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Purification and Characterization of a Shigella Dysenteriae 1- Like Toxin Produced by Escherichia Coli

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Purification and Characterization of a Shigella Dysenteriae 1- Like Toxin Produced by Escherichia Coli CORE Metadata, citation and similar papers at core.ac.uk Provided by UNL | Libraries University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Uniformed Services University of the Health Sciences U.S. Department of Defense 1983 Purification and Characterization of a Shigella dysenteriae 1- Like Toxin Produced by Escherichia coli Alison D. O'Brien Uniformed Services University of the Health Sciences, [email protected] Gerald D. LaVeck Uniformed Services University of the Health Sciences Follow this and additional works at: https://digitalcommons.unl.edu/usuhs Part of the Medicine and Health Sciences Commons O'Brien, Alison D. and LaVeck, Gerald D., "Purification and Characterization of a Shigella dysenteriae 1- Like Toxin Produced by Escherichia coli" (1983). Uniformed Services University of the Health Sciences. 99. https://digitalcommons.unl.edu/usuhs/99 This Article is brought to you for free and open access by the U.S. Department of Defense at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Uniformed Services University of the Health Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. INFECTION AND IMMUNITY, May 1983, p. 675-683 Vol. 40, No. 2 0019-9567/83/050675-09$02.00/0 Copyright C 1983, American Society for Microbiology Purification and Characterization of a Shigella dysenteriae 1- Like Toxin Produced by Escherichia coli ALISON D. O'BRIEN* AND GERALD D. LAVECKt Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 Received 10 January 1983/Accepted 18 February 1983 A toxin from an enteropathogenic strain of Escherichia coli (E. coli H30) was purified to apparent homogeneity from cell lysates. The steps used to isolate the E. coli H30 toxin included French pressure-cell disruption of bacteria grown in iron-depleted media, Affi-Gel Blue chromatography, chromatofocusing, and anti- Shiga toxin affinity chromatography. The mobilities of the subunits of radioiodi- nated E. coli H30 toxin and Shiga toxin observed after the two toxins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identical. In the absence of 2-mercaptoethanol, a narrow band was seen at Mr 31,500 (±1,000), and a wide heavy band was observed between Mr 4,000 and 15,000. In the presence of 2-mercaptoethanol, bands were seen at Mr 31,500 (±1,000), 27,000, and 4,000 to 15,000. Other similarities between purified E. coli H30 and Shiga 60R toxins included identical isoelectric points (7.03 ± 0.02); comparable biological activities, i.e., cytotoxicity, lethality for mice, and entero- toxicity; and the same relative heat stabilities (up to 65°C for 30 min). Neverthe- less, the two toxins had apparently different molecular weights as determined by sucrose gradient analysis, by gel filtration, and by cross-linking experiments with dimethyl suberimidate. The Mr of native E. coli H30 toxin estimated from cross- linking studies was 48,000, whereas the estimated Mr of Shiga 60R toxin was 58,000. These results suggest that like the cholera-E. coli-heat-labile toxin family, a family of Shiga-like toxins exists. Escherichia coli is one of several agents re- enteric isolates (heat-labile and -stable entero- sponsible for acute bacterial gastroenteritis. toxin-positive organisms and an enteroinvasive Two pathogenic mechanisms have been de- strain) make a cell-associated cytotoxin that can scribed by which E. coli can mediate diarrhea be neutralized by antitoxin prepared against the (8). Some strains of E. coli colonize the mucosal purified toxin of Shigella dysenteriae 1 (Shiga). surface of the small bowel and elaborate heat- Moreover, like Shiga extracts, cell lysates of labile or heat-stable enterotoxins or both that certain EPEC strains are also enterotoxic for cause fluid secretion and, hence, diarrhea (30). rabbit ileal segments and lethal and paralytic for In contrast to these noninvasive enterotoxigenic mice. That a cell-associated cytotoxin could be a strains, other E. coli isolates are serologically virulence determinant in EPEC strains is sug- related to the shigellae (10) and, like shigellae, gested by the observation that the destruction of they are able to penetrate and multiply within gut epithelial cell microvilli seen on small bowel colonic epithelial cells (8). These enteroinvasive biopsies of human infants with EPEC diarrhea is E. coli strains are recognized by their ability to confined to regions of dense E. coli adherence evoke keratoconjunctivitis in guinea pigs (Ser- (5, 25). In this investigation, the Shiga-like toxin eny test, reference 27). Strains of a third group from one EPEC strain, E. coli H30, was purified designated by serotype as enteropathogenic E. to apparent homogeneity, and its biological