Isolation of Cholera Toxins by Dextran Sulfate Precipitation

Vol. 96, No. 4 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Oct. 1968, p. 1443-1445 Copyright @ 1968 American Society for Microbiology

Isolation of Cholera Toxins by Dextran Sulfate Precipitation S. H. RICHARDSON AND D. J. EVANS, JR. Department of Microbiology, The Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103

Received for publication 26 July 1968

It has been recently reported (2) that high levels of heat-labile nondialyzable toxin(s) capable of eliciting a lethal choleraic diarrhea in infant rabbits (choleragen; 3) and a delayed indurative skin reaction in guinea pigs (vascular permeability factor or PF; 1) are produced in vitro by Vibrio cholerae strain VC-12 (Ogawa) when it is cultivated under specific conditions quite different from those ordinarily employed for Vibrio toxin production. Quantitative removal of this choleragen/PF activity from culture filtrates by selective precipitation with high molecular weight dextran sulfate (DS) was also reported by Evans and Richardson at the annual meeting of the American Society for Microbiology, Detroit, 1968. Details of the isolation procedure and further findings concerning the semipurified toxin are reported in this paper. An 0.1-ml amount of V. cholerae VC-12 (108 colony-forming units/ml) from an overnight peptone agar slant was inoculated into 2% Difco peptone broth, supplemented with 0.5% NaCl and buffered to pH 6.5 with 0.08 M tris(hydroxymethyl)aminomethane. Cultures (80 ml) were incubated for 10 hr at 29 C in 500-ml baffle flasks (Bellco Glass Inc., Vineland, N.J.) on a Psychrotherm Shaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 250 rev/min. Beyond this point, all manipulations were carried out at 4 to 10 C. The bacteria were removed by centrifugation at 10,000 x g for 30 min, and the pooled supernatant fluids (800 ml) were twice passed through a 0.45-,Mm membrane filter (Millipore Corp., Bedford, Mass.). Disodium ethylenediaminetetraacetate (EDTA), 0.005%, and 10 Mg of crystalline bovine serum albumin per ml were added to stabilize the toxic activities during the dialysis step which is required to lower the salt concentration of the filtrate (high-salt concentrations inhibit complex formation). After overnight dialysis against 0.005 % EDTA (pH 8), CaCI2 was added to 750 ml of filtrate to a concentration of 0.05 M, and solid DS (dextran sulfate 500, molecular weight 500,000; Pharmacia Fine

Chemicals, Piscataway, N.J.) was added slowly with constant stirring to a final concentration of 2 mg/ml. The mixture was allowed to sit at 4 C for 2 hr during which time it became markedly turbid. The precipitate was collected by centrifugation at 10,000 X g for 30 min, and the resultant insoluble pellets were combined and washed three times by homogenization in and centrifugation (4,000 x g, 10 min) from 0.05 M CaC12. The washed toxin-containing complex (DST) was dissolved in 3 ml of 0.15 M NaCl, pH 7.9, containing 0.005% EDTA, and was clarified by centrifugation (4,000 x g, 10 min). The original dialyzed filtrate, the supernatant fluid from the first DS precipitation step, and the final DST preparation were assayed for PF activity in guinea pigs by Craig's procedure (1). The data (Table 1) show that the original filtrate had a total PF activity of 6 x 107 bluing doses (BD) of which 3 x 107 BD were removed as DST. The total BD units in the concentrated DST preparation were of the same order of magnitude as would be expected from a 250-fold concentration of the original ifitrate (750 ml reduced to 3 ml). A total of only 75,000 BD (about 0.1 %) were left in the DS-treated supernatant fluid, indicating that the removal of PF activity is essentially quantitative. Dilutions (1 :100) of DST heated for 60 min at 60 C produced no visible reaction at their intradermal sites of injection, showing that the skin response is due to the heat-labile PF component of the DST. Dilutions of the ifitrate, DST, and suitable controls (heated as above) were prepared in saline, containing 0.1 % gelatin, and were injected directly into the unligated small intestine of suckling rabbits. The control animals exhibited no visible distress during the 10-hr observation period, and on post-mortem examination their intestines appeared normal. These results indicate that the DS per se has no observable effect on the intestine within the time span of the experiment. The original filtrate and DST at dilutions of 1:10 and 1:100 produced the usual choleraic response; namely, marked distension of the small intestine and colon due to the accumulation

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TABLE 1. Dextran sulfate (DS) precipitation of of the DST is 1,300 times that of the ammonium PF and choleragen from a culture filtratea sulfate precipitate. Preliminary attempts to separate PF activity Test material Vomlue BDb/ml Choleragenicityc from DS involved preparative ultracentrifugation in dense salt solutions. This is a standard Dialyzed filprocedure in the purification of serum lipoprotrated ....... 750 80,000 + (2/2. at 1: 2) teins which also form complexes with DS. DS supernaSamples (1 ml) of several different complexes tant fluid. 750 100 ? (Not tested) DS precipiTABLE 2. Relative PF activities of the complex and tate (washed supernatant fluid from a dextran sulfate (DS) 3 10,000,000 + (2/2 at 1:10; three times). treated culture filtrate 1/1 at 1:100) a PF and choleragen activities in the DS precipitate were destroyed by heating for 1 hr at 60 C. b BD (blueing dose), amount of PF producing an 8 by 8 mm vascular permeability skin reaction in a guinea pig. ¢ Choleragenicity, ability to produce choleraic reaction in the infant rabbit when administered

intraintestinally. d From aerated cultures grown in Difco peptone (pH 6.5) at 29 C for 10 hr.

