INFECTION AND IMMUNITY, Oct. 1979, p. 173-177 0019-9567/79/10-0173/05$02.00/0

Vol. 26, No. 1

Isolation and Partial Characterization of Two Different HeatStable Enterotoxins Produced by Bovine and Porcine Strains of Enterotoxigenic Escherichia coli R. A. KAPITANY,'* G. W. FORSYTH,2 A. SCOOT,2 S. F. McKENZIE,3 AND R. W. WORTHINGTON4 Veterinary Infectious Disease Organization,' Department of Physiological Sciences, Western College of Veterinary Medicine, University of Saskatchewan,2 and National Research Council,3 Saskatoon, Saskatchewan S7N OWO; and Department of Physiology, Pharmacology and Toxicology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, 0110, Pretoria, Republic of South Africa4

Received for publication 10 July 1979

Heat-stable enterotoxins (ST-124 and ST-1261) have been isolated from two different enterotoxigenic Escherichia coli of bovine (124) and porcine (1261) origin. The enterotoxin preparations were isolated by ultrafiltration and ionexchange chromatography and were both active in the suckling mouse test and pig ligated loop test in the nanogram range. The bovine (ST-124) enterotoxin was not stable to heating in its isolated form, and significant differences in amino acid composition were observed between the two enterotoxins. Although both toxins were active at similar levels in the suckling mouse and pig ligated loop tests, ST124 lacked the ability to cause the profound secretary responses seen with ST1261 in the weanling pig ligated loop. The role of the enterotoxigenic Escherichia more direct evidence based on differences in coli (EEC) in the etiology of diarrhea of the methanol solubility for different types of heat newborns of humans and domestic food-produc- stable enterotoxins. The present paper provides ing animals is well established. The EEC appear evidence for the isolation of two different lowto possess at least two known virulence factors molecular-weight STs from the broth filtrates of mediating attachment and colonization of the two EEC strains of bovine and porcine origin intestine and toxin synthesis and release. These that were active in both the suckling mouse test virulence factors have been shown to be con- and the weanling pig ligated loop test. trolled by a heterogenous population of transMATERLALS AND METHODS missible plasmids (26). Shortly after the initial demonstration that Culture conditions. EEC-124 (O101:K-:K99:H-) the EEC would dilate ligated intestinal segments is a K99' ST'-only EEC isolated from a natural out(28), two substances (11, 27, 29, 31) a heat-labile break of diarrhea in beef cattle and was received from toxin (LT) and a heat-stable toxin (ST) were S.D. Acres (Veterinary Infectious Disease Organizashown to be present in the cell-free supernatants tion, University of Saskatchewan). EEC-1261 (0138: of EEC. EEC have been shown to produce either H-:K81) is a porcine enteropathogen and was kindly supplied by H.W. Moon (National Animal Disease LT or ST or both (10). Ames, Iowa). The structure and mode of action of LT have Center, A single colony of EEC was taken from a fresh been extensively investigated (5-7, 16); however, blood agar plate and inoculated into 300 ml of syncase the nature of ST and its mechanisms have medium (8) in a 1-liter Erlenmeyer flask and incubated proved quite elusive. Early attempts at the pu- overnight in a shaking water bath at 370C. This was rification of ST produced fractions with molec- used to inoculate either 10 liters of the defined media ular weights ranging from 103 to 104 (3, 12, 17). of Alderete and Robertson (2) supplemented with 1% This past year, Alderete and Robertson (1) pub- sodium lactate to stimulate toxin production (2) or 10 The lished a paper on the purification of ST from an liters of syncase medium in a 12-liter fermentor. with 370C. 20 at to was broth h) (18 grown overnight a molecular that had EEC of porcine origin aeration (4 liters per min) and mixing (300 weight of 4,400 and was active in the suckling vigorous rpm). Cells were removed in a continuous centrifuge mouse test at a few nanograms of protein. (Cepa model LE, New Brunswick Scientific), the culThe question of the heterogeneity of ST has ture supernatant was sterilized through an 0.45-Itm been raised in the past, based on the difference filter, and sodium azide (0.02%) was added to retard in the sensitivity of the various test systems. bacterial growth. Ultrafiltration. The culture supernatant was fracMore recently, Mullan et al. (21) have provided 173

