JOURNAL OF CLINICAL MICROBIOLOGY, June 1990, p. 1314-1320 Vol. 28, No. 6 0095-1137/90/061314-07$02.00/0 Copyright C 1990, American Society for Microbiology

Isolation, Characterization, and Host-Cell-Binding Properties of a Cytotoxin from Campylobacter jejuni SANGEETA MAHAJAN AND FRANK G. RODGERS* Department of Microbiology, Spaulding Life Science Center, University of New 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 had toxic effects on chicken embryo fibroblast, Chinese hamster ovary (CHO), and intestinal 407 (Int407) cells. A monoclonal antibody (CETPMAb4) raised to this eluted toxic protein (ETP) from C. jejuni abolished these toxic activities. Homology between C. jejuni ETP and Vibrio cholerae toxin was not observed in that specific antisera to each did not block their respective toxic activities. In enzyme-linked immunosorbent assays, ETP, unlike cholera enterotoxin, did not bind to GM1 ganglioside. Furthermore, the C. jejuni toxin had cytotoxinlike properties and induced rounding of CHO cells. Binding of ETP to Int407 and primary chicken embryo fibroblast cells was maximal after 2 h as assessed by enzyme-linked immunosorbent assay, and this toxin adherence to host cell membranes was significantly reduced by prior treatment of the cells with proteolytic enzymes, 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 adversely influence uptake of ETP by these cells. These results suggest that the ETP produced by C. jejuni is a cytotoxin which binds to Int407 cells via a protein- or glycoproteinlike receptor on cell membranes and possesses properties dissimilar to those of V. cholerae toxin.

The enteropathogenic bacterium Campylobacter jejuni is acterization of a toxin produced by a virulent strain of C. recognized as one of the major etiologic agents of acute jejuni which was noninvasive in chicken embryo assays. diarrhea (3, 28). Although the disease has a worldwide Many toxins are known to interact with specific receptors on distribution, it is particularly severe in developing countries susceptible cell membranes as a prelude to expression of (4, 5, 10, 21). The invasive nature of this organism has been their toxic potential. This report also presents data on the shown (7, 8, 22, 25); however, induction of secretory diar- nature of the toxin-binding receptor on eucaryotic host cells. rhea points toward production of toxins as an important virulence factor (14, 16, 26). Little is known about the MATERIALS AND METHODS pathogenesis of infection and the mechanism(s) involved in Bacterial strain and culture conditions. Seven strains of C. induction of inflammatory enterocolitis (3, 12) or secretory jejuni biotype 1 were isolated from fecal from diarrhea (10, 26), both of which have been reported in samples patients with acute gastroenteritis or diarrhea. These were association with infections due to C. jejuni. each subsequently passaged once on bacteriological media Since 1983, a number of studies have reported the produc- and maintained in 10% sorbitol in 1% calf serum at -70°C. tion by this organism of an enterotoxin (14, 16, 19, 26) or a Thawed cultures were used to inoculate thioglycolate broth cytotoxin (12, 13, 24, 32). Various eucaryotic cell lines, in 1-liter batches which were incubated at 37°C for 48 h including HeLa, MRC-5, HEp-2, Chinese hamster ovary under microaerobic conditions. In these, the final bacterial and as well as a number of animal (CHO), Vero cells, model cell concentration ranged between 5 x 107 and 4 x 108 systems, have been used for toxigenicity studies (2, 12, 13, CFU/ml. 32). However, the nature of these toxins remains controver- Preparation of a cell-free filtrate. The bacterium-free su- sial. Ruiz-Palacios et al. (26) found a heat sensitive, cholera- pernatant from strain 2483 was obtained by centrifugation of like enterotoxin. Alternatively, McCardell et al. (19) re- broth-grown organisms at 15,300 x g for 10 min at 4°C, ported a similar toxin which was stable after heating at 100°C followed by filtration through a 0.22-,um-pore-size