On leave of absence from Shanghai Hygiene and Anti-. Epidemic Center, Shanghai, the People's Republic of China. acryl S-200 (Pharmacia FineChemicals, ...
INFECTION AND IMMUNITY, July 1983, p. 50-53
Vol. 41, No. 1
0019-9567/83/070050-04$02.00/0 Copyright C 1983, American Society for Microbiology
Analysis of Antigenic Determinants in Cholera Enterotoxin and Heat-Labile Enterotoxins from Human and Porcine Enterotoxigenic Escherichia coli YOSHIFUMI TAKEDA,* TAKESHI HONDA, HUILAN SIMA,t TAKAO TSUJI, AND TOSHIO MIWATANI Department of Bacteriology and Serology, Research Institute for Microbial Diseases, Osaka University, Yamada-oka, Suita, Osaka 565, Japan Received 17 November 1982/Accepted 5 April 1983
Antigenic determinants of cholera enterotoxin (CT) and heat-labile enterotoxin from a human strain (LTh) and a porcine strain (LTp) were analyzed by Ouchterlony double-gel diffusion test against anti-CT, anti-LTh, and anti-LTp, which were treated by immunoaffinity column chromatography. The results showed the existence of the following antigenic determinants: (i) antigenic determinants unique to CT, LTh, and LTp, respectively; (ii) an antigenic determinant common to CT, LTh, and LTp; (iii) an antigenic determinant common to CT and LTh, but not LTp; and (iv) an antigenic determinant common to LTh and LTp, but not CT. On the basis of these results, an antigenic scheme for CT, LTh, and LTp is proposed.
The immunological relatedness of cholera enterotoxin (CT) and heat-labile enterotoxin produced by porcine strains of enterotoxigenic Escherichia coli (LTp) has been well established (1, 5, 8, 9, 15). Recently, we reported the immunological nonidentity of heat-labile enterotoxi'n from human strains of enterotoxigenic E. coli (LTh) and LTp (12). The molecular heterogeneity of CT, LTh, and LTp was also demonstrated (17). These findings were independently confirmed by others (7; R. K. Holmes, E. M. Twiddy, and M. G. Bramucci, 17th Joint Conference of the U.S.-Japan Cooperative Medical Science Program, Cholera Panel, Baltimore, 1981; J. D. Clements and D. C. Flint, Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, B63, p. 28). In this paper we report studies on the antigenic relatedness of CT, LTh, and LTp and propose an antigenic scheme for these three enterotoxins.
acryl S-200 (Pharmacia Fine Chemicals, Uppsala, Sweden) were described previously (16). Preparation of antisera against purified CT and purified LTs. Antisera against purified CT and LT were prepared essentially as described previously (10). Samples (25 F.g) of purified CT or purified LTs in 1 ml of phosphate-buffered saline (pH 7.0) were emulsified with an equal volume of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.), and the emulsion was inoculated intramuscularly into young rabbits weighing about 2 kg. Two booster injections of 25 ,ug of the toxin emulsified with Freund complete and incomplete adjuvants (Difco) were given on days 25 and 50, respectively. In some cases, additional booster injections of 25 ,ug of the toxin emulsified with Freund incomplete adjuvant were given on days 75 and 100. Antisera were obtained 10 days after the last booster
injection. Immunoaffinity column chromatography. Immunoaffinity column chromatography was carried out as described previously (4, 10). Samples of 5 to 7 mg of either purified CT, purified LTh, or purified LTp were coupled with 1 g of cyanogen bromide-activated Sepharose 4B (Pharmacia). The preparations were placed in columns, and the anti-CT, anti-LTh, or antiLTp antiserum was applied to the columns. Each column was washed with phosphate-buffered saline (pH 7.0) until the optical density of the eluate at 280 nm reached a basal level. Then the immunoglobulin bound to the column was eluted with 0.2 M glycinehydrochloride buffer (pH 2.7) containing 0.5 M NaCl. Double-gel diffusion test. The double-gel diffusion test was carried out as described by Ouchterlony (14) with 1.2% Noble agar (Difco) in TEAN buffer (6), which consists of 0.05 M Tris, 1 mM EDTA (disodium salt), 3 mM NaN3, and 0.2 M NaCl (pH 7.5). After the samples had been applied, the plates were placed in a humidified incubator at room temperature for about 16
MATERIALS AND METHODS Purified CT. CT purified from the culture filtrate of
Vibrio cholerae 569B by the method of Ohtomo et al. (13) was purchased from Sanko Junyaku Co., Tokyo. Purified LTh and LTp. LTh and LTp were isolated and purified from E. coli 240-3 and E. coli 0-149-26, respectively, as described by Clements and Finkelstein (3). Details of the procedures for culture of the cells, isolation of crude LT, and purification of LT by successive column chromatographies on Bio-Gel A5m (Bio-Rad Laboratories, Richmond, Calif.) and Seph-
On leave of absence from Shanghai Hygiene and AntiEpidemic Center, Shanghai, the People's Republic of China. '