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tionated over a PM-10 Diaflo membrane (10,000-molecular-weight exclusion limit, Amicon Corp.) in an Amicon TC5E thin-channel system. The PM-10 filtrate was then repartitioned over UM-2 Diaflo membranes (1,000-molecular-weight exclusion limit, Amicon Corp.) as before, and the UM-2 concentrate was collected. The concentrates were washed with several volumes of distilled water and lyophilized. Chromatography. Diethylaminoethyl-Bio-Gel A (2.5 and 5.0 by 75 cm) and O-(carboxymethyl)-BioGel (1.5 x 30 cm) (BioRad Laboratories) columns of various sizes were prepared and used as follows. Columns were packed, equilibrated, and eluted with 2.5 mM HCl (pH 2.8) at a constant flow of 60 ml/h for 5by 75-cm columns, 30 ml/h for 2.5- by 75-cm columns, and 5 ml/h for 1.5- by 30-cm columns. Fractions (10 ml) were collected for the 2.5-cm and 5.0-cm columns, and 2-ml fractions were collected for the 1.5-cm-diameter columns. Samples were lyophilized between steps and applied to the columns suspended in 2.5 mM HCl. Protein determination. Elution of the protein from the columns was monitored at 230 and 260 nm, and protein was estimated by the method of Kalb and Bernlohr (15) based on a calculation with optical densities at 230 and 260 nm. In preliminary studies, the estimates for protein determined on .toxin samples by the method of Kalb and Bernlohr agreed more closely to those estimates based on total amino acids than did estimates based on the Lowry method (18) with bovine serum albumin as the standard. Assay for ST. ST activity was determined in the suckling mouse test (4). A drop of 1% Evans blue dye was added to 0.5 ml of the sample to be tested, and a 0.1-ml portion was placed directly into the stomachs of 2-day-old baby mice. The mice were kept for 3 h at room temperature, weighed, and decapitated, and the entire intestines were removed and immediately weighed. A ratio of gut weight to whole live body weight greater than 0.08 was considered to be a positive response. The titer of the toxin was established as the last of a serial twofold dilution of the sample to give a positive response, and an effective dose (ED) was defined as the minimum amount of protein required to give a positive response. Three mice were used for each determination. ST was also assayed in the porcine ligated loop assay. A dose-related response to purified ST was established with cross-bred weanling pigs of 5 to 8 weeks of age weighing between 5 and 7 kg by methods described by Forsyth et al. (9). Media and other chemical reagents employed throughout were purchased from Sigma Chemical Co. or Fisher Scientific Co. Amino acid analysis. Amino acids were determined after hydrolysis in 6 M HCl at 1000C for 22 h by gas-liquid chromatography (19, 20). Enzyme analysis. Crude (UM-2 concentrate) and isolated ST preparations in 50 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer were incubated with several hydrolytic enzymes (trypsin, elastase, collagenase, and pronase) for 60 min at 371C. Enzyme activity was verified under the same conditions with standard assay techniques (23-25, 30), and appropriate controls were run in the suckling mouse

test. Enzymes were purchased from the Sigma Chemical Co. Samples for heat treatment were suspended in 0.01 M phosphate buffer (pH 7.2) with 0.85% sodium chloride and heated to 60, 80, and 100°C for 10 and 30 min, respectively.

