JOURNAL OF CLINICAL MICROBIOLOGY, June 1990, p. 1314-13200095-1137/90/061314-07$02.00/0Copyright C 1990, American Society for Microbiology

Isolation, Characterization, and Host-Cell-Binding Properties of a

Cytotoxin from Campylobacter jejuniSANGEETA MAHAJAN AND FRANK G. RODGERS*

Department of Microbiology, Spaulding Life Science Center, University ofNew Hampshire,Durham, New Hampshire 03824

Received 10 January 1990/Accepted 19 March 1990

A 68,000-molecular-weight protein was isolated by polyacrylamide gel electrophoresis from the organism-free filtrate of a fully virulent clinical strain of Campylobacterjejuni. The eluted protein was heat labile, was

inactivated at either pH 3.0 or 9.0, was sensitive to trypsin, and was lethal for fertile chicken eggs. It also hadtoxic effects on chicken embryo fibroblast, Chinese hamster ovary (CHO), and intestinal 407 (Int407) cells. Amonoclonal antibody (CETPMAb4) raised to this eluted toxic protein (ETP) from C. jejuni abolished these toxicactivities. Homology between C. jejuni ETP and Vibrio cholerae toxin was not observed in that specific antiserato each did not block their respective toxic activities. In enzyme-linked immunosorbent assays, ETP, unlikecholera enterotoxin, did not bind to GM1 ganglioside. Furthermore, the C. jejuni toxin had cytotoxinlikeproperties and induced rounding of CHO cells. Binding of ETP to Int407 and primary chicken embryofibroblast cells was maximal after 2 h as assessed by enzyme-linked immunosorbent assay, and this toxinadherence to host cell membranes was significantly reduced by prior treatment of the cells with proteolyticenzymes, neuraminidase, or glutaraldehyde but not by treatment with P-galactosidase, lipase, Nonidet P-40,or sodium metaperiodate. In competitive binding assays, sugars, lectins, or GM1 ganglioside did not adverselyinfluence uptake of ETP by these cells. These results suggest that the ETP produced by C. jejuni is a cytotoxinwhich binds to Int407 cells via a protein- or glycoproteinlike receptor on cell membranes and possessesproperties dissimilar to those of V. cholerae toxin.

The enteropathogenic bacterium Campylobacter jejuni isrecognized as one of the major etiologic agents of acutediarrhea (3, 28). Although the disease has a worldwidedistribution, it is particularly severe in developing countries(4, 5, 10, 21). The invasive nature of this organism has beenshown (7, 8, 22, 25); however, induction of secretory diar-rhea points toward production of toxins as an importantvirulence factor (14, 16, 26). Little is known about thepathogenesis of infection and the mechanism(s) involved ininduction of inflammatory enterocolitis (3, 12) or secretorydiarrhea (10, 26), both of which have been reported inassociation with infections due to C. jejuni.

Since 1983, a number of studies have reported the produc-tion by this organism of an enterotoxin (14, 16, 19, 26) or acytotoxin (12, 13, 24, 32). Various eucaryotic cell lines,including HeLa, MRC-5, HEp-2, Chinese hamster ovary(CHO), and Vero cells, as well as a number of animal modelsystems, have been used for toxigenicity studies (2, 12, 13,32). However, the nature of these toxins remains controver-sial. Ruiz-Palacios et al. (26) found a heat sensitive, cholera-like enterotoxin. Alternatively, McCardell et al. (19) re-ported a similar toxin which was stable after heating at 100°Cfor 10 min, and Johnson and Lior (13) found it to be stable at70°C for 30 min. Our previous studies showed that a crudetoxin obtained from cell-free filtrates of C. jejuni was heatlabile (18) and induced cytotoxic effects in primary chickenembryo fibroblast (PCEF) cells (S. Mahajan and F. G.Rodgers, Abstr. Annu. Meet. Am. Soc. Microbiol. 1988,B187, p. 60). Such variations may reflect strain selection,and the possibility that individual isolates produce bothenterotoxin and cytotoxin or only one toxin type cannot beexcluded.

In this report, we describe the isolation and partial char-

* Corresponding author.

acterization of a toxin produced by a virulent strain of C.jejuni which was noninvasive in chicken embryo assays.Many toxins are known to interact with specific receptors onsusceptible cell membranes as a prelude to expression oftheir toxic potential. This report also presents data on thenature of the toxin-binding receptor on eucaryotic host cells.

MATERIALS AND METHODS

Bacterial strain and culture conditions. Seven strains of C.jejuni biotype 1 were isolated from fecal samples frompatients with acute gastroenteritis or diarrhea. These wereeach subsequently passaged once on bacteriological mediaand maintained in 10% sorbitol in 1% calf serum at -70°C.Thawed cultures were used to inoculate thioglycolate brothin 1-liter batches which were incubated at 37°C for 48 hunder microaerobic conditions. In these, the final bacterialcell concentration ranged between 5 x 107 and 4 x 108CFU/ml.

