JOHN D. CLEMENTS AND RICHARD A. FINKELSTEIN*. Department ofMicrobiology .... formed essentially as described by Sack and Sack (29). The adult rabbit ...
Vol. 24, No. 3
INFECTION AND IMMUNITY, June 1979, P. 760-769 0019-9567/79/06-0760/10$02.00/0
Isolation and Characterization of Homogeneous Heat-Labile Enterotoxins with High Specific Activity from Escherichia coli Cultures JOHN D. CLEMENTS AND RICHARD A. FINKELSTEIN* Department of Microbiology, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235 Received for publication 29 January 1979
The heat-labile enterotoxin (LT) has been isolated in homogeneous form with high specific activity from three sources: cell-free supernatant, NaCl extract, and whole-cell lysates of an enterotoxigenic Escherichia coli strain. In vitro immunological assays were used in lieu of tedious and highly variable bioassays to recognize fractions with activity. This revealed that the major portion of the LT remained adherent to columns containing agarose, from which it could be eluted quantitatively in practically homogeneous form by galactose. Isolated LT has remarkable similarities to the cholera enterotoxin (choleragen) in both subunit structure and amino acid composition, although there are also notable differences in these two enterotoxins, which are related immunologically and by mode of action. Unlike choleragen, in which the A region is totally nicked, E. coli LT, depending on its source, is activated by proteolytic processing. The activity of LT is equivalent to that of choleragen in bioassays on adrenal cells, in rabbit skin, and in rabbit ileal loops, especially when, depending on the source of material, the LT has been activated by treatment with trypsin. The whole-cell lysate is the richest source of LT. Attempts to purify the heat-labile enterotoxin (LT) of Escherichia coli have, thus far, met with only limited success. A variety of techniques have been employed to attempt purification, initially with conventional methods such as gel filtration and ion-exchange chromatography (6, 11, 17, 19, 23, 24, 28, 30, 32, 34) and, recently, with approaches such as affinity chromatography with antiserum prepared against the enterotoxin (choleragen) of Vibrio cholerae (4, 5). Reported characterizations of LT have been based on crude and partially purified preparations that vary in physicochemical properties from laboratory to laboratory (6, 8, 11, 13, 17, 21, 22, 27, 28, 30). Molecular weights for LT have been reported to range from 104 to greater than 106 (6, 8, 11, 17, 19, 21, 22, 30, 32, 34), with specific activities that vary from picograms to milligrams within the same test system (6, 11, 17, 19, 24, 32): the products were invariably orders of magnitude less active than choleragen in the same assays. In virtually all of the previous works, various bioassays were used to recognize fractions with biological activity separated during the various purification schemata. Recoveries of activity, when such estimates were made, were invariably poor. Using highly concentrated crude preparations of LT, we recently demonstrated (2, 3) that
LT has antigenic determinants in common with each of the isolated subunits (A and B) of choleragen. Additionally, each of the enterotoxins was shown to possess unique antigenic specificities. In this study, in lieu of biological activity, we screened fractions for antigenic activity. This simple expedient enabled us to devise a simple, practically single-step procedure for isolation of homogeneous E. coli LT from three sources: cell-free supernatants, extracts, and lysates of E. coli cultures. These products and their subunits are described and compared with choleragen and its subunits. The biological activity of E. coli LT, isolated in this manner, is equivalent to choleragen in several assay systems especially when, depending on the source of the material, the LT is proteolytically processed. (This work constitutes a portion of the dissertation of J. D. C., submitted in partial fulfillment of the requirements for the Ph.D. degree, University of Texas Health Science Center at Dallas, Graduate School of Biomedical Sciences.)
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MATERIALS AND METHODS Bacterial strain. The bacterial strain, which we have employed previously (2, 3), was E. coli 711 (FILT) (phe trp pro his Nxr lac), a transformed K-12 derivative bearing LT gene(s) of the Ent plasmid from
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HOMOGENEOUS HIGHLY ACTIVE E. COLI LT
porcine strain P307 (kindly provided by S. Falkow). Culture conditions and preparations of E. coli LT. The culture conditions and preparations of cellfree supernatant (CFS), NaCl extract (EXT), and whole-cell lysate (WCL) were as previously described
(2,3).