RESULTS ST isolation. The ST activity in culture supernatants from both EEC strains passed through the PM10 filters, but was retained by UM2 filters. The material concentrated on the UM2 filters was purified further by ion-exchange chromatography on diethylaminoethyl-Bio-Gel. The elution profiles from this column were identical for both strains. ST activity was present in three of the six peaks (Fig. 1). Seventy to eighty percent of the ST activity eluted from the column was present in peak BII, at an average specific activity of 20 ng/ED, representing a 20fold purification by this procedure. The amount of ST in peak A varied with the degree of desalting of the UM2 concentrate and could reach as high as 30%, but generally was less than 10% of the total eluted activity. Peak BIII was present in the diethylaminoethyl-BioGel eluates from both EEC strains, and contained 20 to 30% of the ST activity applied to the column. Presumably, the toxic material in the BII and BIII peaks was identical, and the differences in peak positions reflect complexing with different material. The protein elution profile from the diethylaminoethyl-Bio-Gel column was not altered quantitatively by growth of EEC strains on syncase medium, with the same six peaks appearing on fractionation, but most of the ST activity was found in peak BI, with less than 30% in BII and only traces in BIII. Rechromatographing the BII peak on diethylaminoethyl-Bio-Gel resulted in a further twofold purification (Fig. 2). However, the toxin D

FRACTION NUMBER

FIG. 1. Diethylaminoethyl-Bio-Gel chromatography elution profile of UM-2 concentrate of culture broth of EEC grown on defined medium.

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(peak II, Fig. 2) eluted in a smaller volume of acid from the column. Further purification of the ST activity was achieved by ion-exchange chromatography on O-(carboxymethyl)-Bio-Gel (Fig. 3 and 4). Although the protein elution profile had two major peaks for both EEC strains, the elution position for ST activity from the O-carboxymethyl columns was strain dependent. ST-1261 was slightly retarded in the column, (peak 3, Fig. 4), whereas ST-124 was eluted with the first protein peak (peak 1, Fig. 3). Overall purifications in the order of 90-fold have been achieved routinely by this procedure, routinely giving specific activities of less than 5 ng of protein per ED. ST properties. ST activity in crude culture supernatants and UM2 concentrates of both EEC strains was stable to heating at 1000C for up to 30 min. ST-1261 retained its heat stability in the isolated state. ST-124, however, when isolated, was largely inactivated by heating at 60'C for 10 min (30% of the activity remaining), and only traces of activity remained after exposure to 1000C for 30 min (6% of the activity remaining). Both ST-1261 and ST-124 were stable to incubation with trypsin, pronase, collagenase, and elastase.

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Equivalent titers of ST-1261 and ST-124 in the suckling mouse test system had relatively similar potencies in the pig ligated jejunal loop assay (10 to 50 mouse ED). Amino acid analysis. Amino acid analyses of ST-1261 and ST-124 are presented in Table 1. ST-1261 lacks methionine and histidine, but otherwise has distinct similarities to the ST-431 data of Alderete and Robertson (1). The relatively high cysteine content of these two STs is most likely involved in stabilizing protein structure. ST-124 differs markedly from ST-1261 in the content of several amino acids. The very low cysteine content and the high amount of glutamic acid distinguish ST-124 from ST-1261 and ST-431.

cE

O 0

z°< 20

40

FRACTION

A~~~~~~~~~~~~~~n 60

80

100

12D

140

160

FRACTION

FIG. 2. Diethylaminoethyl-Bio-Gel rechromatog-

raphy elution profile of peak BII toxic material. E

02o

z

20

33

TABLE 1. Amino acid composition ST-124 ST-1261 16.45 Alanine 3.5a 4.2 10.22 Glycine 2.1 Valine 8.3 Threonine 6.0 3.8 Serine 3.5 2.5 7.1 6.57 Leucine 6.8 1.82 Isoleucine 4.2 7.72 Proline 0.4 11.78 Cysteine (half) Methionine

0\

I0

Aspartic acid Phenylalanine Glutamic acid Lysine Tyrosine 40

50

NUMBER

FIG. 4. O-(carboxymethyl)-Bio-Gel Chromatography elution profile of toxic peak II prepared from EEC-1261 broth ultrafiltration concentrate.

60.

FRACTION NUMBER

FIG. 3. O-(carboxymethyl)-Bio-Gel chromatography elution profile of toxic peak II prepared from EEC-124 broth ultrafiltration concentrate.