Preparation of a cell-free filtrate. The bacterium-free su-pernatant from strain 2483 was obtained by centrifugation ofbroth-grown organisms at 15,300 x g for 10 min at 4°C,followed by filtration through a 0.22-,um-pore-size mem-brane filter. The supernatant was concentrated 50-fold in a

rotary evaporator (R110; Brinkmann Instruments, Inc.,Westbury, N.Y.). The concentrate was dialyzed againstphosphate-buffered saline (pH 7.3) by using 6- to 8-kilodal-ton-cutoff Spectrapor 1 cellulose dialysis tubing (SpectrumMedical Industries, Inc., Los Angeles, Calif.) and furtherconcentrated to one-sixth of its volume by using polyethyl-ene glycol (15 to 20 kilodaltons) by the method of Whitbyand Rodgers (31). The sample was then dialyzed in PBSovernight at 4°C, and the protein concentration was deter-mined by the method of Lowry et al. (17).

Isolation by PAGE. Approximately 2 ml of the concen-trate, together with an equal volume of sample buffer (10%

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glycerol in 0.08 M Tris, pH 6.8), was loaded on a 1.5-mm-thick preparative nondenaturing, discontinuous poly-acrylamide gel without sodium dodecyl sulfate (SDS) byusing a modification of the procedure of Davis (6). Theseparating gel was 10% T with 5% C (10% [wt/vol] acryl-amide-bis in which the bis accounted for 5% of the totalweight of acrylamide), while the stacking gel contained 5%acrylamide in 0.125 M Tris (pH 6.8). For identification, theprotein bands were stained with 0.1% Coomassie blue R250(Sigma Chemical Co., St. Louis, Mo.) and similar unstainedbands were cut from the gels and individually eluted in anElutrap apparatus (Schleicher & Schuell, Inc., Keene,N.H.). Each eluant was filter sterilized and tested for toxic-ity by inoculation into the yolk sacs of 6-day-old fertileLeghorn chicken eggs (University of New Hampshire Poul-try Farms) by previously published methods (18, 30). Theeluted toxic protein (ETP) was subjected to SDS-polyacryl-amide gel electrophoresis (PAGE) using a 5% stacking geland a 10% separating gel (15) and silver stained (23) to revealproteins. Each gel contained molecular weight standards(Bio-Rad Laboratories, Richmond, Calif.).

Polyclonal and monoclonal antibody preparation. Poly-clonal antibody to C. jejuni 2483 was prepared in rabbits bymultiple subdermal injections of a suspension of 108 CFU ofFormalin-fixed whole organisms per ml. Further immuniza-tions were given after 2 and 4 weeks, and the rabbits wereexsanguinated by cardiac puncture 2 weeks after the finalinoculations. Serum was examined for specificity by bothneutralization in chicken embryo lethality assays and immu-nofluorescence of whole organisms using a goat anti-rabbitfluorescein isothiocyanate conjugate (Organon Teknika,Malvern, Pa.).Monoclonal antibodies (MAb) to ETP from strain 2483

were raised by using a modification of the method of Galfréand Milstein (9). Two BALB/c mice were primed by intra-peritoneal injection with 1 to 5 ,ug of ETP in increasing dosesin Freund incomplete adjuvant (Sigma) over 8 weeks. Spleencells from the primed mice were fused with NS1 mousemyeloma cells, and the fusion preparation was plated into96-well plates in RPMI 1640 cell culture medium containing10% hypoxanthine, 1% aminopterin, and 10% thymidine.Clones producing ETP-specific MAb were selected by incu-bating 100 Fil of the medium from each well with a sample ofETP. After incubation at 37°C for 30 min, the mixtures wereinoculated into fertile chicken eggs for a lethality assay (18,30). The ability of the MAb to neutralize the toxic potentialof the toxic filtrates from the six strains was similarlyassayed by egg inoculation. The specificity of the MAb forETP was further confirmed by transfer of ETP to nitrocel-lulose by standard Western blot (immunoblot) techniques(29). Typical transfer conditions were a 100-mA constantcurrent for 18 to 24 h in a Trans-Blot transfer cell (Bio-Rad).Nitrocellulose membranes were then equilibrated in Tris-buffered saline (TBS; pH 7.5) for 30 min. Unoccupied siteson the nitrocellulose were blocked by incubating the blots in3% gelatin-TBS for 30 to 40 min. Immunoblots were washedtwice in TBS and then incubated for 1 h at room temperaturein those primary antisera (MAb) which neutralized the lethalactivity of ETP for chicken embryos. After being washed inTBS, the blots were incubated in goat anti-mouse horserad-ish peroxidase conjugate (Organon Teknika) diluted 1:2,000in 1% gelatin-TBS. Further nitrocellulose transfers wereprepared and manipulated under identical conditions butincubated with the polyclonal antiserum and labeled withgoat anti-rabbit horseradish peroxidase conjugate (OrganonTeknika).