50
ANTIGENIC DETERMINANTS OF CT, LTh, AND LTp
VOL. 41, 1983
B
A
p h
c
aC
p h
h
c
4th)P| l-'
FIG. 1. Ouchterlony double-gel diffusion test of CT, LTh, and LTp against anti-LTh from an immunoaffinity column. Anti-LTh was applied to either a CTcoupled or an LTp-coupled immunoaffinity column, and antiserum not retained by the affinity column was prepared as described in the text. A 20-,ul sample of either anti-LTh from a CT-coupled column (ahC in A) or anti-LTh from a LTp-coupled column (ahP in B) was used after appropriate concentration to give a good precipitin line with 2 to 4 ,ug of CT (c), LTh (h), and LTp (p). The Ouchterlony double-gel diffusion test was carried out as described in the text. to 24 h. Then the plates were washed extensively with a solution of 0.4% NaCl and 0.4% Na2B407. They were then dried, stained with 0.5% Coomassie brilliant blue in 50% methanol containing 10%o acetic acid, and destained with 50% methanol containing 10%o acetic acid.
RESULTS
Antigenic determinants of CT, LTh, and LTp were analyzed by the Ouchterlony double-gel diffusion test with anti-CT, anti-LTh, and antiLTp, which were treated by immunoaffinity column. The existence of the following antigenic determinants was demonstrated: (i) an antigenic determinant unique to CT; (ii) an antigenic determinant unique to LTh; (iii) an antigenic determinant unique to LTp; (iv) an antigenic determinant common to CT, LTh, and LTp; (v) an antigenic determinant common to CT and LTh, but not LTp; and (vi) an antigenic determinant common to LTh and LTp, but not CT. The experimental data from which these conclusions were drawn are presented below. Anti-LTh was applied to a CT-coupled immunoaffinity column, and antiserum not retained by the affinity column was examined by the Ouchterlony double-gel diffusion test. As shown in Fig. 1A, the serum gave precipitin lines against LTh and LTp, but not against CT. It was shown that the precipitin line between LTh and anti-LTh (LTh-aLTh) and that between LTp and anti-LTh (LTP-aLTh) formed a spur. (Hereafter the precipitin line between antigen X and anti- Y antiserum is referred to as X-a Y.) This indicates the existence of a common antigenic determinant between LTh and LTp that is not found in CT, as well as an antigenic determinant unique to LTh. When anti-LTh was applied to an LTpcoupled immunoaffinity column and antiserum not retained by the affinity column was exam-
51
ined by the Ouchterlony double-gel diffusion test, the serum gave precipitin lines against LTh and CT, but not against LTp (Fig. 1B). Spur formation between CT-otLTh and LTh-aLTh (Fig. 1B) indicates the existence of a common antigenic determinant between CT and LTh that is not found in LTp. The spur confirmed the existence of an antigenic determinant unique to LTh. Anti-LTp was applied to a CT-coupled immunoaffinity column, and antiserum not retained by the affinity column was examined by the Ouchterlony double-gel diffusion test. As shown in Fig. 2A, the serum gave precipitin lines against LTh and LTp, but not against CT. Spur formation between LTh-aLTP and LTp-aLTp indicated the existence of a common antigenic determinant between LTh and LTp that is not found in CT, as well as an antigenic determinant unique to LTp. When anti-LTp was applied to an LTh-coupled immunoaffinity column and antiserum not retained by the affinity column was examined by the Ouchterlony double-gel diffusion test (Fig. 2B), the serum did not give any precipitin line against either LTh or CT. This result confirmed the existence of an antigenic determinant unique to LTp that is not found in either CT or LTh and also suggested the absence of a common antigenic determinant between CT and LTp that is not found in LTh. The absence of a common antigenic determinant between CT and LTp that is not found in LTh was also suggested by the experimental data shown in Fig. 3A. In this experiment, antiCT was applied to an LTh-coupled immunoaffinity column, and antiserum not retained by the affinity column was examined by the Ouchterlony double-gel diffusion test. The serum did not give any precipitin line against both LTh and LTp. This result also indicates the existence of an antigenic determinant unique to CT that is not found in either LTh or LTp. When anti-CT was applied to an LTp-coupled immunoaffinity colB
A h
pr~ac
h
C
p
p
c
t p9 P
FIG. 2. Ouchterlony double-gel diffusion test of CT, LTh, and LTp against anti-LTp from an immunoaffinity column. Anti-LTp was applied to either a CTcoupled or an LTh-coupled immunoaffinity column, and antiserum not retained by the column (apC in A and apH in B, respectively) was prepared and used. Other experimental conditions and abbreviations are as described in the legend to Fig. 1.
