (250 ml). The flask was incubated at 20°C, shaking at 150 rpm until the population reached approximately 50,000 staged nematodes. After centrifugation at 2000 × g for 2 min, the supernatant was removed to a minimal volume and the culture was split into 15-ml tubes. The nematodes were exposed in S-basal to 0, 100, 500, and 2000μM CuSO4 for 24 h with five biological replicates at each concentration. Total RNA was extracted using a standard Tri-reagent protocol (Sigma, Gillingham, UK), purified and concentrated by ethanol precipitation, and 2 μg was used to generate complementary DNA (cDNA).

Gene-specific primers were used to amplify cutc-1 (ZK353.7) and rla-1 (Y37E3.7) from C. elegans cDNA (Supplementary Table 1). The PCR product was visualized by agarose gel electrophoresis, gel extracted and cloned into pGEM-T (Promega, Southampton, UK). The inserts of the plasmids were sequenced using universal M13 primers to confirm the identity of the insert. Quantitative PCR (qPCR) amplifications were performed using TaqMan probe technology on the ABI Prism 7700 Sequence Detection System exploiting the primers and probes provided in Supplementary Table 1. Calibration standards and samples were quantified in parallel. Five biological replicates were prepared for each test sample, and for each biological replicate, the cDNA samples were analyzed in triplicate.

Preparation of target genes for RNAi

Following cDNA synthesis, cutc-1 was amplified using specific primers (Supplementary Table 1) with EcoR1 adapters at the 5′ end. The PCR product was purified by gel extraction, ligated into the pGEM-T vector, and transformed into DH5α competent cells. The plasmid was purified, and positive clones were identified by restriction digest with EcoR1. The digested DNA was purified by gel extraction and subcloned into the RNAi vector pPD129.36 (an AmpR plasmid containing an IPTG-inducible T7 RNA promoter). The cutc-1–containing plasmid was confirmed by sequencing and transformed into competent HT115 (F-, mcrA, mcrB, IN (rrnD-rrnE)1, lambda-, rnc14::Tn10 (DE3 lysogen:lacUV5 promoter—T7 polymerase)) which are deficient in RNase III, allowing the stable expression of double stranded RNA. For all RNAi experiments, the empty pPD129.36 vector was also transformed into HT115 cells and used as a control.

Caenorhabditis elegans RNAi exposure and sample preparation

Nematodes were washed off agar plates using M9 buffer, centrifuged for 2 min at 2000 × g, and the supernatant removed. After pooling the nematodes, they were distributed onto agar plates containing 0, 20, 100, and 500μM CuSO4 streaked with HT115 containing either the empty RNAi vector (pPD129.36) or the cutc-1 RNAi clone. Once gravid, adults were synchronized using the standard egg preparation protocol and the F1 generation placed back onto the agar plates. When the nematodes reached L4 stage, they were either used to determine the life history parameters or frozen at − 80°C for subsequent RNA extraction.

Total brood size, generation time, life span, and lethality assay

The life cycle of C. elegans was assessed over a range of copper concentrations in the presence or absence of cutc-1 RNAi. Typically, staged nematodes were placed on NGM agar plates and replica plated every 24/36 h to a new plate. Brood size (n = 20 per dose and RNAi exposure), generation time, the time an egg takes to develop into a reproducing adult (n = 30 per dose and RNAi exposure), and life span, the timeframe between egg and death (n = 100 per dose and RNAi exposure) were measured by manual counting and observation. As E. coli OP50 does not grow on agar plates containing in excess of 1mM CuSO4, the lethality assay was performed in liquid culture. The lethal concentration of CuSO4cutc-1 RNAi) was determined using staged nematodes placed in M9 buffer (to maintain the pH and minimize the risk of a shift in Cu speciation) supplemented with OP50. The nematodes were rotated at 20°C and the titer determined every 24 h (three biological replicates were used with four titers performed for each exposure), and the average number of nematodes was calculated.