All immunoblots were developed in 4-chloro-1-naphthol(Bio-Rad). Substrate buffer was made by dissolving 60 mg of4-chloro-1-naphthol in 20 ml of ice-cold methanol, which wasthen added to TBS containing 0.015% H202. The reactionwas stopped by rinsing the blots in distilled water, followedby TBS.

Nature of toxin. (i) Cell culture assays. Human intestinal407 (Int407) cells obtained from the American Type CultureCollection, Rockville, Md., and PCEF cells were used todetermine the cytotoxic activities of the bacterium-freesupernatant fluids and ETP. Cells were grown to confluentmonolayers in 96-well plates (Costar, Cambridge, Mass.) inEagle minimum essential medium (Irvine Scientific, SantaAna, Calif.) supplemented with 200 mM-glutamine (10 ml/liter), 7.5% sodium bicarbonate (29.4 ml/liter), and 10%newborn calf serum (Sigma). ETP (0.1 pg/ml) and bacterium-free supernatant fluids (30 ,ug/ml) diluted in Hanks balancedsalt solution (Irvine Scientific) were added as 0.1-ml volumesto the plates, and the cells were observed for 48 h forcytotoxic effects. For neutralization of the toxic activity incell cultures, the supernatant and ETP were incubated at37°C for 30 min with a 1:10 dilution of the polyclonalantiserum. Similar incubations were done with the ETPMAb (CEPTMAb4), which both neutralized the toxicity ofETP in lethality assays and showed the highest reactivity inenzyme-linked immunosorbent assays (ELISA). These wereadded to Int407 and PCEF cells in 96-well plates andexamined for toxic activity.As described by Guerrant et al. (11), the CHO cell assay

was performed with ETP to establish the nature of the toxin.The percentage of elongated cells was calculated after incu-bation of the cells with 0.01 or 0.1 ,ug of ETP per ml for 24 hat 37°C. CHO cells were grown as confluent monolayers insix-well plates (Costar) in supplemented Eagle minimumessential medium as described for Int407 and PCEF cells.Assays were repeated by using the bacterial cell-free filtratesof the remaining six strains of C. jejuni at a concentration of30 pg/ml. Heat-labile enterotoxins were obtained similarlyfrom Escherichia coli H10407 (Stanley Falkow, StanfordUniversity, Stanford, Calif.) and Vibrio cholerae 2868 (RitaColwell, University of Maryland, College Park) grown over-night in tryptic soy broth to a bacterial concentration ofapproximately 5 x 107 CFU/ml. The cell-free filtrates wereobtained by filtration through a 0.22-,um-pore-size mem-brane filter and used as controls at concentrations of 0.01,0.1, and 30 ,ug/ml.

(il) Binding specificity. An ELISA was used to calculatethe binding of ETP to Int407 and PCEF cells. To determinethe time for maximum binding, cell monolayers were incu-bated with 0.1 ml of ETP (0.01 ,ug of protein) for varioustimes. The cell monolayers were washed three times with0.05% Tween 20 in phosphate-buffered saline, and for eachtime interval, 0.1 ml of CETPMAb4 was added to each welland the plates were incubated at 37°C for 2 h. After threefurther washings with 0.05% Tween 20-phosphate-bufferedsaline, the cell monolayers were incubated with 0.1 ml ofgoat anti-mouse horseradish peroxidase conjugate at 37°Cfor 1 h. After three washings with 0.05% Tween 20-phos-phate-buffered saline, 3',3',5,5'-tetramethyl benzidine sub-strate (Sigma) was added and the reaction was stopped with2 M sulfuric acid. The color intensity was read by using anenzyme immunoassay reader at 405 nm (Whittaker MABioproducts, Walkersville, Md.).

It is a characteristic feature that E. coli and V. choleraeenterotoxins bind to the monosialoganglioside GM1, andusing GM1 as a specific sorbent for enterotoxins has been an

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1316 MAHAJAN AND RODGERS

alternative approach for identification of enterotoxins (1,27). Therefore, similar ELISA experiments were performedin which the wells of microtiter plates were each coated with100 ,ul of a 1.5 ,uM solution of GM1 (Sigma) and the bindingof ETP and the six cell-free filtrates to GM1 was assayed. V.cholerae enterotoxin was used as a positive control in thesebinding studies. Antitoxin to V. cholerae was kindly pro-

vided by James Kaper (Center for Vaccine Development,University of Maryland, Baltimore).To cleave potential protein, glycoprotein, or lipid recep-

tors for ETP, Int407 cell monolayers were washed and thentreated with 0.005 U of protease XIV from Streptomycesgriseus per ml, 0.1 U of chymotrypsin per ml, 50 U of trypsinper ml, 100 U of pepsin per ml, 1 U of neuraminidase per ml,1 U of P-galactosidase per ml, 100 U of lipase per ml, or