52
TAKEDA ET AL.
A
INFECT. IMMUN.
reported. These studies, however, were done before the demonstration of the immunological hh p . nonidentity of LTh and LTp, which was reported (2, 12). The present study demonstrated :cP s l-recently c( c c C £ acP the antigenic relatedness of the three enterotoxins, CT, LTh, and LTp. Ouchterlony double-gel diffusion tests with column-treated anti-CT, antiimmunoaffinity FIG. 3. Ouchterlony doublle-gel diffusion test of CT, LTh, and LTp against anti -CT from an immunoaf- LTh, and anti-LTp demonstrated the existence finity column. Anti-CT was ap)plied to either an LTh- of the following antigenic determinants: (i) an coupled or an LTp-coupled ihmmunoaffinity column, antigenic determinant unique to CT ([C1]) (Fig. and antiserum not retained by the column (acH in A 3A and B); (ii) an antigenic determinant unique and acP in B, respectively) w{as prepared and used. to LTh ([H1]) (Fig. 1A and B); (iii) an antigenic Other experimental conditionss and abbreviations are determinant unique to LTp ([P1]) (Fig. 2A and as described in the legend to FFig. 1. B); (iv) an antigenic determinant common to CT, LTh, and LTp ([C2-H2-P2) (Fig. 4A through D); (v) an antigenic determinant common to CT and umn and antiserum not retrained by the affinity LTh, but not LTp ([C3-H31) (Fig. 1B, 3B, and column was examined by t]he Ouchterlony dou- 4D); and (vi) an antigenic determinant common ble-gel diffusion test (Fig. 3B), spur formation to LTh and LTp, but not CT ([H4-P4]) (Fig. lA, was observed between CT-(xLTp and LTh-aLTp, 2A, and 4C). From these results, the following indicating the existence of a common antigenic antigenic scheme for CT, LTh, and LTp is prodeterminant between CT and LTh that is not posed: CT = [C1] [C2-H2-P2] [C3-H3], LTh = found in LTp and an anitigenic determinant [H1] [C2-H2-P2] [C3-H3] [H4-P4], and LTp = [P1] unique to CT that is not found in either LTh or [C2-H2-P2] [H4-P4]. LTp. No antigenic determinant common to CT and The existence of an arntigenic determinant LTp, but not LTh ([C-P]), was demonstrated in common to the three enteroitoxins, CT, LTh, and this study. Several different batches of anti-CT LTp, is shown in Fig. 4. Forr Fig. 4A anti-CT was and anti-LTh were prepared, and several atapplied to an LTp-coupled immunoaffinity col- tempts were made to demonstrate [C-P], but umn, and the serum retaine d by the column was without success. It is possible that [C-P], if it eluted and examined by the Ouchterlony double- exists, is only weakly antigenic and thus cannot diffusion test."If The se rum gave a line of be demonstrated. Geary et al. (7) reported that gel IT, * 1 _ w-w and LIilp. Simllarly, identnty agalnsts 'Cl, L'Ih, when anti-LTp was applied to a CT-coupled immunoaffinity column and serum retained by A B h C the column was examined, the serum gave a line of identity against all three enterotoxins (Fig. 4B). For Fig. 4C, anti-LTh was applied to an P .apCI p C C LTp-coupled column, and serum retained by the P h column was examined. Spur formations were observed between LTp-aLTh and CT-aLTh and between LTh-aLTh and CT-aLTh. These results D indicate that CT, LTh, and LTp share a common P C antigen and that in addition there is an antigenic P c determinant in LTh that is not found in CT. h ahC h h ahP hSimilarly, when anti-LTh was applied to a CTcoupled column and serum retained by the column was examined, results showed the exisFIG. 4. Ouchterlony double-gel diffusion test of tence of an antigen common to all three CT, LTh, and LTp against antisera from an immunoafenterotoxins and an antigenic determinant in finity column. Anti-CT was applied to an LTp-coupled LTh that is not found in LTp (Fig. 4D). column, antiserum not retained by the column was washed out, and then the immunoglobulin bound to DISCUSSION the column was eluted as described in the text. This serum, ac'P, was used in A after appropriate concenIt has been well established by Ouchterlony tration. Anti-LTp from a CT-coupled column (ap'C in double-gel diffusion tests that CT and LT of B), anti-LTh column (ah'P in C), from an enterotoxigenic E. coli share a common antigen- and anti-LTh from a LTp-coupled CT-coupled column (ah'C in D) ic determinant (1, 8). The existence of unique were prepared similarly. Other experimental condiand shared antigenic determinants in CT and tions and abbreviations are as described in the legend LTp (2) and in CT and LTh (11) have been to Fig. 1. B