and coli (EPEC) have been incriminated as agents of biochemical characteristics were compared with epidemic and endemic diarrheal disease in in- those of Shiga toxin. fants (1, 26), but they are not enteroinvasive, nor do they produce heat-labile or -stable enterotox- MATERIALS AND METHODS ins (12). Recent studies from this laboratory (20) Bacterial strains. E. coli H30, an 026 EPEC isolate revealed that most EPEC and some other E. coli (14), was used as the source of Shiga-like toxin (20). Shiga toxin was prepared from Shigella dysenteriae 1 t Present address: Laboratory of Human Carcinogenesis, strain 60R (7). National Institutes of Health, Bethesda, MD 20814. Antitoxin. Monospecific antitoxin against purified 675 676 O'BRIEN AND LAVECK INFECT. IMMUN. Shiga 60R toxin was raised in rabbits by injection of toxin or E. coli Shiga-like toxin activities were neutral- toxin-antitoxin complexes, as previously described ized by rabbit anti-Shiga toxin (21). (19). Radioiodination. Purified and partially purified prep- Preparation of bacterial extracts. Iron-depleted syn- arations of toxin from Shiga 60R and E. coli H30 were case broth was prepared by a previously described labeled by a modification of the chloramine T method method (18, 20). Organisms were incubated for 48 h (13). Carrier-free sodium iodide (1 mCi; Amersham with shaking (280 rpm) at 37°C. Culture volumes were Corp., Arlington Heights, Ill.) was added to 10 to 100 typically 8 to 12 liters. Bacteria were harvested by ,ug of toxin in 500 ,ul of 140 mM NaCl in 10 mM centrifugation, washed, and disrupted in a French phosphate (pH 7.4) buffer. The solution was then made pressure cell, and extracts were clarified by ultracen- 0.2 mM chloramine T by the addition of appropriate trifugation as detailed elsewhere (19). quantities of concentrated reagent. After a 5-min incu- Toxin purification. French press extracts were dia- bation on ice, the mixture was made 0.5 mM sodium lyzed against 100 volumes of 10 mM sodium phosphate metabisulfite and 10 mM Nal. One minute later, 100 pAl buffer (pH 7.4) and then were applied to a column (2.5 of a 1 mg/ml solution of myoglobin was added to by 23 cm) that contained 50 ml of Affi-Gel Blue (Bio- prevent nonspecific absorption of the radiolabeled Rad Laboratories, Richmond, Calif.), a chromato- toxin to the glassware. The mixture was then dialyzed graphic procedure originally used by Olsnes et al. (23). extensively against 140 mM NaCl in 10 mM phosphate The column was then washed with 10 mM sodium (pH 7.4). phosphate buffer (pH 7.4) until the optical density at Sucrose gradients. Radiolabeled toxin samples were 280 nm (OD280) was <0.050. Material which had layered onto 4-ml, 10 to 40%0o sucrose gradients in 140 bound to the column was eluted with 500 mM NaCl in mM NaCI-10 mM phosphate (pH 7.4) and centrifuged 10 mM sodium phosphate (pH 7.4). A pool of this in a Beckman SW-60 rotor at 485,000 x g (60,000 rpm) eluate was then dialyzed against 200 volumes of 25 for 20 h. Fractions of 200 p.l were collected from the mM Tris (pH 8.3). Next, the partially purified toxin tops of the tubes with a Buchler Auto Densi Flow II C was concentrated to a volume of 5 ml on an Amicon (Buchler Instruments, Fort Lee, N.J.), and the refrac- concentrator with a YM-10 membrane (Amicon Corp., tive index of each fraction was measured to quantitate Lexington, Mass.). The concentrated toxin was then the percent sucrose of the sample. To locate toxin, the applied to a column (0.9 by 30 cm) that contained 20 ml radioactivity and cytotoxic activity of each fraction of polybuffer exchanger PBE 94 (Pharmacia, Uppsala, was determined. The OD280 reading and the refractive Sweden) which had been equilibrated with 25 mM Tris index of each fraction from a separate tube that (pH 8.3). This ion-exchange column was eluted with a contained bovine serum albumin and myoglobin were mixture of polybuffer 96 and polybuffer 74 as de- used to determine the position of the toxin relative to scribed by Pharmacia ("Chromatofocusing with Poly- these protein standards. buffer and PBE," technical brochure) to generate a pH Immunoprecipitation. Immunoprecipitations of 125I_ gradient between pH 8.0 and 5.0. Increments of 0.02 labeled toxin samples were carried out as previously pH units were formed per 2-ml column fraction. By described (19). this chromatofocusing procedure, the HeLa cell cyto- Polyacrylamide gel electrophoresis. Immunopre- toxic peak fraction occurred between pH 6.9 and 7.1. cipitates or samples of toxins were mixed with an The peak sample and the two fractions on either side equal volume of disruption buffer (100 mM Tris [pH of it were pooled and applied to a 40-ml column of 7.0], 4% [wt/vol], sodium dodecyl sulfate [SDS], 10% rabbit anti-Shiga toxin linked to Sepharose 4B (Phar- [wt/vol] sucrose, and 0.04% [wt/vol] bromophenol macia) that had been prepared as previously described blue).
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