Arginine Histidine Tryptophan

0.7 16.3 2.6 28.6 2.6 3.3 1.8 1.2

NDb a Expressed as moles percent. b ND, Not determined.

0

11.68 4.38 13.28 0.78 5.69 1.18 0 ND

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Biological potency. The relationship be- 1261 showed a linear increase in secretion over a 5-log range of toxin dose, which showed no loops for both ST-124 and ST-1261 is presented tendency to plateau within the dose range used, in Fig. 5. The pig jejunum was quite sensitive to whereas ST-124 showed a definite tendency to ST-1261 and generally demonstrated a linear reach a maximal secretary response at a relaincrease in secretion rate relative to the log of tively lower toxin dose (Fig. 5). Both toxins were the ST dose. There was no tendency for secre- equally stable to incubation with the hydrolytic tion rates to plateau even with the very high enzymes used. ST-1261 showed distinct similarities to the dose of 50,000 mouse ED per jejunal loop. However, the fluid secretion patterns were different toxin (ST-431) isolated from a porcine EEC by with ST-124 and showed a marked tendency to Alderete and Robertson (1). Although the speplateau at relatively low toxin levels, and little cific activities reported by these workers were further increase in secretion was observed over slightly higher (1 ng/ED versus 3 ng/ED), a two- to fourfold variation inherent in the sucka further 2-log increase in dose. ling mouse assay and methods for the estimation DISCUSSION of protein makes exact comparisons difficult. The finding of two different types of ST based The virtual absence of methionine and histidine in ST-1261 would suggest that it was actually on amino acid composition, heat stability propmore purified than ST-431 (1). slightly erties, and biological activity is a major point of The secretary response versus ST-1261 dose interest. The isolation by very similar procedures implies that the two STs do, however, in the pig jejunum appeared to deviate from have some similar physical and chemical prop- direct linear relationship. This deviation may erties in common. As well, both toxins were relate to different secretary mechanisms operaactive in the suckling mouse test (1 to 5 ng of tive in the pig jejunum. Hamilton et al. (12, 13) protein per ED) and the weanling pig test (10 to found that the components giving rise to net 50 ng of protein per ED) at the same level of secretion of fluid and sodium in the proximal jejunum of the pig differed with exposure time biological potency. ST-124 lacked the six half-cysteine residues to ST. After 20 min the effect was due to dethat appear to confer the heat stability on ST- creased absorption; after 40 min, both decreased 1261 and ST-431 (1) and showed significantly absorption and increased secretion were observed; and a 20-min measurement after a 60more glutamate and aspartate. That these amino acids possibly exist in the acid form is evidenced min incubation period detected only increased by ST-124 being excluded from the O-(carboxy- secretion. The level of crude toxin used in their methyl)-Bio-Gel column (Fig. 3), whereas ST- study elicited fluid responses that fell in the 1261 was slightly retarded (Fig. 4). ST-124 was exponential stage of the first phase of the dosealmost completely heat labile in the isolated response curve for purified toxin. It is possible form and was observed to lack the potency to that an effect similar to that which was seen with time may be seen with dose. cause the profound secretary responses seen in The exact relationship of ST-124 and ST-1261 the pig ligated loops with ST-1261 (Fig. 5). STdescribed here and ST-431 (1) reported earlier to the STa and STb reported by Mullan et al. ST-1261 (21) is unclear, and exact comparisons are diffio cult. ST-124 and ST-1261 (unpublished data) and ST-431 (1) are acetone soluble and may be ii similar to STa (21). All four of these toxins (ST124, ST-1261, ST-431, and STa) were also active in suckling mouse tests, in contrast to STb, 12/0 ST -124 which was not (21). In addition, STa was also partially heat labile (21). Moon et al. (22) have classified the EEC into L two categories. The class I enteropathogens secrete a toxin that is active in the weanling pig and suckling mouse tests. Therefore, it appears that ST-124, ST-1261, ST-431, and STa all fall ODD 1IP00 10OD 5 into this class I category. However, on the basis MOUSE E.D!s FIG. 5. Fluid fluxes produced by ST-124 and ST- of the data presented here, these class I entero1261 in porcine jejunal ligated loops. Values shown toxins appear to exist in two forms, a heat-stable are fluxes above control and are the mean + standard form such as ST-431 and ST-1261, and a form of deviation of three loops in three different animals. ST that is heat labile when purified, such as STtween ST dose and fluid secretion in pig jejunal

.