mem- for 10 min, and Johnson and Lior (13) found it to be stable at brane filter. The supernatant was concentrated 50-fold in a 70°C for 30 min. Our previous studies showed that a crude rotary evaporator (R110; Brinkmann Instruments, Inc., toxin obtained from cell-free filtrates of C. jejuni was heat Westbury, N.Y.). The concentrate was dialyzed against labile (18) and induced cytotoxic effects in primary chicken phosphate-buffered saline (pH 7.3) by using 6- to 8-kilodal- embryo fibroblast (PCEF) cells (S. Mahajan and F. G. ton-cutoff Spectrapor 1 cellulose dialysis tubing (Spectrum Rodgers, Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, Medical Industries, Inc., Los Angeles, Calif.) and further B187, p. 60). Such variations may reflect strain selection, concentrated to one-sixth of its volume and the possibility that individual isolates produce both by using polyethyl- ene glycol (15 to 20 kilodaltons) by the method of Whitby enterotoxin and cytotoxin or only one toxin type cannot be excluded. and Rodgers (31). The sample was then dialyzed in PBS overnight at 4°C, and the protein concentration was deter- In this we report, describe the isolation and partial char- mined by the method of Lowry et al. (17). Isolation by PAGE. Approximately 2 ml of the concen- * Corresponding author. trate, together with an equal volume of sample buffer (10% 1314 VOL. 28, 1990 TOXIC ACTIVITY OF C. JEJUNI 1315 glycerol in 0.08 M Tris, pH 6.8), was loaded on a 1.5- All immunoblots were developed in 4-chloro-1-naphthol mm-thick preparative nondenaturing, discontinuous poly- (Bio-Rad). Substrate buffer was made by dissolving 60 mg of acrylamide gel without sodium dodecyl sulfate (SDS) by 4-chloro-1-naphthol in 20 ml of ice-cold methanol, which was using a modification of the procedure of Davis (6). The then added to TBS containing 0.015% H202. The reaction separating gel was 10% T with 5% C (10% [wt/vol] acryl- was stopped by rinsing the blots in distilled water, followed amide-bis in which the bis accounted for 5% of the total by TBS. weight of acrylamide), while the stacking gel contained 5% Nature of toxin. (i) Cell culture assays. Human intestinal acrylamide in 0.125 M Tris (pH 6.8). For identification, the 407 (Int407) cells obtained from the American Type Culture protein bands were stained with 0.1% Coomassie blue R250 Collection, Rockville, Md., and PCEF cells were used to (Sigma Chemical Co., St. Louis, Mo.) and similar unstained determine the cytotoxic activities of the bacterium-free bands were cut from the gels and individually eluted in an supernatant fluids and ETP. Cells were grown to confluent Elutrap apparatus (Schleicher & Schuell, Inc., Keene, monolayers in 96-well plates (Costar, Cambridge, Mass.) in N.H.). Each eluant was filter sterilized and tested for toxic- Eagle minimum essential medium (Irvine Scientific, Santa ity by inoculation into the yolk sacs of 6-day-old fertile Ana, Calif.) supplemented with 200 mM-glutamine (10 ml/ Leghorn chicken eggs (University of New Hampshire Poul- liter), 7.5% sodium bicarbonate (29.4 ml/liter), and 10% try Farms) by previously published methods (18, 30). The newborn calf serum (Sigma). ETP (0.1 pg/ml) and bacterium- eluted toxic protein (ETP) was subjected to SDS-polyacryl- free supernatant fluids (30 ,ug/ml) diluted in Hanks balanced amide gel electrophoresis (PAGE) using a 5% stacking gel salt solution (Irvine Scientific) were added as 0.1-ml volumes and a 10% separating gel (15) and silver stained (23) to reveal to the plates, and the cells were observed for 48 h for proteins. Each gel contained molecular weight standards cytotoxic effects. For neutralization of the toxic activity in (Bio-Rad Laboratories, Richmond, Calif.). cell cultures, the supernatant and ETP were incubated at Polyclonal and monoclonal antibody preparation. Poly- 37°C for 30 min with a 1:10 dilution of the polyclonal clonal antibody to C. jejuni 2483 was prepared in rabbits by antiserum. Similar incubations were done with the ETP multiple subdermal injections of a