Vol. 58, No. 8INFECTION AND IMMUNITY, Aug. 1990, p. 2414-24190019-9567/90/082414-06$02.00/0Copyright © 1990, American Society for Microbiology

Partial Purification and Characterization of the EnterotoxinProduced by Campylobacter jejuni

TOHRU DAIKOKU,t MARIKO KAWAGUCHI, KOZO TAKAMA, AND SATORU SUZUKI*Department ofFood Science and Technology, Faculty of Fisheries,

Hokkaido University, Hakodate, Hokkaido 041, JapanReceived 18 October 1989/Accepted 16 April 1990

Campylobacterjejuni enterotoxin was partially purified from culture supernatant. The purified fraction aftergel filtration indicated three bands at 68, 54, and 43 kilodaltons on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). This fraction enhanced the adenylate cyclase activity of HeLa cell membranes by1.5-fold over that of the control. The study with anti-cholera toxin immunoglobulin G (IgG) and gangliosideaffinity column chromatographies revealed that the eluent from the anti-cholera toxin IgG column chroma-tography exhibited a single band (68 kDa) on SDS-PAGE and native PAGE, whereas the eluent fromganglioside column chromatography exhibited two bands (68 and 54 kDa) on SDS-PAGE. These suggest thatthe 68-kDa polypeptide should have an immunological relationship with cholera toxin, and the 68- and 54-kDapolypeptides might be responsible for the recognition of ganglioside.

Campylobacterjejuni recently has been recognized as animportant pathogen in human diarrheal disease. With theadvent of improved methods (5), C. jejuni is now isolatedfrom human diarrheal stools frequently worldwide.Symptoms of C. jejuni infection are generally those of

gastrointestinal disorder, for example, watery diarrhea and adysenterylike syndrome, or rarely those of an extraintestinalinfection. Noteworthy characteristics of C. jejuni are asfollows: the latent period is long (1 to 7 days) compared withthose of other bacteria, and the diarrhea caused by thisbacteria usually continues for 2 to 7 days. The organismspends from 2 weeks to 3 months in the host before beingexcreted without any treatment with antibiotics. The mech-anism of the various symptoms of diarrhea caused by C.jejuni has been unclear despite the many studies that havebeen done on this bacteria (20).However, one important mechanism causing diarrhea is

thought to be derived from the production of potent toxinsby the bacteria. It is now known that C. jejuni produces atleast two exotoxins: a heat-labile enterotoxin and a cyto-toxin (8, 12). The former is immunologically related tocholera toxin (CT) and Escherichia coli heat-labile entero-toxin (LT), i.e., it can be neutralized with antiserum for CTand LT (10, 11). The latter has cytotoxic effect againstcultured mammalian cells, including Vero and HeLa cells (8,12, 21). These two exotoxins are considered to relatestrongly to the bacterial pathogenesis of C. jejuni. However,the biochemical properties of the toxins are still unclear,although there are two reports on the purification of entero-toxin (11, 16).To reveal the expression mechanisms of the toxins and to

clarify the mechanism of pathogenesis, it is necessary toobtain a lot of purified toxin. Moreover, examination of thebiochemical properties of the purified toxin will give usefulinformation for immunotherapy and chemotherapy forcampylobacteriosis.

In this study, we attempted to purify and characterize the

* Corresponding author.t Present address: Nagoya University School of Medicine, Nagoya

466, Japan.

enterotoxin produced by C. jejuni as the first step to achiev-ing the results described above.

MATERIALS AND METHODSAll experiments were done at 0 to 4°C unless otherwise

noted.Bacterial strain and growth conditions. The C. jejuni strain

used in this study was isolated from the stool of a child inHakodate Chuou Hospital. It was grown at 37°C in theCampy Pak system (BBL Microbiology Systems) for 48 h ona Skirrow plate without antibiotics. Harvested cells wereinoculated into 600 ml of Casamino Acids-yeast extractbroth (CYE) (Difco Laboratories) containing 1.0 Fg of ferricchloride per ml and incubated at 37°C in the presence of 10%CO2 for 24 h with shaking (17). This liquid culture was theninoculated into 12 liters of CYE broth and grown under theconditions mentioned above.These cultures were centrifuged at 10,000 x g for 30 min.