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124 and STa. It is tempting to speculate that the heat-labile forms are unique to the bovine EEC and the heat-stable forms are unique to the porcine EEC; however, the ST purified from EEC-B44 (09: K30:K99), a bovine EEC, was heat stable in the isolated form (submitted for publication). It therefore seems unlikely that such a simple solution exists. On the other hand, the isolation of two different STs secreted by different class I enteropathogens may suggest that these toxins mediate different diarrheal syndromes, and this may account for some of the variability in mortality seen clinically. The isolation scheme reported here has replaced the technically complex procedures of solvent fractionation and preparative gel electrophoresis (1) by ion-exchange chromatography with negligible effects on the yield and purity of the isolated ST. It provides a more rapid and direct method of isolating milligram quantities of ST and is suitable for the preparative separation of STs of different composition. ACKNOWLEDGMENTS We acknowledge the financial support of the Veterinary Infectious Disease Organization, Saskatoon, Saskatchewan. We are also gratified for the excellent technical assistance of S. Feschuk and M. Kumph and to P. Zoerb and P. Platel for their help in preparing the manuscript.

LITERATURE CITED 1. Alderete, J. F., and D. C. Robertson. 1978. Purification

2.

3.

4.

5.

6.

7.

8.

9.

and chemical characterization of the heat-stable enterotozin produced by porcine strains of enterotozigenic Escherichia coli. Infect. Immun. 19:1021-1030. Alderete, J. F., and D. C. Robertson. 1977. Nutrition and enterotoxin synthesis by enterotozigenic strains of Escherichia coli: defined medium for production of heat-stable enterotozin. Infect. Immun. 15:781-788. Bywater, R. J. 1971. Dialysis and ultrafiltration of a heat-stable enterotoxin from Escherichia coli. J. Med. Microbiol. 5:337-343. Dean, A. G., Y. C. Ching, R. G. Williams, and L. B. Harder. 1972. Test for Escherichia coli enterotozin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407411. Dorner, F., H. Jaksehe, and W. Stockl. 1976. Escherichia coli enterotoxin: purification, partial characterization and immunological observations. J. Infect. Dis. 133:S142-S156. Evans, D. J. Jr., L C. Chen, G. T. Curlin, and D. G. Evans. 1972. Stimulation of adenyl cyclase by Escherichia coli enterotoxin. Nature (London) New Biol. 236:137-138. Evans, D. J. Jr., D. G. Evans, S. IL Richardson, and S. L Gorbach 1976. Purification of the polymyxinreleased, heat-labile enterotoxin of Escherichia coli. J. Infect. Dis. 131:S97-S102. Finkelatein, R. A., P. Atthaaampunna, M. Chulasamaya, and P. Charunmethee. 1966. Pathogenesis of experimental cholera: biologic activities of purified procholeragen A. J. Immunol. 96:440-449. Forsyth, G. W., D. L. Hamilton, K. E. Goertz, and M. R. Johnson. 1978. Cholera toxin effects on fluid secretion, adenylate cyclase, and cyclic AMP in porcine small intestine. Infect. Immun. 21:373-380.