suspension of 108 CFU of MAb (CEPTMAb4), which both neutralized the toxicity of Formalin-fixed whole organisms per ml. Further immuniza- ETP in lethality assays and showed the highest reactivity in tions were given after 2 and 4 weeks, and the rabbits were enzyme-linked immunosorbent assays (ELISA). These were exsanguinated by cardiac puncture 2 weeks after the final added to Int407 and PCEF cells in 96-well plates and inoculations. Serum was examined for specificity by both examined for toxic activity. neutralization in chicken embryo lethality assays and immu- As described by Guerrant et al. (11), the CHO cell assay nofluorescence of whole organisms using a goat anti-rabbit was performed with ETP to establish the nature of the toxin. fluorescein isothiocyanate conjugate (Organon Teknika, The percentage of elongated cells was calculated after incu- Malvern, Pa.). bation of the cells with 0.01 or 0.1 ,ug of ETP per ml for 24 h Monoclonal antibodies (MAb) to ETP from strain 2483 at 37°C. CHO cells were grown as confluent monolayers in were raised by using a modification of the method of Galfré six-well plates (Costar) in supplemented Eagle minimum and Milstein (9). Two BALB/c mice were primed by intra- essential medium as described for Int407 and PCEF cells. peritoneal injection with 1 to 5 ,ug of ETP in increasing doses Assays were repeated by using the bacterial cell-free filtrates in Freund incomplete adjuvant (Sigma) over 8 weeks. Spleen of the remaining six strains of C. jejuni at a concentration of cells from the primed mice were fused with NS1 mouse 30 pg/ml. Heat-labile enterotoxins were obtained similarly myeloma cells, and the fusion preparation was plated into from Escherichia coli H10407 (Stanley Falkow, Stanford 96-well plates in RPMI 1640 cell culture medium containing University, Stanford, Calif.) and Vibrio cholerae 2868 (Rita 10% hypoxanthine, 1% aminopterin, and 10% thymidine. Colwell, University of Maryland, College Park) grown over- Clones producing ETP-specific MAb were selected by incu- night in tryptic soy broth to a bacterial concentration of bating 100 Fil of the medium from each well with a sample of approximately 5 x 107 CFU/ml. The cell-free filtrates were ETP. After incubation at 37°C for 30 min, the mixtures were obtained by filtration through a 0.22-,um-pore-size mem- inoculated into fertile chicken eggs for a lethality assay (18, brane filter and used as controls at concentrations of 0.01, 30). The ability of the MAb to neutralize the toxic potential 0.1, and 30 ,ug/ml. of the toxic filtrates from the six strains was similarly (il) Binding specificity. An ELISA was used to calculate assayed by egg inoculation. The specificity of the MAb for the binding of ETP to Int407 and PCEF cells. To determine ETP was further confirmed by transfer of ETP to nitrocel- the time for maximum binding, cell monolayers were incu- lulose by standard Western blot (immunoblot) techniques bated with 0.1 ml of ETP (0.01 ,ug of protein) for various (29). Typical transfer conditions were a 100-mA constant times. The cell monolayers were washed three times with current for 18 to 24 h in a Trans-Blot transfer cell (Bio-Rad). 0.05% Tween 20 in phosphate-buffered saline, and for each Nitrocellulose membranes were then equilibrated in Tris- time interval, 0.1 ml of CETPMAb4 was added to each well buffered saline (TBS; pH 7.5) for 30 min. Unoccupied sites and the plates were incubated at 37°C for 2 h. After three on the nitrocellulose were blocked by incubating the blots in further washings with 0.05% Tween 20-phosphate-buffered 3% gelatin-TBS for 30 to 40 min. Immunoblots were washed saline, the cell monolayers were incubated with 0.1 ml of twice in TBS and then incubated for 1 h at room temperature goat anti-mouse horseradish peroxidase conjugate at 37°C in those primary antisera (MAb) which neutralized the lethal for 1 h. After three washings with 0.05% Tween 20-phos- activity of ETP for chicken embryos. After being washed in phate-buffered saline, 3',3',5,5'-tetramethyl benzidine