RESULTS

Lethality of Caenorhabditis elegans Exposed to CuSO4 in Liquid Culture

A distinct dose-responsive effect causing detrimental effects on the survival of nematodes with increasing CuSO4 concentrations was observed (Figure 1) where survival was reduced to 50% within 76 h at a nominal concentration of 0.25mM CuSO4. At CuSO4 concentrations of 0.5, 1, 2, and 3mM, the timeframe was reduced to 46, 42, 16, and 13 h, respectively. LC50 concentrations for C. elegans were obtained from three independent repeats ± SEM, resulting in an LC50 (24 h) of 1.59 ± 0.04mM CuSO4, an LC50 (48 h) of 0.47 ± 0.01mM CuSO4, an LC50 (72 h) of 0.29 ± 0.01mM CuSO4, and an LC50 (96 h) of 0.02 ± 0.01mM CuSO4.

FIG. 1.

The effect of copper (0, 0.25, 0.5, 1, 2, and 3mM CuSO4) on the lethality of nematodes maintained in liquid culture over a 4-day exposure period. The data presented are averages from five independent experiments and expressed as mean survival ± SEM.

FIG. 1.

The effect of copper (0, 0.25, 0.5, 1, 2, and 3mM CuSO4) on the lethality of nematodes maintained in liquid culture over a 4-day exposure period. The data presented are averages from five independent experiments and expressed as mean survival ± SEM.

Bioinformatic Analysis of Caenorhabditis elegans cutc-1

The putative copper homeostasis protein CUTC-1 (ZK353.7) is 250 amino acids in length and contains two exons. It is located in the center of an operon containing an ubiquitin regulatory protein and leucine aminopeptidase, a zinc metalloprotease. C. elegans CUTC-1 was aligned to human, rat, fly, nematode, and bacterial orthologs using the ClustalW algorithm (see Supplementary Fig. 1). The protein family does not contain classic copper-binding motifs such as MXM or MXXXM but instead displays a highly conserved motif, VTFHRAFD, of unknown function. Pair wise comparison, using the bl2seq option of the NCBI BLAST suite, revealed a consistent identity score of 38–39% and similarity score of 58–60% in relation to human, rat, fly, and bacterial counterparts which increased to 82% identity and 92% similarity when compared to the nematode Caenorhabditis briggsae, a conservation that is not dissimilar to the conservation between rat and human orthologs (Figure 2, Panel A). Further analysis included a hydrophobicity plot, which indicated that CUTC-1 does not contain transmembrane-bound domains and therefore is unlikely to be located in the membrane (data not shown). Likewise, it was deduced that the C. elegans CUTC-1 protein does not have an N-terminal signal peptide, a mitochondrial or nuclear localization site, or an ER retention motif as defined by the EXPASY bioinformatics tools (http://www.expasy.org/tools) but is predicted to be of cytoplasmic nature (Figure 2, Panel B).

FIG. 2.

Identity/similarity scores of Caenorhabditis elegans CUTC-1 (Ce) and corresponding CutC orthologs in Homo sapiens (Hs), Rattus norvegicus (Rn), Drosophila melanogaster (Dm), Escherichia coli (Ec), and Caenorhabditis briggsae (Cb) were calculated using NCBI's bl2seq algorithm (Panel A). Prediction of the cellular location of cutc-1 was performed using the ExPASy k-NN program (Panel B). The 3D molecular structure of CUTC-1 was modeled from the x-ray crystallography of the protein from Shigella flexneri (Zhu et al., 2005) using SWISS-MODEL (Guex and Peitsch, 1997; Peitsch, 1995; Schwede et al., 2003) and visualized using Swiss-pdbViewer 3.7 (Panel C).

FIG. 2.

Identity/similarity scores of Caenorhabditis elegans CUTC-1 (Ce) and corresponding CutC orthologs in Homo sapiens (Hs), Rattus norvegicus (Rn), Drosophila melanogaster (Dm), Escherichia coli (Ec), and Caenorhabditis briggsae (Cb) were calculated using NCBI's bl2seq algorithm (Panel A). Prediction of the cellular location of cutc-1 was performed using the ExPASy k-NN program (Panel B). The 3D molecular structure of CUTC-1 was modeled from the x-ray crystallography of the protein from Shigella flexneri (Zhu et al., 2005) using SWISS-MODEL (Guex and Peitsch, 1997; Peitsch, 1995; Schwede et al., 2003) and visualized using Swiss-pdbViewer 3.7 (Panel C).