0.005% Nonidet P-40. In addition, similar monolayers were

treated with either 5 mM sodium metaperiodate to oxidizecarbohydrate moieties or 0.1% glutaraldehyde to immobilizeactive proteins on cell surfaces. The lectins wheat germ

agglutinin and concanavalin A were each used at 100 ,ug/mlto saturate N-acetylglucosamine and mannose-binding sites,respectively. After treatment, the cells were washed toremove the agents and binding of ETP from strain 2483 was

assessed by ELISA.Adherence-blocking studies were done in a competitive

manner by using mono- and disaccharide sugars commonlyfound on mammalian mucosal surfaces. Glucose, galactose,mannose, fucose, arabinose, maltose, sucrose, N-acetylglu-cosamine N-acetylgalactosamine, and N-acetylneuraminicacid (NurNAc), each at 100 mM, were used. Besides sugars,binding was also assessed in the presence of GM1 at 100p.g/mi and the lectins. In these studies, the blocking agentswere added to the test cells 10 min before addition of ETPand the mixture was incubated for 2 h. Hanks balanced saltsolution was used as a control in place of sugars, GM1, andlectins. ETP binding was assessed by ELISA, as describedabove. Except for glutaraldehyde (Electron MicroscopySciences, Fort Washington, Pa.), all of the agents listed wereobtained from Sigma.

All results were generated from experiments performed intriplicate.

RESULTS

Electrophoretic pattern of the concentrate. The four bandsobtained by preparative PAGE of the concentrated cell-freefiltrate are shown in Fig. 1 (lane A). After each was electro-eluted and tested in eggs, one only proved to be lethal forchicken embryos. Pieces of acrylamide were electroelutedand 0.1-ml samples were injected into eggs as controls. Nolethality was observed. Whether reducing or nonreducingconditions were used, SDS-PAGE of this lethal ETP yieldedtwo bands of 65 and 68 kilodaltons (Fig. 1, lanes B and C).

The lethality of ETP was abolished by 0.5 mg each of trypsinand protease per ml, changes in pH, and heating at 60 or100°C for 15 min. Similar findings were reported previouslyfor crude cell-free filtered broth (18).MAb screening. Immunoperoxidase probes of ETP re-

solved by SDS-PAGE and transferred to nitrocellulose were

positive for both bands when the polyclonal antibody wasused. However, monoclonal antibody CETPMAb4 showedonly the 68-kilodalton band (Fig. 1, lanes D and E). WhenETP or crude filtrates were incubated with CETPMAb4,lethality for fertile chicken eggs was abolished (Table 1).

Cell culture assays. By inverted microscopy, the toxiceffects of ETP on Int407 and PCEF cells appeared as

A r-, B C D E

-97.4

ET P

*25.7

FIG. 1. Lanes: A, nondenaturing, discontinuous PAGE of theconcentrated, dialyzed bacterial cell-free filtrate of C. jejuni 2483showing ETP; B, SDS-PAGE of ETP from lane A; C, molecular sizemarkers of 200, 97.4, 68, 43, 25.7, 18.4, and 14.3 kilodaltons; D,Western blot of the ETP profile from lane B incubated withpolyclonal antibodies to whole organisms; E, Western blot of theETP profile from lane B incubated with monoclonal antibodyCETPMAb4.

cytotoxic (rounding) and cytolytic (lysis) changes in cellswithin 12 h posttreatment (Fig. 2). At that time, approxi-mately 80% of the intact cells remaining in the monolayerswere rounded, and of these, 78% were nonviable in trypanblue exclusion assays. However, these cytopathic effectswere absent if ETP was incubated for 30 min with eitherCETPMAb4 or the polyclonal antibody. The effects on CHOcells of ETP from C. jejuni 2483 or the six crude unconcen-trated toxic filtrates and cholera toxin and E. coli enterotoxinwere examined. By 24 h postinoculation, the seven C. jejunisamples added to the CHO cell monolayers had causedrounding in 68 to 82% of the cells, and of these rounded cells,72% were nonviable by trypan blue assay. The enterotoxinsof E. coli and V. cholerae caused elongation in approxi-

TABLE 1. Percent mortality of fertile chicken eggs due toC. jejuni, V. cholerae, or E. coli toxin after

incubation with antisera

% Mortalityafter incubation with":

Toxina

CETPMAb4 CholeraCETPMAb4 ~antitoxin

C. jejuni ETP O 100Filtrates from six additional strains 0 100V. cholerae enterotoxin 100 0E. coli enterotoxin 100 0

a Fertile chicken eggs were inoculated via the yolk sacs at 6 days ofincubation. Inocula were 0.1 ml per egg, and toxin protein concentrationswere 0.01 ,ug for ETP and 3 pug for all filtrates.

b Incubation of toxins and antisera was at 37°C for 30 min. Ten eggs wereused for each inoculum, and lethality was assayed after 24 h. These resultswere generated by three separate experiments. Incubation with phosphate-buffered saline resulted in 100% mortality.