sub- TBS, the blots were incubated in goat anti-mouse horserad- strate (Sigma) was added and the reaction was stopped with ish peroxidase conjugate (Organon Teknika) diluted 1:2,000 2 M sulfuric acid. The color intensity was read by using an in 1% gelatin-TBS. Further nitrocellulose transfers were enzyme immunoassay reader at 405 nm (Whittaker MA prepared and manipulated under identical conditions but Bioproducts, Walkersville, Md.). incubated with the polyclonal antiserum and labeled with It is a characteristic feature that E. coli and V. cholerae goat anti-rabbit horseradish peroxidase conjugate (Organon enterotoxins bind to the monosialoganglioside GM1, and Teknika). using GM1 as a specific sorbent for enterotoxins has been an 1316 MAHAJAN AND RODGERS J. CLIN. MICROBIOL. alternative approach for identification of enterotoxins (1, A r-, B C D E 27). Therefore, similar ELISA experiments were performed in which the wells of microtiter plates were each coated with 100 ,ul of a 1.5 ,uM solution of GM1 (Sigma) and the binding of ETP and the six cell-free filtrates to GM1 was assayed. V. -97.4 cholerae enterotoxin was used as a positive control in these binding 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 then treated with 0.005 U of protease XIV from Streptomyces griseus per ml, 0.1 U of chymotrypsin per ml, 50 U of trypsin per 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 ET P 0.005% Nonidet P-40. In addition, similar monolayers were treated with either 5 mM sodium metaperiodate to oxidize *25.7 carbohydrate moieties or 0.1% glutaraldehyde to immobilize active proteins on cell surfaces. The lectins wheat germ agglutinin and concanavalin A were each used at 100 ,ug/ml to saturate N-acetylglucosamine and mannose-binding sites, respectively. After treatment, the cells were washed to remove the agents and binding of ETP from strain 2483 was FIG. 1. Lanes: A, nondenaturing, discontinuous PAGE of the assessed by ELISA. concentrated, dialyzed bacterial cell-free filtrate of C. jejuni 2483 Adherence-blocking studies were done in a competitive showing ETP; B, SDS-PAGE of ETP from lane A; C, molecular size manner by using mono- and disaccharide sugars commonly markers of 200, 97.4, 68, 43, 25.7, 18.4, and 14.3 kilodaltons; D, found on mammalian mucosal surfaces. Glucose, galactose, Western blot of the ETP profile from lane B incubated with mannose, fucose, arabinose, maltose, sucrose, N-acetylglu- polyclonal antibodies to whole organisms; E, Western blot of the cosamine N-acetylgalactosamine, and N-acetylneuraminic ETP profile from lane B incubated with monoclonal antibody acid (NurNAc), each at 100 mM, were used. Besides sugars, CETPMAb4. binding was also assessed in the presence of GM1 at 100 p.g/mi and the lectins. In these studies, the blocking agents were added to the test cells 10 min before addition of ETP cytotoxic (rounding) and cytolytic (lysis) changes in cells and the mixture was incubated for 2 h. Hanks balanced salt within 12 h posttreatment (Fig. 2). At that time, approxi- solution was used as a control in place of sugars, GM1, and mately 80% of the intact cells remaining in the monolayers lectins. ETP binding was assessed by ELISA, as described were rounded, and of these, 78% were nonviable in trypan above. Except for glutaraldehyde (Electron Microscopy blue exclusion assays. However, these cytopathic effects Sciences, Fort Washington, Pa.), all of the agents listed were were absent if ETP was incubated for 30 min with either obtained from Sigma. CETPMAb4 or the polyclonal antibody. The effects on CHO All results were generated from experiments performed in cells of ETP from C. jejuni 2483 or the six crude unconcen- triplicate. trated toxic filtrates and cholera toxin and E. coli enterotoxin were examined. By 24 h postinoculation, the seven C. jejuni RESULTS samples added to the CHO cell monolayers had caused rounding in 68 to 82% ofthe cells, and of these rounded cells, Electrophoretic pattern of the concentrate. The four bands 72% were nonviable by trypan