A 3D representation of CUTC-1 was generated by homology modeling (SWISS-MODEL; Guex and Peitsch, 1997; Peitsch, 1995; Schwede et al., 2003) using the x-ray crystallography of the CUTC of Shigella flexneri as a template (Zhu et al., 2005) and visualized using Swiss-pdbViewer 3.7 (Figure 2, Panel C). The 3D structure of CUTC-1 consists of a series of parallel β-sheets arranged in a barrel formation surrounded by α-helices. The α-helices connect the parallel strands of the β-sheets and run antiparallel to the β-sheets, an overall structure that closely resembles a TIM or α/β barrel protein. The conserved VTFHRA motif is located within the hydrophobic core of the protein with conserved histidine and charged residues reside inside the TIM barrel (see Supplementary Fig. 1) which have previously been predicted to be functionally important for copper binding (Zhu et al., 2005). In addition, two evolutionarily conserved cysteine residues (cys15 and cys32) lie inside the barrel and therefore may potentially be involved in metal binding.

Expression of cutc-1 in Wild-type Caenorhabditis elegans and Efficiency of cutc-1 RNAi

The transcriptional expression of cutc-1 was quantified in Caenorhabditis elegans exposed to different CuSO4 concentrations using TaqMan probe technology and normalized against the control gene rla-1. Compared to control nematodes, the relative expression of cutc-1 was reduced in nematodes exposed for 24 h to 100μM CuSO4 and statistically significantly reduced at 500μM CuSO4 (p ≤ 0.012) and 2000μM CuSO4 (p ≤ 0.006) (Figure 3, Panel A). In addition, qPCR was used to determine the efficiency of the transcript suppression following the RNAi silencing of cutc-1 by bacterial feeding. The comparison of cutc-1 expression in the presence and absence of RNAi demonstrated that cutc-1 was effectively silenced by 75% (see Supplementary Fig. 2A).

FIG. 3.

The relative gene expression of cutc-1 is reduced in nematodes exposed to copper. * denotes a significant difference (p ≤ 0.05) from control (0 CuSO4) and # denotes a significant difference from nematodes exposed to 100μM CuSO4 (Panel A). Total (nitric acid extractable) body burden of copper increases with environmental concentrations. Note that there is no statistically significant difference between wild type and the knock down of cutc-1 (Panel B).

FIG. 3.

The relative gene expression of cutc-1 is reduced in nematodes exposed to copper. * denotes a significant difference (p ≤ 0.05) from control (0 CuSO4) and # denotes a significant difference from nematodes exposed to 100μM CuSO4 (Panel A). Total (nitric acid extractable) body burden of copper increases with environmental concentrations. Note that there is no statistically significant difference between wild type and the knock down of cutc-1 (Panel B).

Copper Accumulation in the Presence or Absence of RNAi Silencing of cutc-1

Caenorhabditis elegans was routinely maintained on standard NGM agar which was shown to contain a basal level of 6μM copper when extracted with concentrated nitric acid. Assessment of the nitric acid–extractable concentration of copper in the agar plates was observed to be 40% less than the nominal concentration of CuSO4 added to the agar plates (see Supplementary Table 2). Due to the small physical size of nematodes, it was necessary to tool 1000 individuals to allow the quantification of total, nitric acid–extractable, copper concentrations. A dose-dependent increase was observed, and 100μM CuSO4 caused a statistically significant increase in copper levels. The RNAi of cutc-1 caused a similar increase that, though marginally lower at the highest concentration tested, was statistically not different to wild-type nematodes (see Figure 3B). This implies that C. elegans cutc-1 is not the major exporter of excess copper.

Life Cycle Traits Following the Knockdown of cutc-1 by RNAi

Total brood size decreased with increasing CuSO4 concentrations in both the control nematodes and those exposed to cutc-1 RNAi. In the presence of RNAi of cutc-1, nematodes exposed to 100μM CuSO4 produced significantly less progeny than the respective wild-type nematodes (p ≤ 0.05). It is remarkable that at 500μM CuSO4, the reproductive output was decimated to below 5% of control plates (Figure 4A). Generation time increased with increasing concentrations of CuSO4. The average generation time was 66 h at 0μM CuSO4, increasing to 78 h at 500μM CuSO4. There was no significant difference in the generation time between wild-type nematodes and those exposed to the RNAi of cutc-1 (see Supplementary Fig. 2B).

FIG. 4.

Total brood size is reduced in nematodes exposed to increasing doses of copper. Following the RNAi of cutc-1 (black bars), no statistical difference in brood size was observed under control and low copper exposure conditions, but deemed statistically different while exposed to 100μM CuSO4 (Panel A). After a 5-day exposure period, the length of the nematode is reduced in a copper dose-dependent manner, a trend that is amplified in nematodes subjected to the RNAi of cutc-1 (Panel B). Replicates per dose and condition: brood size n = 20; length n = 15.

