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Cytotoxin from Campylobacterjejuni

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Cytotoxin from Campylobacterjejuni 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).
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