Then, the supernatants were filtered through a 0.45-,m-poresize membrane filter and used as the starting material fortoxin purification.

Ultrafiltration. Ultrafiltration with a PT membrane (30,000-Mr cut-off; Millipore Corp.) was used to concentrate a cell-free supernatant. Precipitation of proteins was performed inan 80% saturated solution of ammonium sulfate. The precip-itate was centrifuged at 10,000 x g for 15 min after incuba-tion at 4°C for 4 h. The precipitate was suspended in bufferA (50 mM Tris hydrochloride [Tris-HCl, pH 7.8], 1 mMEDTA, 10 mM 2-mercaptethanol) containing 1 mM phenyl-methylsulfonyl fluoride. This was dialyzed against buffer Acontaining 1 mM benzamidine and 30% glycerol and storedat -200C.

Gel filtration. The ammonium sulfate fraction was appliedto a Sephadex G-100 (Pharmacia, Uppsala, Sweden) column(2.5 by 85 cm) equilibrated with buffer A containing 100 mMNaCl and 10% glycerol. Eluents were collected (1.5 ml pertube) at a flow rate of 30 ml/h.ELISA. C. jejuni enterotoxin could be neutralized by

anti-CT serum and bound to ganglioside (16). Based on thisproperty, the enterotoxin assay was performed by enzyme-linked immunosorbent assay (ELISA) with a goat antiserum(LBL) for CT with gangliosides (SRL) in the solid phase.

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ENTEROTOXIN PRODUCED BY C. JEJUNI 2415

Protein assay. Protein concentration was estimated by themethod of Bradford (1). Bovine serum albumin (BSA) wasused as the standard.PAGE. The purity of the toxin in each step of purification

was examined by sodium dodecyl sulfate-polyacrylamideslab gel electrophoresis (SDS-PAGE), as described byLaemmli (14). Non-reductive PAGE (native PAGE) wascarried out without SDS and heat treatment. Gels werestained with Coomassie brilliant blue. Molecular mass mark-ers used were phosphorylase b (97.4 kilodaltons [kDa]), BSA(66 kDa), egg albumin (45 kDa), trypsinogen (24 kDa), andlactalbumin (14.2 kDa). Whether the sample had glycopro-tein was determined by periodic acid-Schiff (PAS) stain ofthe gel (9).Treatment of HeLa cells with toxins. HeLa cells were

grown in 35-mm (inner diameter) tissue culture dishes toconfluence. Cells were incubated at 37°C for 24 h after theaddition of an appropriate amount of the purified enterotoxinor CT (LBL).

Preparation of HeLa cell membranes. Monolayers of HeLacells were washed twice with phosphate-buffered saline(PBS), and cells were harvested by scraping them off into 0.2ml of PBS and pelleted by centrifugation at 200 x g for 5min. The cell membrane fraction was prepared by sonication(50W for 15 s, four times) of the pellet in 1 ml of buffer B (50mM Tris-HCl [pH 7.5], 0.25 M sucrose, 5 mM MgCl2). Thismixture was centrifuged at 500 x g for 5 min, and then thesupernatant was centrifuged at 25,000 x g for 10 min. Thepellet was washed twice with buffer B and dispersed in thesame buffer (protein concentration, ca. 4 mg/ml) (2).Assay for adenylate cyclase activity (assay 1). Adenylate

cyclase activity was assayed by the method of Chang et al.(2) with some modifications. The total volume of the reactionmixture was 100 ,lI which contained 25 mM Tris-HCl (pH7.5), 0.5 mM [a-32P]ATP (9 x 104 cpm/nmol) (New EnglandNuclear Corp.), 6 mM MgCl2, 1 mM EDTA, 20 mM creatinephosphate (Sigma Chemical Co.), 100 U of creatine phos-phate kinase (Sigma) per ml; 1 mM 3',5'-cyclic AMP (cAMP)(Yamasa, Choshi, Japan), 20 ,uM GTP (Yamasa), 1 mMNAD (Oriental, Tokyo, Japan), and an appropriate amountof HeLa cell membranes. Cocktail without cell membranewas preincubated at 30°C for 5 min. Then the membranefraction of HeLa cells was added to the reaction mixture,and incubation was continued at 30°C for 30 min. Thereaction was stopped by the addition of 100 ,ul of stopsolution containing 2% SDS, 40 mM ATP, and 1.4 mMcAMP.