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TABLE 2. Cytological changes in CHO cells due to the toxins ofC. jejuni, V. cholerae, and E. coli after incubation with antisera

% Effect on cells after incubation with':Toxin(concn) No CETPMAb4 Cholera

antiserum antitoxin

C. jejuni ETP (0.1 ,ug/mI) 80 4 75C. jejuni ETP (0.01 p.g/ml) 70 2 68Filtrates from six additional 68-82 <10 68-82

strains (30 ,ug/ml)V. cholerae enterotoxin 75 72 9

(30 p.g/ml)V. cholerae enterotoxin 62 68 0

(0. 1 ,ug/ml)V. cholerae enterotoxin 10 8 0

(0.01 p.g/ml)E. coli enterotoxin (30 ,ug/ml) 75 72 15E. coli enterotoxin (0.1 ,ug/ml) 60 68 0E. coli enterotoxin (0.01 ,ug/ 15 10 0

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a Incubation of toxins and antisera was at 37°C for 30 min, and cytopathicchanges were recorded after 24 h. The data represent percent cell rounding for

*lF-& % t, C. jejuni toxin and percent cell elongation for the toxins of V. cholerae and E.t. ,-X*k; ét 0 f cofi. These results were generated by three separate experiments.

tv.>:.:̂«'s lately 75% of the CHO cells (Fig. 3). When ETP from strain2483 and toxic cell-free filtrates from the remaining six C.

b i 2 S, . jejuni strainswere incubated with CETPMAb a ndtheneoo.8e_-t `.N"-' {*j<§ ,t ts tS added to CHO cells, no cytotoxic activity was seen.

b. ..f -v 'A CETPMAb4did notneutralize cholera toxin or E. coli toxin,.f:Z Z ^ » *.<> rX ;WZ t and cholera antitoxin had no effect on the toxin from C.

FIG. 2. Toxic effects of C. jejuni ETP on Int407 cells. (a) jejuni as determined by fertile chicken egg lethality (Table 1)Untreated control showing a confluent monolayer. (b) Cells treated and CHO cell assays (Table 2).iith 0.1 ,ug of ETP per mil for 12 h. Note cell rounding and lysis as Binding specificity of the toxin. Int407 cells avidly boundshown by loss of monolayer integrity. Magnification, x 1,980. ETP, and this was maximal after 2 h of incubation at 37°C

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FIG. 3. Toxic effects of C. jejuni ETP on CHO cells. (a) Untreated control showing a confluent monolayer. (b) Cells exposed to 0.1 ,ugof V. cholerae enterotoxin per ml for 24 h. Note cell elongation characteristic of the activity of cholera toxin. (c) Cells treated with 0.1 ,ugof ETP per ml for 24 h. In contrast to the activity of the cholera toxin, note rounding and lysis of affected cells. Magnification, x 1,980.

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1318 MAHAJAN AND RODGERS

5 10 15 20I

25 30 35

GM1

Con A

WGA

NurNAc

GaNAc

GINAcI . . I I I I I I

0 10 20 30 40 50 60 70 80 90 100

40

time/hFIG. 4. Binding of C. jejuni ETP to Int407 cells as assessed by

ELISA. Maximum binding occurred after 2 h, and similar bindingdynamics were observed for primary chicken embryo fibroblastcells. The results shown are averages of three separate evaluations.

% reductionFIG. 6. Competitive binding of C. jejuni ETP to Int407 cells as

assessed by ELISA. Percent reduction of ETP binding followingcoincubation of the toxin and eucaryotic cells in the presence oflectins, acetylated sugars, and GM1. Except for NurNAc, nonesignificantly affected binding of ETP. The results shown are aver-ages of three separate trials. Con A, Concanavalin A; WGA, wheatgerm agglutinin; GaNAc, N-acetylgalactosamine; GlNAc, N-acetyl-glucosamine.

(Fig. 4). By 24 h, the cells were rounded or lysing. Incontrast to V. cholerae enterotoxin, ETP and crude filtratesfrom the six C. jejuni strains showed no binding to GM1.ETP binding to Int407 cells following enzymatic and other

glutaraldehyde

sod. metaper.

nonidet P40

neuraminidase

beta-gal

lipase

protease

pepsin

chymotrypsin

trypsin

r

-10 0 1 0 20 30 40 50 60 70 80 90 100

% reduction

FIG. 5. Binding of C. jejuni ETP to Int407 cells as assessed byELISA. Percent reduction in adherence of toxin after treatment ofhost cells with potential eucaryotic receptor-modifying agents isshown. The results shown are averages of three separate trials. Sod.metaper., Sodium metaperiodate; beta-gal, ,-galactosidase.