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10. Gyles, C. L 1970. Heat-labile and heat-stable forms of enterotoxin from Escherichia coli strains enterotoxigenic for pigs. Ann. N.Y. Acad. Sci. 176:314-322. 11. Gyles, C. L, and D. A. Barnum. 1969. A heat-labile enterotoxin from strains of Escherichia coli enterotoxigenic for pigs. J. Infect. Dis. 120:419-426. 12. Hamilton, D. L, ML R. Johnson, G. W. Forsyth, W. E. Roe, and N. 0. Nielsen. 1978. The effect of cholera toxin and heat labile and heat stable Escherichia coli enterotoxin on cyclic AMP concentrations in small intestinal mucosa of pig and rabbit. Can. J. Comp. Med. 42:327-331. 13. Hamilton, D. L, W. E. Roe, and N. 0. Nielsen. 1977. Effect of heat-stable and heat-labile Escherichia coli enterotoxins, cholera toxin and theophylline on unidirectional sodium and chloride fluxes in the proximal and distal jejunum of weanling swine. Can. J. Comp. Med. 41:306-317. 14. Jacks, T. W., and B. J. Wu. 1974. Biochemical properties of Escherichia coli low-molecular-weight, heat-stable enterotoxin. Infect. Immun. 9:342-347. 15. Kalb, V. F., and F. W. Bernlohr. 1977. A new spectrophotometric assay for protein in cell extracts. Anal. Biochem. 82:362-371. 16. Kantor, IL S., P. Tao, and S. L. Gorbach. 1974. Stimulation of intestinal adenyl cyclase of Escherichia coli enterotoxin: comparison of strains from an infant and an adult with diarrhea. J. Infect. Dis. 129:1-9. 17. Klipstein, F. A., C.-S. Lee, and R. F. Engert. 1976. Assay of Escherichia coli enterotoxins by in vivo perfusion of the rat jejunum. Infect. Immun. 14:1004-1010. 18. Lowry, 0. 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. 19. MacKenzie, S. L., and T. Tenasehuk. 1974. Gas-liquid chromatography of N-heptafluorobutyryl isobutyl esters of amino acids. J. Chromatogr. 97:19-24. 20. MacKenzie, S. L, and T. Tenaschuk. 1975. Rapid formation of amino acid Iso-butyl esters for gas chromatography. J. Chromatogr. 111:413-415. 21. Mullan, N. A., M. N. Burgess, and P. M. Newsome. 1978. Characterization of a partially purified, methanol soluble heat-stable Escherichia coli enterotoxin in infant mice. Infect. Immun. 19:779-784. 22. Moon, H. W., and S. C. Whipp. 1970. Development of resistance with age by swine intestine to effects of enteropathogenic Escherichia coli. J. Infect. Dis. 122: 220-223. 23. Narahashi, Y. 1970. Proteolytic enzymes. Methods Enzymol. 19:651-665. 24. Seifter, S., and E. Harper. 1970. Proteolytic enzymes. Methods Enzymol. 19:613-635. 25. Shatton, D. M. 1970. Proteolytic enzymes. Methods Enzymol. 19:113-139. 26. Skerman, F. J., S. B. Formal, and S. Falkow. 1972. Plasmid-associated enterotoxin production in a strain of Escherichia coli isolated from humans. Infect. Immun. 5:622-624. 27. Smith, IHL W., and C. L Gyles. 1970. The relationship between two apparently different enterotoxins produced by enterotoxigenic strains of Escherichia coli of porcine origin. J. Med. Microbiol. 3:387-401. 28. Smith, IL W., and S. Halls. 1967. Observations by ligated intestinal segment and oral inoculation methods on Escherichia coli infection in pigs, calves, lambs, and rabbits. J. Pathol. Bacteriol. 93:499-529. 29. Smith, IL W., and S. Halls. 1967. Studies on Escherichia coli enterotoxin. J. Pathol. Bacteriol. 93:531-543. 30. Walsh, K. A. 1970. Proteolytic enzymes. Methods Enzymol. 19:41-63. 31. Whipp, S. C., IL W. Moon, and N. C. Lyon. 1975. Heatstable Escherichia coli enterotoxin production in vivo. Infect. Immun. 12:240-244.

Isolation and partial characterization of two different heat-stable enterotoxins produced by bovine and porcine strains of enterotoxigenic Escherichia coli.

INFECTION AND IMMUNITY, Oct. 1979, p. 173-177 0019-9567/79/10-0173/05$02.00/0 Vol. 26, No. 1 Isolation and Partial Characterization of Two Different...
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