This mixture was applied to a DEAE-cellulose column (5by 45 mm) (bicarbonate form). Nucleotides were eluted by alinear gradient of 0 to 0.3 M triethylamine bicarbonate buffer(pH 8.3).

Detection of [32PJcAMP converted from [a-32P]ATP. A100-,u1 amount was taken from the fractionated sample, andthe radioactivity was counted with an Aloka 673 liquidscintillation counter in a toluene-based scintillator. In paral-lel, cold nucleotides such as cAMP, AMP, ADP, and ATPwere applied to the DEAE-cellulose column. Their elutionprofile was monitored by the A260Radioimmunoassay of cAMP in HeLa cells (assay 2). To

confirm the activation of adenylate cyclase in the toxin-treated cells, cAMP in the cells was determined by radioim-munoassay. HeLa cells treated with enterotoxin or CT wereharvested and homogenized with 6% (wt/vol) trichloroaceticacid. Quantitative analysis of cAMP was performed with a[125 ]cAMP radioimmunoassay kit (New England Nuclear).Anti-CT IgG affinity column chromatography. A 20-mg

-4

-

-JLi

Vo

150 200Elution vol. (ml)

000

r4

FIG. 1. Elution profile of C. jejuni enterotoxin on SephadexG-100 gel filtration chromatography. Symbols: O-O, enterotoxinactivity monitored by ELISA; - - - , A280. A 22-ml amount of crudetoxin was applied to the column (2.5 by 85 cm). Markers, bluedextran 200 (VO [void volume]) and BSA monomer, are indicated byarrows.

amount of anti-CT goat immunoglobulin (IgG) (LBL), dis-solved in 0.1 ml ofNaHCO3 buffer (pH 8.5) containing 0.1 MNaCl, was reacted overnight with 1 ml of activated Affi-Gel10 (Bio-Rad Laboratories) at 4°C with shaking (13). Theremaining active sites of the resin were blocked with 0.1 Methanolamine for 30 min at 4°C. Approximately 75% of theIgG coupled to the gel. The coupling rate was monitored byA280. The partially purified enterotoxin was applied to thecolumn. After being washed with a 10- to 20-fold bed volumeof buffer A, the column was eluted with 50 mM Tris-HCl (pH10.5) by the stepwise method.

Ganglioside affinity column chromatography. A 1-mgamount of gangliosides (SRL) dissolved in distilled water (1mg/ml) and 30 mg of EDC [N-ethyl-N'-(3-dimethyl amino-propyl)carbodiimide hydrochloride] were added to 1 ml ofAF-Amino Toyopearl (Toso, Tokyo, Japan), which waskindly provided by Y. Nagashima, Toso Co. After incuba-tion overnight at 4°C with shaking, the column was washedwith 10 ml of distilled water, 1 M NaCl, distilled water, and50 mM Tris-HCl (pH 7.8) in that order. The column was thenequilibrated with buffer A. The partially purified enterotoxinwas applied to the column. After being washed with a 10-foldbed volume of buffer A, the column was eluted with 7 M ureain 50 mM Tris-HCl buffer (pH 7.8) by the stepwise method(3).

RESULTS

Partial purification of the enterotoxin. Enterotoxin activitywas measured in the cell-free supernatant, polymyxin-treated supernatant, and sonicated supernatant fractions.The highest total activity was observed in the cell-freesupernatant (3.84 x 10' ELISA units), followed by thesupernatant of polymyxin-treated cells (0.94 x 106 ELISAunits), and the lowest activity was found in the supernatantobtained from sonicated cells (0.22 x 10' ELISA units).However, the opposite order was found in the amount oftotal protein. Therefore, the cell-free supernatant had thehighest specific activity (41.3 x 103 ELISA units/mg). Forthis reason, the cell-free supernatant was used as the startingmaterial for purification of the enterotoxin.Crude enterotoxin was concentrated twofold after ammo-

nium sulfate precipitation. This concentrated sample wasapplied to a Sephadex G-100 column. A typical elution

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2416 DAIKOKU ET AL.