blue assay. The enterotoxins obtained by preparative PAGE of the concentrated cell-free of E. coli and V. cholerae caused elongation in approxi- filtrate are shown in Fig. 1 (lane A). After each was electro- eluted and tested in eggs, one only proved to be lethal for chicken embryos. Pieces of acrylamide were electroeluted TABLE 1. Percent mortality of fertile chicken eggs due to and 0.1-ml samples were injected into eggs as controls. No C. jejuni, V. cholerae, or E. coli toxin after lethality was observed. Whether reducing or nonreducing incubation with antisera conditions were used, SDS-PAGE of this lethal ETP yielded % Mortality two bands of 65 and 68 kilodaltons (Fig. 1, lanes B and C). after incubation with": The lethality of ETP was abolished by 0.5 mg each of trypsin Toxina Cholera and protease per ml, changes in pH, and heating at 60 or CETPMAb4CETPMAb4 ~antitoxin 100°C for 15 min. Similar findings were reported previously for crude cell-free filtered broth (18). C. jejuni ETP O 100 MAb screening. Immunoperoxidase probes of ETP re- Filtrates from six additional strains 0 100 solved by SDS-PAGE and transferred to nitrocellulose were V. cholerae enterotoxin 100 0 positive for both bands when the polyclonal antibody was E. coli enterotoxin 100 0 used. However, monoclonal antibody CETPMAb4 showed a Fertile chicken eggs were inoculated via the yolk sacs at 6 days of only the 68-kilodalton band (Fig. 1, lanes D and E). When incubation. Inocula were 0.1 ml per egg, and toxin protein concentrations ETP or crude filtrates were incubated with CETPMAb4, were 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 were lethality for fertile chicken eggs was abolished (Table 1). used for each inoculum, and lethality was assayed after 24 h. These results Cell culture assays. By inverted microscopy, the toxic were generated by three separate experiments. Incubation with phosphate- effects of ETP on Int407 and PCEF cells appeared as buffered saline resulted in 100% mortality. VOL. 28, 1990 TOXIC ACTIVITY OF C. JEJUNI 1317

TABLE 2. Cytological changes in CHO cells due to the toxins of C. 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 75 C. jejuni ETP (0.01 p.g/ml) 70 2 68 Filtrates 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 15 E. coli enterotoxin (0.1 ,ug/ml) 60 68 0 E. coli enterotoxin (0.01 ,ug/ 15 10 0 mI) a Incubation of toxins and antisera was at 37°C for 30 min, and cytopathic changes 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 strain 2483 and toxic cell-free filtrates from the remaining six C. b i 2 S, . jejuni strainswere incubated with CETPMAb a ndthen eoo.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) UUntreated control showing a confluent monolayer. (b) Cells treated and CHO cell assays (Table 2). wiith 0.1 ,ug of ETP per mil for 12 h. Note cell rounding and lysis as Binding specificity of the toxin. Int407 cells avidly bound SIshown 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 ,ug of V. cholerae enterotoxin per ml for 24 h. Note cell elongation characteristic of the activity of cholera toxin. (c) Cells treated with 0.1 ,ug of 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. 1318 MAHAJAN AND RODGERS J. CLIN. MICROBIOL.

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0.0 -T- I o 5 10 15 20 25 30 35 40 % reduction FIG. 6. Competitive binding of C. jejuni ETP to Int407 cells as time/h assessed by ELISA. Percent reduction of ETP binding following coincubation of the toxin and eucaryotic cells in the presence of FIG. 4. Binding of C. jejuni ETP to Int407 cells as assessed by lectins, acetylated sugars, and GM1. Except for NurNAc, none ELISA. Maximum binding occurred after 2 h, and similar binding significantly affected binding of ETP. The results shown are aver- dynamics were observed for primary chicken embryo fibroblast ages of three separate trials. Con A, Concanavalin A; WGA, wheat cells. The results shown are averages of three separate evaluations. germ agglutinin; GaNAc, N-acetylgalactosamine; GlNAc, N-acetyl- glucosamine.