treatments is shown in Fig. 5. Trypsin, protease, chymo-trypsin, pepsin, neuraminidase, or aldehyde treatment re-duced toxin binding to host cells by greater than 50%.Treatment of the cells with lipase,"-galactosidase, NonidetP-40, or sodium metaperiodate had no significant effect ontoxin binding. Of the lectins, neither wheat germ agglutininnor concanavalin A affected ETP attachment. Furthermore,no reduction in binding of ETP to Int407 cells in the presenceof GM1 was observed, suggesting that this C. jejuni toxin,unlike cholera toxin, did not bind to the GM1 receptor.Although incubation of ETP in the presence of N-acetylglu-cosamine or N-acetylgalactosamine did not adversely affectattachment to host cells, treatment with NurNAc effected a50% reduction in binding (Fig. 6). In addition, the remainingmonosaccharides and disaccharides (glucose, galactose,mannose, fucose, arabinose, maltose, and sucrose) all re-

sulted in less than 7% reduction in binding of ETP to Int407cells.

DISCUSSION

The pathogenesis of C. jejuni has been widely investi-gated, but the nature of the bacterial virulence factors andthe mechanisms by which this organism initiates entericdisease are not clear. Invasion of the bloodstream of the hosthas been reported as an important virulence factor in variousstudies (3, 10). However, production of toxin by C. jejuni,first reported by Ruiz-Palacios et al. (26), has gained impor-tance, and in noninvasive strains, toxin production probablyconstitutes a major virulence factor (18). Enterotoxin pro-duction undoubtedly causes the mucus- and blood-free,watery diarrhea often observed in C. jejuni infections, andthis would be particularly so for infections caused by strainslacking invasive properties. C. jejuni also produces a cyto-toxin (12, 13, 24, 32) which may be responsible for inflam-matory diarrhea. It seems that C. jejuni resembles E. coli in

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TOXIC ACTIVITY OF C. JEJUNI 1319

that some strains are enteroinvasive and some are toxic innature, with virulence a multifactorial process. Belbouri andMegraud (2) reported that 64% of the strains tested hadenterotoxinlike activity, while Lindblom et al. (16) reportedthat only 32% of the strains isolated from humans with acuteenteritis and healthy egg-laying chickens were toxigenic. It ispossible that failure to identify toxin production in appar-ently nontoxigenic strains reflects variations in storage andgrowth conditions before assay. It has been observed inother toxin-producing organisms that after several in vitropassages, strains revert to nontoxigenic forms, especially ifthe toxin production is plasmid mediated. This may be trueof C. jejuni. Alternatively, as has been shown by Klipstein etal. (14), strains of the organism may be independentlytoxigenic, invasive or, for some strains, both.The molecular size of the toxin isolated in this study was

lower than that of cholera toxin (84 kilodaltons) or the E. coliheat-labile enterotoxin (91.5 kilodaltons), although it approx-imates the single 70-kilodalton band reported by McCardellet al. (19) for C. jejuni. The 65-kilodalton protein band alsopresent in ETP from the discontinuous nondenaturing gellacked toxic activity. In embryo protection studies, the toxicactivity of ETP was neutralized by incubation withCETPMAb4, and furthermore, the degree of ETP binding toInt407 cells as measured by ELISA was the same whetherCETPMAb4 or the polyclonal antibody was used. The tox-icity of ETP resided solely in the 68-kilodalton band whichdid not further cleave in the presence of P-mercaptoethanol;therefore, this C. jejuni toxin, unlike those of V. choleraeand E. coli, did not possess subunits. In addition, it has beenreported that low concentrations of GM1 inhibited the toxicactivity of the V. cholerae and E. coli toxins, and thisbinding was known to be a function of the B subunits. Incontrast to V. cholerae enterotoxin, ETP did not bind toGM1 and induced rounding and lysis of CHO cells. In thesestudies, it appeared that the ETP of C. jejuni differedmarkedly from V. cholerae enterotoxin. Furthermore, theirlack of immunological relatedness was confirmed by theabsence of cross-neutralizing reactivity between ETP and V.cholerae toxin and their respective antisera. Indeed, thecharacteristics of this toxin lead us to believe that it is apotent cytotoxin rather than an enterotoxin.

Similarities between the cytotoxin described in this reportand the crude cytotoxic C. jejuni filtrate reported by Guer-rant et al. (12) included molecular weight, heat and trypsinsensitivity, and CHO cell rounding. However, the cytotox-icity of our toxin was lost on freeze-thawing but it retainedactivity for up to 4 weeks at 4°C and was unaffected bytreatment with polymyxin B.Most toxins are known to interact with specific receptors