M A M B

97.4k

132k

66k} - _ -68k

54k

45k -43k

661 -

.*:.:.4 5k _ : :: :.s ss..))..: iP,,:

... .. ...

........... ;..

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FIG. 2. SDS-PAGE and native PAGE of the fraction from gelfiltration. Lanes: M, marker proteins (sizes in kilodaltons); A,SDS-PAGE (10% gel); B, native PAGE (10% gel). S, Band stuck oncondensation gel. Arrowhead indicates tracking dye.

pattern of enterotoxin is shown in Fig. 1. The enterotoxinactivity, monitored by ELISA, was detected between frac-tions 140 and 155 (1-ml fractions) of eluent. Fractions 140 to155 were combined, and the purity was checked by SDS-PAGE and native PAGE (Fig. 2). In native PAGE, thissample stuck on the stacking gel. However, SDS-PAGEgave three main bands with molecular masses of 68, 54, and43 kDa. Moreover, glycoproteins were not detected in thePAGE by the method of PAS staining (data not shown).These results suggest that the halotoxins might aggregate.The overall purification results are summarized in Table 1.The enterotoxin was purified 8.7-fold from the crude

supernatant to the gel filtration step. The final fractionshowed a specific ELISA titer of 35.82 x 104 ELISAunits/mg. ELISA units were defined as described previously(4).

Adenylate cyclase activity of the enterotoxin. The purifiedC. jejuni enterotoxin and CT were not toxic to HeLa cells,and no morphological changes were observed. Chang et al.

TABLE 2. cAMP content in HeLa cells treated with C. jejunienterotoxin or CT

cAMP assay ia cAMP assay 2bToxin (Rg/ml) (nmol/mg of protein) (pmol/106 cells)

None (control) 2.6 1.9 ± 0.355C. jejuni

18 4.0 NTC21 NT 3.0 ± 0.044

CT91 5.5 NT96 NT 3.3 ± 0.043a Cells were incubated with no toxin or the indicated toxin for 24 h at 37°C.

Assay conditions are described in the text. Values are for a single experiment.b Cells were incubated with no toxin or the indicated toxin for 24 h at 37°C.

Assay conditions are described in the text. Values are means ± standarddeviations for triplicate experiments.

c NT, Not tested.

(2) reported that type II heat-labile enterotoxin of E. coli(LT-II), which is similar to CT, increased the cAMP amountby activating adenylate cyclase through the GTP-dependentADP-ribosylation of specific membrane proteins. In thisexperiment, the enzymatic activity of enterotoxin was deter-mined by the same method. Results are shown in Table 2.Enterotoxin was found to enhance adenylate cyclase activityin HeLa cells. Adenylate cyclase activity increased in mem-branes prepared from HeLa cells incubated with enterotoxinby 1.5-fold over that in the control under these conditions,i.e., 4.0 nmol of cAMP was converted per mg of membraneprotein, whereas the control fraction converted 2.6 nmol ofcAMP per mg. After treatment with CT, adenylate cyclaseactivity in the HeLa membranes was enhanced 2.0-fold overthat in the control. We could not compare the specificactivity of both toxins because the amounts used were notequal. However, the findings in this experiment suggest thatthe enterotoxin has a mechanism similar to that of CT, whichcauses induction of secretory diarrhea by stimulating ade-nylate cyclase activity in intestinal cells.Enhancement of adenylate cyclase activity in HeLa cells

was confirmed by quantitation of cAMP (Table 2). As in theadenylate cyclase assay (assay 1), the results of assay 2revealed that treatment with the enterotoxin or CT elevatedthe cAMP content in HeLa cells.