of massive quantities of a straw-colored fluid in the lumen. The reaction in the animal injected with the 1:100 dilution was as intense as the responses of those injected with 1:10 dilutions, suggesting that at least another 10-fold dilution would be required to reach an end point in the reaction. A portion of the accumulated fluid from the intestine of an experimental animal, which was injected with a 1:10 dilution of DST, was diluted 1:2 with gelatin-saline, and replicate 0.1-ml samples were injected intradermally into a guinea pig. At 18 to 24 hr, there were no discernible responses to these injections, suggesting that PF was selectively bound to or absorbed from the intestine during fluid accumulation or that it was destroyed by the intestinal enzymes of the rabbit. In considering PF binding to the intestinal surface, the similar cation exchange properties of DS and the sulfated mucopolysaccharides on the lumenal surface of the microvilli should be kept in mind. When the nondialyzable (protein) material remaining in supernatant fluids from DS precipitates was concentrated by salting out with ammonium sulfate or by ultrafiltration, results typified by the data contained in Table 2 are obtained. Although in the example given the PF recovery is quite low (less than 10%), the selectivity of the DS precipitation is evident in that a 10-fold increase in PF specific activity is observed in the DST complex, whereas less than 0.08% of the starting PF activity is recoverable from the supernatant fluid. The specific activity

Volume

Test material

BD/ml

Total BD

Dialyzed filtrate... 132 160,000 21,000,000 DS complex....... 1.5 1,300,000 1,900,000 Ammonium sulfate precipitate-. 1.5 1,000 1,500 a Obtained by bringing the DS-treated supernatant fluid to 80%o saturation with solid ammonium sulfate. This procedure quantitatively removes protein (including PF activity) from untreated dialyzed filtrates.

0.

_

:1 r .05

~200 ,

%

E

Top

0

12

9 6 3 Fraction

0

FIG. 1. Dissociation of the DST complex by ultracentrifugation in a KBr-NaCl gradient. Symbols: (0) optical density at 272 nm (protein); (A) area of bluing in PF skin test (64 mm2 or larger considered positive);

(0) Toluidine Blue 0 positive. DST (I ml) in I M NaCI, pH 8, layered on 11 ml of 35.4% KBr. Centrifugation was 100,000 X g for 15.5 hr at 12 C. Fractions (I ml). Toluidine Blue 0 detects DS.

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in 1 M NaCl (pH 8) containing 0.005% EDTA were layered over 11-ml volumes of 35.4% (w/v) KBr (d = 1.32 g/cc) of the same pH and EDTA concentration. The samples were centrifuged at 100,000 x g for 15.5 hr at 12 C in a preparative ultracentrifuge (800, Arden Instrument Co., Silver Spring, Md.). Twelve consecutive 1-ml fractions were removed from each tube employing capillary pipettes with bent tips. Complete ultraviolet spectra (220 to 320 nm against an appropriate KBr blank) were run on each sample, and a portion of each fraction was diluted 1:10 and was assayed for PF activity. A typical result from these experiments (Fig. 1) shows that PF activity (proportional to the area of bluing) is limited to the upper half of the salt gradient and is associated with proteincontaining material (absorption maximum 272 nm). When each fraction was tested for the presence of DS with Toluidine Blue 0 (a sensitive test for dextran), DS was found only at the bottom of the tube (fraction 12) where there is no significant absorbancy at 272 nm and no PF activity. These experiments offer clear-cut evidence that the DST complex can be dissociated (presumably by the high ionic strength of the centrifugation media) into DS and a toxic fraction which still maintains toxic activity. The data presented here plus the fact that similar DST complexes were prepared in our laboratory employing a variety of culture media and other strains of choleragenic vibrios suggest that DS precipitation offers a rapid yet gentle method for quantitative concentration of choleragen/PF activity from culture filtrates.

Because of its interaction with DS, the buoyant nature of the dissociated toxin, and the detection of fatty acids in DST complexes by gas chromatography (R. St. Clair, personal communication), it is believed that the toxic fraction contains lipoprotein material. This conclusion is consistent with electrophoretic studies carried out by J. Kaur and W. Burrows (personal communication); the toxin they used contained about 50% lipid. Our current efforts are aimed at dissociating the complex and purification of the toxic moiety. ACKNOWLEDGMENTS We thank Alayne Noftle, Dolores Evans, and Richard St. Clair for their help and suggestions. This project was supported by Public Health Service grants AI 07772-02 and Al 268-04 from the National Institute of Allergy and Infectious Diseases and Public Health Service Research Career Development Award (Al 09018) to S. H. Richardson. LITERATURE CITED

1. Craig, J. P. 1966. Preparation of the vascular permeability factor of Vibrio chloerae. J. Bacteriol. 92:793-795. 2. Evans, D. J., Jr., and S. H. Richardson. 1968. In vitro production of choleragen and vascular permeability factor by Vibrio choler-ae. J. Bacteriol. 96:126-130. 3. Finkelstein, R. A., H. Norris, and N. Dutta. 1964. Pathogenesis of experimental cholera in infant rabbits. I. Observations on the intraintestinal infection and experimental cholera produced with cell-free products. J. Infect. Diseases 114:203-216