on susceptible cell membranes as a prelude to cytoplasmicuptake or initiation of their toxic responses. Others actuallydamage cell membranes per se. Both Int407 and PCEF cellscarry receptors on their membranes specific for ETP, andthese showed maximal binding after 2 h of treatment, fol-lowed by rounding and lysis of the host cells with a concom-itant decrease in ETP binding by 24 h. Toxin-binding recep-tors may be delineated on eucaryotic host cells by usingspecific blocking agents. This process of investigating func-tion through loss of function was used to assess the reduc-tion or loss in toxin uptake due to inhibitory agents. Degra-dation and immobilization of surface proteins significantlyreduced ETP adherence to Int407 cells, while oxidation ofcarbohydrate moieties by periodate marginally increasedbinding. This indicated that the toxin-binding receptor is asurface protein and that some stearic hindrance from carbo-

hydrate moieties occurs. Indeed, stearic and hydrophobicinteractions between different membrane components ofeucaryotic host cells may have a role in the recognition ofthe specific receptors of ETP. Since the carbohydratespresent during competitive blocking studies and the lipid-modifying agents used to pretreat host cells did not signifi-cantly reduce binding, a glycolipidlike receptor does notseem likely.

It was interesting that coincubation of ETP with NurNAc,a parent acid of a family of amino sugars, did reduce bindingto Int407 cells by 50%. Treatment of the cells with neuramin-idase, a receptor-destroying enzyme that liberates NurNAcresidues from cell membranes, reduced binding by greaterthan 90%. However, commercially available neuraminidasehas residual protease activity, and this might explain thehigher reduction in binding of ETP compared with treatmentwith protease alone. These studies indicated that the aminoterminal of NurNAc acts as a receptor for C. jejuni ETP perse. Alternatively, it is possible that the toxin receptor onhost cells is a surface protein either linked directly to oracting in consort with a neuraminic acid moiety. McSweeganand Walker (20) have shown that C. jejuni lipopolysaccha-ride molecules were involved in adhesion of the organisms.toInt407 cells and that adherence was reduced by 50 to 60% byfucose or mannose. These two monosaccharides had noeffect on the binding of C. jejuni ETP to Int 407 cells. It doesnot seem unreasonable that the organism and its toxin havedeveloped different receptors and thereby avoid competitionfor the same host cell-binding sites.That C. jejuni virulence is multifactorial, with toxin pro-

duction as one facet, is established. Indeed, mechanisms fortoxin-induced disease have been proposed (13, 18). How-ever, the role of toxin binding to host cells in the pathogen-esis of disease is less certain. This study indicates thatproduction of a cytoxin which binds to proteinlike receptorson host cell membranes is potential virulence trait that maybe involved in the inflammatory diarrhea caused by many C.jejuni isolates. Elucidation of the mechanisms involved inthe binding of this bacterial cytotoxin to cell membranes andthe role of host cell receptors in initiating disease will betterdefine the infectious process at the cellular and molecularlevels.

ACKNOWLEDGMENTS

This work was supported in part by Biomedical Research Supportgrant 2-S07-RR07-108-14 from the National Institutes of Health andby the University of New Hampshire Research Office.

LITERATURE CITED1. Back, E., A. Svennerholm, J. Holmgren, and R. Moilby. 1979.

Evaluation of a ganglioside immunosorbent assay for detectionof Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol.10:791-795.

2. Belbouri, A., and F. Megraud. 1988. Enterotoxin-like activityproduced by Campylobacter jejuni and Campylobacter coliisolated from patients and healthy controls in Algeria. FEMSMicrobiol. Lett. 51:25-28.

3. Blaser, M. J., I. D. Berkowitz, F. M. LaForce, J. Cravens, L. B.Reller, and W. I. Wang. 1979. Campylobacter enteritis: clinicaland epidemiologic features. Ann. Intern. Med. 91:179-185.

4. Blaser, M. J., R. I. Glass, M. Imdadul Huq, B. Stoll, G. M.Kibriya, and A. R. M. A. Alim. 1980. Isolation of Campylobac-ter fetus subsp. jejuni from Bangladeshi children. J. Clin.Microbiol. 12:744-747.

5. Calva, J. J., G. M. Ruiz-Palacios, A. B. Lopez-Vidal, A. Ramos,and R. Bojalil. 1988. Cohort study of intestinal infection withCampylobacter in Mexican children. Lancet i:503-506.

VOL. 28, 1990

on August 23, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

1320 MAHAJAN AND RODGERS

6. Davis, B. J. 1964. Disc electrophoresis-11: method and applica-tion to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427.

7. Fauchere, J. L., M. Veron, A. Lellouch-Tubiana, and A. Pfister.1985. Experimental infection of gnotobiotic mice with Campylo-bacter jejuni: colonization of intestine and spread to lymphoidand reticuloendothelial organs. J. Med. Microbiol. 20:215-224.

8. Field, L. H., V. L. Headley, J. L. Underwood, S. M. Payne, andL. J. Berry. 1986. The chicken embryo as a model for campylo-bacter invasion: comparative virulence of human isolates ofCampylobacterjejuni and Camphylobacter coli. Infect. Immun.54:118-125.