Elution profiles of the enterotoxin in the affinity columnchromatography. Elution profiles of enterotoxin from anti-CT IgG affinity column chromatography are shown in Fig. 3.The enterotoxin bound to the column was eluted with 50 mMTris-HCl buffer (pH 10.5). The elution pattern was moni-tored by ELISA. Fractions 80 to 95 were collected andsubjected to SDS-PAGE and native PAGE. The material

TABLE 1. Summary of purification of enterotoxina

Total Total Activity (ELISA unitsb) Fold YieldStep vol protein Total (106 Specific (104 purification (%)

(ml) (mg) units) units/mg)

Cell-free supernatant 12,000 93.0 3.84 4.13 1.00 100Ultrafiltration 1,800 79.0 3.46 4.38 1.06 9080% saturated 22 39.8 3.60 9.04 2.19 94(NH4)2S04

Gel filtration 16 5.5 1.97 35.82 8.67 51

a Fractions from affinity columns (see text) are not shown because the amount of protein in these fractions was too small for the assay.b ELISA units were calculated as ELISA titer (the amount of antigen giving a positive reaction in the ELISA [>0.1 optical density unit] at the highest dilution

of each fraction) times the total volume of the fraction (4).

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.1

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FIG. 3. Elution profile of C. jejuni enterotoxin on anti-CT IgG affinity column chromatography. Inset is SDS-PAGE of pooled fraction (p);tracking dye is indicated by the arrowhead. Elution (e) was performed with 50 mM Tris-HCl (pH 10.5).

97.4k -

66k - _ - 68k- 54k

14.2k -

$DS-PA!

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FIG. 4. Elution profile of C. jejuni enterotoxin on ganglioside affinity column chromatography. Inset is SDS-PAGE of pooled fraction (p);tracking dye is indicated by the arrowhead. Elution (e) was performed with 7 M urea in buffer A.

0.6

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2418 DAIKOKU ET AL.

gave a single band at 68 kDa on SDS-PAGE (Fig. 4) and alsoon native PAGE (data not shown). The purity of this fractionwas determined by scanning of the stained gel with a Flyingspot scanner (Shimadzu Co., Kyoto, Japan), and it wasfound to be homogeneous. This suggests that the immuno-logical crossing site of the enterotoxin to CT should bepresent in the 68-kDa polypeptide.

In the case of ganglioside affinity column chromatography,elution was performed with 7 M urea in 50 mM Tris-HCl (pH7.8). The elution profile and the SDS-PAGE pattern of theeluent are shown in Fig. 4. Unlike the anti-CT IgG column,the material obtained from the ganglioside column gave twobands at 68 and 54 kDa in SDS-PAGE. This might have beencaused by the different specificity of the subunits of the toxinfor binding to the affinity resins. The homogeneity of thisfraction monitored by gel scanning was greater than 73%.The fractions from the two affinity columns were not able tobe examined for cAMP because not enough of the toxin wasobtained for the cAMP assay in these steps.

DISCUSSION

The enterotoxin produced by C. jejuni is similar to CT andLT in many ways, such as immunological properties andenzymatic activity. This enterotoxin is also known to be heatlabile and to induce accumulation of fluid and electrolytes inboth rat (19) and rabbit (15) ileal loops. The enterotoxin alsocauses cytotoxic changes in confluent-monolayer cell lines,such as elongation of CHO cells (6, 7) and rounding of Y-1cells (15, 19). These findings are consistent with evidencethat the mechanism of action of the toxin is mediated by theincrease in cAMP in the cells (19).We purified the enterotoxin and examined the enzymatic

activity and the structure of the toxin. We also examined thebinding of the toxin to affinity columns. The crude toxin waspurified 8.7-fold by three steps, including gel filtration chro-matography. This purified toxin gave three bands on SDS-PAGE, although it stuck on native PAGE. From this appar-ent discrepancy, it was hypothesized that the holotoxinmight aggregate by itself. PAS staining showed that therewere no detectable sugars in the toxin protein. Therefore, itis suggested that the holotoxins of the enterotoxin wouldaggregate each other and form oligomers as the native form.