(Fig. 4). By 24 h, the cells were rounded or lysing. In contrast to V. cholerae enterotoxin, ETP and crude filtrates treatments is shown in Fig. 5. Trypsin, protease, chymo- from the six C. jejuni strains showed no binding to GM1. trypsin, pepsin, neuraminidase, or aldehyde treatment re- ETP binding to Int407 cells following enzymatic and other duced toxin binding to host cells by greater than 50%. Treatment of the cells with lipase,"-galactosidase, Nonidet P-40, or sodium metaperiodate had no significant effect on toxin binding. Of the lectins, neither wheat germ agglutinin glutaraldehyde nor concanavalin A affected ETP attachment. Furthermore, r no reduction in binding of ETP to Int407 cells in the presence sod. metaper. of GM1 was observed, suggesting that this C. jejuni toxin, unlike cholera toxin, did not bind to the GM1 receptor. nonidet P40 Although incubation of ETP in the presence of N-acetylglu- cosamine or N-acetylgalactosamine did not adversely affect neuraminidase attachment to host cells, treatment with NurNAc effected a 50% reduction in binding (Fig. 6). In addition, the remaining monosaccharides and disaccharides beta-gal (glucose, galactose, mannose, fucose, arabinose, maltose, and sucrose) all re- sulted in less than 7% reduction in binding of ETP to Int407 lipase cells.

protease DISCUSSION pepsin The pathogenesis of C. jejuni has been widely investi- gated, but the nature of the bacterial virulence factors and chymotrypsin the mechanisms by which this organism initiates enteric disease are not clear. Invasion of the bloodstream of the host trypsin has been reported as an important virulence factor in various studies (3, 10). However, production of toxin by C. jejuni, -10 0 1 0 20 30 40 50 60 70 80 90 100 first reported by Ruiz-Palacios et al. (26), has gained impor- tance, and in noninvasive strains, toxin production probably constitutes a major virulence factor (18). Enterotoxin pro- % reduction duction undoubtedly causes the mucus- and blood-free, watery diarrhea often observed in C. jejuni infections, and FIG. 5. Binding of C. jejuni ETP to Int407 cells as assessed by ELISA. Percent reduction in adherence of toxin after treatment of this would be particularly so for infections caused by strains host cells with potential eucaryotic receptor-modifying agents is lacking invasive properties. C. jejuni also produces a cyto- shown. The results shown are averages of three separate trials. Sod. toxin (12, 13, 24, 32) which may be responsible for inflam- metaper., Sodium metaperiodate; beta-gal, ,-galactosidase. matory diarrhea. It seems that C. jejuni resembles E. coli in VOL. 28, 1990 TOXIC ACTIVITY OF C. JEJUNI 1319 that some strains are enteroinvasive and some are toxic in hydrate moieties occurs. Indeed, stearic and hydrophobic nature, with virulence a multifactorial process. Belbouri and interactions between different membrane components of Megraud (2) reported that 64% of the strains tested had eucaryotic host cells may have a role in the recognition of enterotoxinlike activity, while Lindblom et al. (16) reported the specific receptors of ETP. Since the carbohydrates that only 32% of the strains isolated from humans with acute present during competitive blocking studies and the lipid- enteritis and healthy egg-laying chickens were toxigenic. It is modifying agents used to pretreat host cells did not signifi- possible that failure to identify toxin production in appar- cantly reduce binding, a glycolipidlike receptor does not ently nontoxigenic strains reflects variations in storage and seem likely. growth conditions before assay. It has been observed in It was interesting that coincubation of ETP with NurNAc, other toxin-producing organisms that after several in vitro a parent acid of a family of amino sugars, did reduce binding passages, strains revert to nontoxigenic forms, especially if to Int407 cells by 50%. Treatment ofthe cells with neuramin- the toxin production is plasmid mediated. This may be true idase, a receptor-destroying enzyme that liberates NurNAc of C. jejuni. Alternatively, as has been shown by Klipstein et residues from cell membranes, reduced binding by greater al. (14), strains of the organism may be independently than 90%. However, commercially available neuraminidase toxigenic, invasive or, for some strains, both. has residual