9. Galfrè, G., and C. Milstein. 1981. Preparation of monoclonalantibodies: strategies and procedures. Methods Enzymol. 73:1-45.

10. Glass, R. I., B. J. Stoll, M. I. Huq, M. J. Blaser, and A. K. M.Kibriya. 1983. Epidemiologic and clinical features of endemicCamphylobacter jejuni infection in Bangladesh. J. Infect. Dis.148:292-296.

11. Guerrant, R. L., L. L. Brunton, T. C. Schnaitman, L. I. Rebhun,and A. G. Gilman. 1974. Cyclic adenosine monophosphate andalteration of Chinese hamster ovary cell morphology: a rapid,sensitive in vitro assay for enterotoxins of Vibrio cholerae andEscherichia coli. Infect. Immun. 10:320-327.

12. Guerrant, R. L., C. A. Wanke, R. A. Pennie, L. A. Barrett,A. A. M. Lima, and A. D. O'Brien. 1987. Production of a uniquecytotoxin by Camphylobacter jejuni. Infect. Immun. 55:2526-2530.

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

14. Klipstein, F. A., R. F. Engert, and H. B. Short. 1986. Enzyme-linked immunosorbent assays for virulence properties of Cam-pylobacter jejuni clinical isolates. J. Clin. Microbiol. 23:1039-1043.

15. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

16. Lindblom, G. B., B. KaiJser, and E. Sjogren. 1989. Enterotoxinproduction and serogroups of Campylobacter jejuni and Cam-pylobacter coli from patients with diarrhea and from healthylaying hens. J. Clin. Microbiol. 27:1272-1276.

17. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

18. Mahajan, S., and F. G. Rodgers. 1989. Virulence of Camphylo-bacter jejuni for chicken embryos. J. Clin. Microbiol. 27:1377-1379.

19. McCardell, B. A., J. M. Madden, and E. C. Lee. 1984. Cam-pylobacter jejuni and C. coli production of a cytotonic toxinimmunologically similar to cholera toxin. J. Food Prot. 47:943-949.

20. McSweegan, E., and R. I. Walker. 1986. Identification andcharacterization of two Campylobacter jejuni adhesins for cel-lular and mucous substrates. Infect. Immun. 53:141-148.

21. Neogi, P. K. B., and N. S. Shahid. 1987. Serotypes of Campylo-bacterjejuni isolated from patients attending a diarrheal diseasehospital in urban Bangladesh. J. Med. Microbiol. 24:303-307.

22. Newell, D. G., H. McBride, F. Saunders, Y. Dehele, and A. D.Pearson. 1985. The virulence of clinical and environmentalisolates of Campylobacterjejuni. J. Hyg. 94:45-54.

23. Oakley, B. R., D. R. Kisch, and N. R. Morris. 1980. A simplifiedstain for detecting proteins in polyacrylamide gels. Anal. Bio-chem. 105:361-363.

24. Perez-Perez, G. I., D. L. Cohn, R. L. Guerrant, C. M. Patton,L. B. Reller, and M. J. Blaser. 1989. Clinical and immunologicsignificance of cholera-like toxin and cytotoxin production byCampylobacter species in patients with acute inflammatorydiarrhea in the USA. J. Infect. Dis. 160:460-467.

25. Ruiz-Palacios, G. M., E. Escamilla, and N. I. Torres. 1981.Experimental Campylobacter diarrhea in chickens. Infect. Im-mun. 34:250-255.

26. Ruiz-Palacios, G. M., J. Torres, N. I. Torres, E. Escamilla, B.Ruiz-Palacios, and J. Tamayo. 1983. Cholera-like enterotoxinproduced by Campylobacterjejuni: characterization and clinicalsignificance. Lancet ii:250-251.

27. Sack, D. A., S. Hudas, P. K. B. Neagl, R. R. Daniel, and W. R.Spira. 1980. Microtiter ganglioside enzyme-linked immunosor-bent assay for Vibrio cholerae and Escherichia coli heat-labileenterotoxins and antitoxin. J. Clin. Microbiol. 11:35-40.

28. Svedhem, A., and B. Kaiser. 1980. Campylobacterfetus subsp.jejuni: a common cause of diarrhea in Sweden. J. Infect. Dis.142:353-359.

29. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

30. Tzianabos, A. O., and F. G. Rodgers. 1989. Pathogenesis andchemotherapy of experimental Legionella pneumophila infec-tion in the chick embryo. Zentralbl. Bakteriol. Hyg. Abt. Orig.A 271:293-303.

31. Whitby, H. J., and F. G. Rodgers. 1980. Detection of virusparticles by electron microscopy with polyacrylamide hydrogel.J. Clin. Pathol. 33:484-487.

32. Yeen, W. I., S. D. Puthucheary, and T. Pang. 1983. Demonstra-tion of a cytotoxin from Campylobacterjejuni. J. Clin. Pathol.36:1237-1240.

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