It is known that CT and LT ADP-ribosylate a guaninenucleotide-binding regulatory protein (G.) (16, 18), whereaspertussis toxin ADP-ribosylates an inhibitory guanine nucle-otide-binding regulatory protein (Gi) (2). In this study, weexamined the adenylate cyclase activity of HeLa cellstreated with C. jejuni enterotoxin. Ruiz-Palacios et al. (19)reported on the production of cAMP in CHO cells treatedwith C. jejuni enterotoxin and observed that the cAMPconcentration in the cell increased. The results of this studyshowed that the enterotoxin purified by gel chromatographyincreased adenylate cyclase activity in HeLa cells. Fromthese findings, the enzymatic activity of enterotoxin shouldresemble that of CT.The subunit structure of the enterotoxin was reported as a

single polypeptide by McCardell et al. (16). However, ourexperiment and the report by Klipstein and Engert (11)indicate that the holotoxin of the enterotoxin would beconstituted of several subunits. Klipstein and Engert (11)found that the holotoxin treated with guanidine showed threeprotein peaks in gel filtration with Sephadex G-75, althoughtheir precise molecular sizes were not indicated. They alsoreported that the largest subunit among the three cross-reacted with LT. In this study, it was found that a single

band at 68 kDa was observed on SDS-PAGE in the fractionfrom the anti-CT IgG affinity column; however, two bands at68 and 54 kDa were detected by ganglioside affinity columnchromatography. These results suggest that the holotoxinshould consist of at least two polypeptides of 68 and 54 kDaand that the large subunit should have antigenic sites similarto those of CT and LT. Both subunits might be responsiblefor binding the toxin to the ganglioside receptors on thesurface of the cell.A vaccine for enterotoxigenic E. coli is currently being

developed (11). One such candidate vaccine for E. coliconsists of the heat-labile toxin cross-linked to the LT Bsubunit. C. jejuni enterotoxin is known to have a closeimmunological relationship with LT and CT. Therefore,precise knowledge of the structure of the toxin is needed inorder to develop an effective vaccine for campylobacters.

ACKNOWLEDGMENTS

We thank T. Eguchi (Hakodate Chuou Hospital) for kindlysupplying the bacterial strains used in this work, Y. Nishiyama(Nagoya University) and T. Suzutani (Asahikawa Medical College)for the gift of the cultured cell lines, and T. Yamaguchi (ResearchLaboratory, Yamasa Shoyu Co.) for kindly supplying the nucleo-tides used in this work. We thank T. Suzuki and Y. Ezura,Hokkaido University, for useful suggestions.

LITERATURE CITED

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3. Cuatrecasas, P., I. Parikh, and M. D. Holienbelg. 1973. Affinitychromatography and structural analysis of Vibrio cholerae en-terotoxin-ganglioside-agarose and the biological effects of gan-glioside-containing soluble polymers. Biochemistry 12:4253-4264.

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8. Johnson, W. M., and H. Lior. 1986. Cytotoxic and cytotonicfactors produced by Campylobacterjejuni, Campylobacter coli,and Campylobacter laridis. J. Clin. Microbiol. 24:275-281.

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12. Klipstein, F. A., R. F. Engert, H. Short, and E. A. Schenk. 1985.Pathogenic properties of Campylobacter jejuni: assay and cor-relation with clinical manifestations. Infect. Immun. 50:43-49.

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15. Lecce, J. G. 1958. Some biochemical characteristics of Vibriofetus and other related vibrios isolated from animals. J. Bacte-riol. 76:312-316.

16. McCardell, B. A., J. M. Madden, and E. C. Lee. 1984. Cam-pylobacterjejuni and Campylobacter coli production of a cyto-tonic toxin immunologically similar to cholera toxin. J. FoodProt. 47:943-949.

17. McCardell, B. A., J. M. Madden, and J. T. Stanfield. 1986.Effect of iron concentration on toxin production in Campylo-

bacter jejuni and Campylobacter coli. Can. J. Microbiol. 32:395-401.

18. Moss, J., D. L. Burns, J. A. Hsia, E. L. Hewlett, R. L. Guerrant,and M. Vaughan. 1984. Cyclic nucleotides: mediators of bacte-rial toxin action in disease. Ann. Intern. Med. 101:653-666.

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