Purification of E. coli LT. At no time was any preparation derived from E. coli exposed to a column, membrane, or vessel that had been exposed to choleragen or supernatants derived from cultures of V. cholera. Crude preparations of CFS were concentrated by ultrafiltration with N2 pressure on Amicon PM-10 membranes (Amicon Corp., Lexington, Mass.). EXT, WCL, and concentrated CFS were precipitated with 60%o saturated (NH4)2SO4, and the precipitate was harvested and dialyzed against TEAN buffer (2). After concentration by ultrafiltration, preparations were applied to a column of agarose A-5m (Bio-Rad Laboratories, Richmond, Calif.), equilibrated with TEAN at 4VC. TEAN was applied to the column until the optical density (280 nm, LKB Uvi-cord II) of the effluent returned to base line. LT, which our studies have shown was still adherent to the column, was then eluted with 0.2 M D-(+)-galactose (Eastman Kodak Co., Rochester, N. Y., or Pfanstiehl Laboratories, Inc., Waukegan, Ill.) in TEAN. Absorbance was recorded and fractions were collected automatically (LKB, Uvicord II), and the peaks were pooled. The essentially pure material eluted by galactose was concentrated by ultrafiltration on Amicon PM-10 membrane filters and applied to a column of Sephacryl S-200 (Pharmacia Fine Chemicals, Uppsala, Sweden) for molecular size approximation and to remove any residual galactose, aggregated material, and other trace contaminants. The S-200 column, equilibrated with TEAN, was previously calibrated with proteins of various molecular weights. Apo-ferritin (molecular weight 480,000), aldolase (molecular weight 158,000), bovine serum albumin (molecular weight 68,000), ovalbumin (molecular weight 45,000), chymotrypsinogen A (molecular weight 25,000), and cytochrome c (molecular weight 12,400) served as markers. Bioassays. Skin tests (7) were performed in pairs of rabbits inoculated intracutaneously on shaved backs with serial dilutions of products in 0.1-ml amounts. The next day, the rabbits were inoculated intravenously with 5 ml of a 2%o solution of trypan blue in 0.85% NaCl and depilated with commercial depilatory, and areas of edema, induration, and bluing were measured. Assays in mouse Y-1 adrenal cells were performed essentially as described by Sack and Sack (29). The adult rabbit intestinal loop assay was performed as described by Pierce and Wallace (25). New Zealand albino rabbits, weighing approximately 2 kg, were sedated, and the ilea were ligated in 5- to 7-cm segments. Each product was tested in two rabbits in which the proximal-to-distal orientation of serial dilutions was alternated. Samples were serially diluted in TEAN, and each segment was inoculated with 1.0 ml. Animals were killed by injection of sodium pentobarbitol after 18 h and the volume-of-fluid-to-length-of-segment (milliliters to centimeters) ratio was determined. Immunodiffusion. Immunodiffusion experiments were performed in 1% Noble agar (Difco), with 1% sodium azide added as a preservative. Wells contained
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50 tl and were 9 mm apart, center to center. Immunodiffusion reactions were allowed to develop overnight at room temperature. Trypsin treatment. Trypsin (Sigma Chemical Co., St. Louis, Mo.) was added to samples in TEAN at a final concentration of 1 ,ug/ml, and the samples were incubated at 370C for 1 h. For polyacrylamide gel electrophoresis (PAGE) in sodiumn dodecyl sulfate (SDS), 2% SDS-1% 2-mercaptoethanol was added to each sample and then the samples were heated to 1000C for 3 min in a boiling water bath. For other determinations, soybean trypsin inhibitor (type 1-S, Sigma Chemical Co., St. Louis, Mo.) was added in TEAN at a final concentration of 1 pg/ml. Electrophoresis. SDS-PAGE was performed by the technique of Weber and Osborn (35) or by a modification of the technique of Laemmli (20). PAGE was performed by a modification of the technique of Laemmli (20). Cholera enterotoxin subunits. The cholera entertoxin subunits, cholera A and cholera B, were isolated from pure choleragen by gel filtration under dissociating conditions as described previously (18). Antisera. Antisera to cholera enterotoxin subunits and to E. coli LT were prepared as described previously (2, 3). Protein determinations. Protein determinations were made by the method of Bradford (1). Amino acid analysis. Choleragen, choleragenoid, and LT from CFS, EXT, and WCL were subjected to amino acid analysis after hydrolysis in 6 M hydrochloric acid in vacuo for 20 h at 110'C. All amino acid analyses were performed on a Durrum D-500 amino acid analyzer. RESULTS Typical profiles of concentrated CFS, EXT, and WCL, on elution from the agarose column, are shown in Fig. 1A. Fractions from each separation were pooled, concentrated to approximately the original volume applied by ultrafiltration on Amicon PM-10 membrane filters, and examined by immunodiffusion for the presence of specific antigen. These tests, employing monospecific antiserum to E. coli LT, antiserum to cholera A, and antiserum to cholera B, revealed that little or no specific antigen eluted from the column with TEAN, although precipitating antigens were clearly evident in the original preparations (2, 3). Galactose was then applied, and
the resulting elution profiles for each of the preparations are shown in Fig. lB. A single 280nm absorbing peak eluted just prior to the leading edge of the applied galactose (which has slight absorbance at 280 nm). This peak was pooled, concentrated by ultrafiltration on Amicon PM-10 membrane filters, and examined for specific antigen by immunodiffusion. In each case, the eluted material reacted with the specific antisera, giving a reaction indistinguishable from that previously observed with crude preparations (3) (Fig. 2). Thus, antisera prepared
CLEMENTS AND FINKELSTEIN
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INFECT. IMMUN.
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