FIG. 4.

Total brood size is reduced in nematodes exposed to increasing doses of copper. Following the RNAi of cutc-1 (black bars), no statistical difference in brood size was observed under control and low copper exposure conditions, but deemed statistically different while exposed to 100μM CuSO4 (Panel A). After a 5-day exposure period, the length of the nematode is reduced in a copper dose-dependent manner, a trend that is amplified in nematodes subjected to the RNAi of cutc-1 (Panel B). Replicates per dose and condition: brood size n = 20; length n = 15.

Nematode length decreased with increasing CuSO4 concentrations in wild-type and RNAi exposures. Reduction in growth was most marked in nematodes exposed to the two highest concentrations (100 and 500μM CuSO4) during the knock down of cutc-1 and significantly different to wild-type (p < 0.000 and 0.002 for nematodes exposed to 100 and 500μM CuSO4, respectively) (Figure 4, Panel B). The average life span of nematodes generally decreased with increasing CuSO4 concentrations, being significantly shorter at 500μM CuSO4 compared to 0, 20, and 100μM CuSO4 (p < 0.001). There was also a significant decrease in the life span from 0 to 100μM CuSO4 and from 20 to 100μM CuSO4 (p < 0.001). No differences in life span were observed between wild type and the nematodes exposed to the RNAi of cutc-1 (see Supplementary Fig. 2C).

Phenotypic Effects of CuSO4 Following RNAi of cutc-1

The phenotypes protruding vulva (pvl) and bag of worms (egl) were frequently encountered in C. elegans exposed to CuSO4. The prevalence of these phenotypes over a range of CuSO4 concentrations was observed, and the number of nematodes that perished from bag of worms was determined from over 100 nematodes per dose. Nematodes exposed to the RNAi of cutc-1 displayed the bagged phenotype at all CuSO4 concentrations with an increasing incidence at elevated CuSO4 concentrations, with 15% of the nematodes displaying the phenotype at 0μM CuSO4, increasing to 74% at 500μM CuSO4. In contrast, bagging in wild-type nematodes occurred only at 100μM CuSO4 and 500μM CuSO4, with 10% and 70%, respectively, of the nematodes developing bag of worms (Figure 5, Panel A). PD4251 (ccls4251 l; dpy-20 (e1282) IV) a strain that produces green fluorescent protein in all body wall and vulval muscles (with a combination of mitochondrial and nuclear localization) was used to visualize the presence of both phenotypes during CuSO4 exposure and RNAi of cutc-1 (Figure 5, Panel B). It is, at this stage, not known if the pvl/egl phenotype is an indirect or direct effect of copper toxicosis, a notion that clearly warrants further investigations.

FIG. 5.

Copper induces an egg laying–defective (egl) phenotype (leading to the offspring hatching internally, bagging). RNAi of cutc-1 increases the percentage of bagging, notably also in the absence of CuSO4 (Panel A; n = 100). Bagging was frequently caused by a protruding vulva (pvl) phenotype. Panel B illustrates this using PD4251, a transgenic strain that expresses a vulval and body wall muscle green fluorescent protein, in the presence and absence of cutc-1 RNAi and CuSO4.

FIG. 5.

Copper induces an egg laying–defective (egl) phenotype (leading to the offspring hatching internally, bagging). RNAi of cutc-1 increases the percentage of bagging, notably also in the absence of CuSO4 (Panel A; n = 100). Bagging was frequently caused by a protruding vulva (pvl) phenotype. Panel B illustrates this using PD4251, a transgenic strain that expresses a vulval and body wall muscle green fluorescent protein, in the presence and absence of cutc-1 RNAi and CuSO4.

DISCUSSION

Metal analysis highlighted that basal amounts (prior to the addition of CuSO4) approximated 6μM copper in control agar. In C. elegans, concentrations of excess CuSO4 induced detrimental effects on brood size (Table 1), life span and an increase in generation time. In addition to the development of phenotypic abnormalities, development (manifested as a reduction in body size and growth) was impaired. The results of whole-organism copper toxicity in nematodes is of course not novel and has been exquisitely documented for C. elegans, Pristionchus pacificus, and Panagrellus redivivus (Boyd and Williams, 2003), results that are concurrent with other soil-inhabiting organisms such as the springtail Proisotoma minuta (Nursita et al., 2005) and the earthworm Lumbricus rubellus (Spurgeon et al., 2005).