p
h
-
T
(ac'P')
VOL. 41, 1983
ANTIGENIC DETERMINANTS OF CT, LTh, AND LTp
LTh seem to be more closely related than LTp to CT. The antigenic scheme described above supports this idea. In the present study, holotoxins of CT, LTh, and LTp were used for immunization. Thus it is not clear whether the immunological relatedness reported is of B subunits, of A subunits, or of both. However, since the preparations of antiCT, anti-LTh, and anti-LTp did not give any precipitin line against homologous A subunits, the results are probably due to the B subunits of CT, LTh, and LTp. This problem can be settled by preparing anti-B subunits and anti-A subunits of each enterotoxin. ACKNOWLEDGMENTS We thank S. Taga for skilful technical assistance. This work was supported by a Grant-in-Aid for scientific Research from the Ministry of Education, Science and Culture of Japan and a World Health Organization Research Training Grant to H.S. LITERATURE CITED 1. Clements, J. D., and R. A. Finkelstein. 1978. Immunological cross-reactivity between a heat-labile enterotoxin(s) of Escherichia coli and subunits of Vibrio cholerae enterotoxin. Infect. Immun. 21:1036-1039. 2. Clements, J. D., and R. A. Finkelstein. 1978. Demonstration of shared and unique immunological determinants in enterotoxins from Vibrio cholerae and Escherichia coli. Infect. Immun. 22:709-713. 3. Clements, J. D., and R. A. Flnkelstein. 1979. Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli culture. Infect. Immun. 24:760-769. 4. Cuatrecasas, P., and C. B. Anfinsen. 1971. Affinity chromatography. Methods Enzymol. 22:345-378. 5. Evans, D. G., D. F. Evans, Jr., and S. L. Gorbach. 1973. Identification of enterotoxigenic Escherichia coli and serum antitoxin activity by the vascular permeability factor
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assay. Infect. Immun. 8:731-735. 6. Finkelstein, R. A., and J. J. LoSpalluto. 1969. Pathogenesis of experimental cholera. Preparation and isolation of choleragen and choleragenoid. J. Exp. Med. 130:185-202. 7. Geary, S. J., B. A. Marchlewicz, and R. A. Finkelstein. 1982. Comparison of heat-labile enterotoxins from porcine and human strains of Escherichia coli. Infect. Immun. 36:215-220. 8. Gyles, C. L. 1974. Immunological study of the heat-labile enterotoxins of Escherichia coli and Vibrio cholerae. Infect. Immun. 9:564-570. 9. Gyles, C. L., and D. A. Barnum. 1969. A heat-labile enterotoxin from strains of Escherichia coli enteropathogenic for pigs. J. Infect. Dis. 120:419-426. 10. Honda, T., S. Taga, Y. Takeda, and T. Miwatani. 1981. A modified Elek test for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 13:1-5. 11. Honda, T., Y. Takeda, and T. Mlwatani. 1981. Isolation of special antibodies which react only with homologous enterotoxins from Vibrio cholerae and enterotoxigenic Escherichia coli. Infect. Immun. 34:333-336. 12. Honda, T., T. Tsuji, Y. Takeda, and T. Miwatani. 1981. Immunological nonidentity of heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli. Infect. Immun. 34:337-340. 13. Ohtomo, N., T. Muraoka, H. Inoue, H. Sasaoka, and H. Takahashi. 1974. Preparation of cholera toxin and immunization studies with cholera toxoid, p. 132-142. In Proceedings of the 9th Joint Conference of U.S.-Japan Cooperative Medical Science Program, Cholera Panel. National Institutes of Health, Bethesda, Md. 14. Ouchterlony, 0. 1949. Antigen-antibody reactions in gels. Acta Pathol. Microbiol. Scand. 26:507-515. 15. Smith, N. W., and R. B. Sack. 1973. Immunologic crossreactions of enterotoxins from Escherichia coli and Vibrio cholerae. J. Infect. Dis. 127:164-170. 16. Takeda, Y., T. Honda, S. Taga, and T. Miwatani. 1981. In vitro formation of hybrid toxins between subunits of Escherichia coli heat-labile enterotoxin and those of cholera enterotoxin. Infect. Immun. 34:341-346. 17. Tsuji, T., S. Taga, T. Honda, Y. Takeda, and T. Miwatani. 1982. Molecular heterogeneity of heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli. Infect. Immun. 38:444 448.