protease activity, and this might explain the The molecular size of the toxin isolated in this study was higher reduction in binding of ETP compared with treatment lower than that of cholera toxin (84 kilodaltons) or the E. coli with protease alone. These studies indicated that the amino heat-labile enterotoxin (91.5 kilodaltons), although it approx- terminal of NurNAc acts as a receptor for C. jejuni ETP per imates the single 70-kilodalton band reported by McCardell se. Alternatively, it is possible that the toxin receptor on et al. (19) for C. jejuni. The 65-kilodalton protein band also host cells is a surface protein either linked directly to or present in ETP from the discontinuous nondenaturing gel acting in consort with a neuraminic acid moiety. McSweegan lacked toxic activity. In embryo protection studies, the toxic and Walker (20) have shown that C. jejuni lipopolysaccha- activity of ETP was neutralized by incubation with ride molecules were involved in adhesion of the organisms.to CETPMAb4, and furthermore, the degree of ETP binding to Int407 cells and that adherence was reduced by 50 to 60% by Int407 cells as measured by ELISA was the same whether fucose or mannose. These two monosaccharides had no CETPMAb4 or the polyclonal antibody was used. The tox- effect on the binding of C. jejuni ETP to Int 407 cells. It does icity of ETP resided solely in the 68-kilodalton band which not seem unreasonable that the organism and its toxin have did not further cleave in the presence of P-mercaptoethanol; developed different receptors and thereby avoid competition therefore, this C. jejuni toxin, unlike those of V. cholerae for the same host cell-binding sites. and E. coli, did not possess subunits. In addition, it has been That C. jejuni virulence is multifactorial, with toxin pro- reported that low concentrations of GM1 inhibited the toxic duction as one facet, is established. Indeed, mechanisms for activity of the V. cholerae and E. coli toxins, and this toxin-induced disease have been proposed (13, 18). How- binding was known to be a function of the B subunits. In ever, the role of toxin binding to host cells in the pathogen- contrast to V. cholerae enterotoxin, ETP did not bind to esis of disease is less certain. This study indicates that GM1 and induced rounding and lysis of CHO cells. In these production of a cytoxin which binds to proteinlike receptors studies, it appeared that the ETP of C. jejuni differed on host cell membranes is potential virulence trait that may markedly from V. cholerae enterotoxin. Furthermore, their be involved in the inflammatory diarrhea caused by many C. lack of immunological relatedness was confirmed by the jejuni isolates. Elucidation of the mechanisms involved in absence of cross-neutralizing reactivity between ETP and V. the binding of this bacterial cytotoxin to cell membranes and cholerae toxin and their respective antisera. Indeed, the the role of host cell receptors in initiating disease will better characteristics of this toxin lead us to believe that it is a define the infectious process at the cellular and molecular potent cytotoxin rather than an enterotoxin. levels. Similarities between the cytotoxin described in this report and the crude cytotoxic C. jejuni filtrate reported by Guer- ACKNOWLEDGMENTS rant et al. (12) included molecular weight, heat and trypsin sensitivity, and CHO cell rounding. However, the cytotox- This work was supported in part by Biomedical Research Support icity of our toxin was lost on freeze-thawing but it retained grant 2-S07-RR07-108-14 from the National Institutes of Health and activity for up to 4 weeks at 4°C and was unaffected by by the University of New Hampshire Research Office. treatment with polymyxin B. Most toxins are known to interact with specific receptors LITERATURE CITED on susceptible cell membranes as a prelude to cytoplasmic 1. Back, E., A. Svennerholm, J. Holmgren, and R. Moilby. 1979. or initiation of their toxic Others Evaluation of a ganglioside immunosorbent assay for detection uptake responses. actually of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. damage cell membranes per se. Both Int407 and PCEF cells 10:791-795. carry receptors on their membranes specific for ETP, and 2. Belbouri, A., and F. Megraud. 1988. 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