TABLE 1

The Brood Size of Caenorhabditis Elegans Exposed to Copper (0–1mM CuSO4) on Standard NGM Plates

CuSO4 concentration (μM) Brood size 0–24 h (±SEM) Brood size 25–48 h (±SEM) Brood size 49–72 h (±SEM) Brood size cumulative (±SEM) Total brood size (% of control) 
59 ± 5.8 94 ± 8.2 94 ± 5.4 247 ± 11.8 100 
36 ± 6.8 82 ± 7.5 89 ± 1.0 207 ± 14.9 84 
33 ± 6.5 85 ± 6.1 106 ± 4.3 224 ± 11.5 91 
10 41 ± 5.8 67 ± 11.3 107 ± 5.0 215 ± 19.4 87 
25 43 ± 13.4 77 ± 8.0 98 ± 0.5 218 ± 11.5 88 
50 41 ± 7.1 83 ± 6.5 78 ± 2.0 202 ± 11.3 82 
75 39 ± 3.9 70 ± 10.1 57 ± 4.7 166 ± 14.2 67 
100 41 ± 8.9 48 ± 13.2 37 ± 3.5 126 ± 30.3 51 
125 31 ± 6.3 51 ± 6.6 35 ± 3.0 117 ± 12.3 47 
150 41 ± 5.9 51 ± 3.2 18 ± 2.7 113 ± 9.3 46 
200 44 ± 4.4 18 ± 1.6 10 ± 0.0 69 ± 6.2 28 
400 48 ± 5.3 16 ± 4.1 3 ± 0.0 67 ± 8.5 27 
500 40 ± 5.9 17 ± 7.5 2 ± 0.0 59 ± 12.0 24 
600 30 ± 8.9 15 ± 4.8 0 ± 0.0 45 ± 13.6 18 
800 30 ± 7.2 20 ± 2.6 0 ± 0.0 50 ± 9.2 20 
1000 34 ± 0.6 19 ± 1.5 0 ± 0.0 53 ± 1.9 21 
CuSO4 concentration (μM) Brood size 0–24 h (±SEM) Brood size 25–48 h (±SEM) Brood size 49–72 h (±SEM) Brood size cumulative (±SEM) Total brood size (% of control) 
59 ± 5.8 94 ± 8.2 94 ± 5.4 247 ± 11.8 100 
36 ± 6.8 82 ± 7.5 89 ± 1.0 207 ± 14.9 84 
33 ± 6.5 85 ± 6.1 106 ± 4.3 224 ± 11.5 91 
10 41 ± 5.8 67 ± 11.3 107 ± 5.0 215 ± 19.4 87 
25 43 ± 13.4 77 ± 8.0 98 ± 0.5 218 ± 11.5 88 
50 41 ± 7.1 83 ± 6.5 78 ± 2.0 202 ± 11.3 82 
75 39 ± 3.9 70 ± 10.1 57 ± 4.7 166 ± 14.2 67 
100 41 ± 8.9 48 ± 13.2 37 ± 3.5 126 ± 30.3 51 
125 31 ± 6.3 51 ± 6.6 35 ± 3.0 117 ± 12.3 47 
150 41 ± 5.9 51 ± 3.2 18 ± 2.7 113 ± 9.3 46 
200 44 ± 4.4 18 ± 1.6 10 ± 0.0 69 ± 6.2 28 
400 48 ± 5.3 16 ± 4.1 3 ± 0.0 67 ± 8.5 27 
500 40 ± 5.9 17 ± 7.5 2 ± 0.0 59 ± 12.0 24 
600 30 ± 8.9 15 ± 4.8 0 ± 0.0 45 ± 13.6 18 
800 30 ± 7.2 20 ± 2.6 0 ± 0.0 50 ± 9.2 20 
1000 34 ± 0.6 19 ± 1.5 0 ± 0.0 53 ± 1.9 21 

Averages were calculated from six replicates (±SEM).

As the minimum inhibitory concentration of E. coli on agar medium is 1mM (Spain and Alm, 2003), LC50 experiments were carried out in liquid culture to ensure the bacterial food source was not a limiting factor. The 24-h and 72-h LC50 of CuSO4 in C. elegans was determined to be 1.59 and 0.29mM, respectively. This concurs with a previous study in C. elegans, where the 24-h LC50 of CuSO4 was predicted to be 1.57mM (Tatara et al., 1998).

The essential yet toxic nature of copper highlights that regulation via a simple copper exclusion system is unlikely. While information is available regarding the handling of metals in prokaryotes and yeast, the precise mechanisms by which multicellular eukaryotes regulate copper are complex and thus not well understood. In eukaryotes, the control of heavy metals is believed to be multifactorial, consisting of several different pathways, encompassing a whole scope of chaperones, metalloenzymes, permeases, reductases, and transcription factors. The knowledge base has recently expanded significantly through the application of microarray technology, including experimental systems such as yeast (Yasokawa et al., 2008), earthworms (Bundy et al., 2008), and mice (Eun et al., 2008); however, a meaningful and exhaustive bioinformatic analysis remains the major challenge to facilitate the identification of new genes and pathways involved in copper trafficking.

Bioinformatic analysis of C. elegans CUTC-1 confirmed its putative role as a cytosolic copper chaperone. The sequence alignment highlighted a conserved VTFHRA motif of unknown function that was conserved in all species. 3D structure prediction placed this motif in the hydrophobic core of the protein, indicating a likely role of importance. Noteworthy is the observation that histidine residues, frequently associated with copper binding (Rogers et al., 1991), are located on either side of the protein pore. The overall structure of the modeled protein closely resembles an α/β barrel typical (but not exclusive) of enzymes that catalyze a very wide range of different reactions but can also have nonenzymatic roles in the cell including binding and transport proteins (Nagano et al., 1999; Reardon and Farber, 1995). CUTC-1 is located in the center of an operon containing the ubiquitin regulatory protein, UBXD2, and lap-1, a leucine aminopeptidase. Given that operons are not random gene assemblages but coregulate genes that make proteins with related functions (Blumenthal et al., 2002) provides circumstantial evidence that cutc-1 may indeed be part of the protein degradation pathway, a notion that is clearly worthy of further investigation.

In E. coli, cutC mutants are copper sensitive and accumulate copper but display normal kinetics of copper uptake. The gene was therefore postulated to encode for an efflux protein responsible for the removal of excess copper from the cytoplasm (Gupta et al., 1995). This is in contrast to the data observed for C. elegans. Firstly, the concentration of total copper did not change significantly upon the knock down of cutc-1. Secondly, transcription of cutc-1 was downregulated at elevated CuSO4 concentrations, a finding that is in partial agreement with Rodrigues et al. (2008) who found that cutC was downregulated in biofilm cells of Xylella fastidiosa a plant pathogen at 7mM CuSO4 compared to 3 and 5mM. This suggests that cutc-1 is, at least in the nematode, unlikely to be a positive regulator of bulk copper efflux, per se.

By means of RNAi, it was possible to characterize the involvement of cutc-1 in CuSO4-induced changes in life cycle parameters. Prior to performing the RNAi experiments, the efficiency of cutc-1 knock down was assessed by qPCR, which showed that cutc-1 expression could be reduced by over 75% using RNAi. Nematodes exposed to cutc-1 RNAi were significantly smaller than wild-type nematodes at elevated CuSO4. High levels of copper were also shown to induce vulval abnormalities (pvl and resultant bagging), a phenotype that was more frequently encountered when cutc-1 was knocked down and was also present at basal copper concentrations. In conclusion, this work suggests that cutc-1 may have a complex role in the trafficking of copper from the cell or in intracellular storage of free copper ions. Furthermore, it provides the first tantalizing insights into the biological processes and molecular functions associated with this novel copper metallochaperone within a eukaryotic organism and reveals a substantial diversity in function over its prokaryotic counterparts.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

FUNDING

UK Natural Environmental Research Council (NER/T/S/2001/00021); Brixham Environmental Laboratory (AstraZeneca); Royal Society to S.R.S.

We thank the Caenorhabditis Genetics Centre, which is funded by the National Institutes of Health National Centre for Research Resources, for the supply of N2 and PD4251. In addition, we are grateful to Wendy Williams for her contribution during the early stages of the project and Mr M. O'Riley (Cardiff University) and Dr T. Blackall (King's College London) for metal analyses.

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