0019-9567/78/0019-0066$02.00/0 Vol. 19, No. 1
INFECTION AND IMMUNITY, Jan. 1978, p. 66-70 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Purification of Pseudomonas aeruginosa Exotoxin by Affinity Chromatography NANCY S. TAYLOR AND MATTHEW POLLACK* Department of Microbiology, Naval Medical Research Institute, Bethesda, Maryland 20014 Received for publication 1 September 1977
Pseudomonas aeruginosa exotoxin A was purified by affinity chromatography from culture supernatants by elution of toxin from antitoxin immunoglobulin GSepharose 4B with 3 M NaSCN. The purity, toxicity, and enzymatic activity of exotoxin obtained were comparable to those of toxin purified by previously reported multiple-step procedures. Exotoxin A, produced by most clinical strains of Pseudomonas aeruginosa (1, 21), is highly toxic for animals and tissue cultures and inhibits protein synthesis in vitro (16) and in vivo (18) by the enzymatic addition of adenosine 5'-diphosphate ribose to elongation factor 2 (9). Exotoxin A has been purified from culture supernatants of a nonproteolytic P. aeruginosa strain, PA 103, by several physicochemical methods (2, 3, 11, 13). These procedures are complex and yield toxin with variable purity and potency. We describe a fast and simple one-step procedure for P. aeruginosa exotoxin purification utilizing affinity chromatography (AC) with good recovery of highly pure, toxic, and enzymatically active material.
and to crude culture supernatant (19). Preparation of AT-IgG. To prepare antitoxin immunoglobulin G (AT-IgG), sheep antitoxin serum was absorbed with Formalin-killed whole cells of strain PA 103 to eliminate antibodies to cellular antigens (17); this absorption did not affect the antitoxin titer of the serum. The absorbed serum was then subjected to three successive precipitations with 50, 40, and 33% solutions of saturated ammonium sulfate (4). The final precipitate was redissolved in normal saline, dialyzed against 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5) adjusted to a conductivity of 8,000 Q with 17% NaCl, and applied to a diethylaminoethyl-cellulose column (1 by 20 cm), and the IgG was eluted with the same buffer. AC. Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) was activated with cyanogen bromide (CNBr) (15). Activated Sepharose was reacted with purified AT-IgG in the proportion of 1 or 5 mg of AT-IgG per ml of Sepharose (8). The AT-IgGSepharose was suspended in equilibration buffer, and columns were poured and equilibrated with several volumes of buffer. Culture supernatant, dialyzed against equilibration buffer, was applied to the ATIgG-Sepharose columns, and equilibration buffer was added until no more material with an absorbance of 280 nm was detected in the elutate. Columns were then eluted with 3 M sodium thiocyanate (NaSCN), followed by extensive washing with equilibration buffer. Protein-containing fractions eluted with the NaSCN were dialyzed as soon as possible against equilibration buffer without azide and assayed for toxin. All of the above procedures were perfonned at
MATERIALS AND METHODS Culture preparation. The toxigenic, nonproteolytic P. aeruginosa strain PA 103 (12) was donated by P. V. Liu, University of Louisville, Louisville, Ky. Cultures were grown without nitrilotriacetic acid (21). After centrifugation at 10,000 rpm for 30 min, culture supernatants were filter sterilized, concentrated with a PM-30 membrane (Amicon Corp., Lexington, Mass.), and dialyzed overnight against several changes of 0.05 M phosphate buffer (pH 7.0) with 0.02% sodium azide. All procedures were performed at 4°C. Antitoxin serum. Sheep antitoxin serum was prepared against toxin lot 11475 (2, 3, 20). This toxin preparation showed one major and several minor protein bands after electrophoresis in 10% sodium dodecyl sulfate (SDS) gels and had a 50% lethal dose (LD5o) of 62 ng for 20-g mice (3). The sheep antitoxin serum yielded a single precipitin line to purified toxin (100 ,ug/ml) and PA 103 culture filtrate by immunodiffusion, and demonstrated a passive hemagglutination titer of 1:32,000 (20). One milliliter of the antitoxin serum neutralized 768 ,ug of purified toxin, based on mouse lethality (2), or 640 Mug, based on cytotoxicity for L cells (10, 19), and it reacted with 1,088 Mg in a hemagglutination inhibition (HI) test (see below). Rabbit antisera were prepared to AC-purified toxin
40C.
Protein measurement. Protein concentrations were determined by the method of Lowry et al. (14),
using Dade Lab-trol (American Hospital Supply Corp., Miami, Fla.) as the standard. HI assay for exotoxin. A microtiter hemagglutination assay for P. aeruginosa exotoxin antibody (21) was used to measure toxin by HI. Four hemagglutinating units of sheep antitoxin serum in 25 p1 of Veronal buffer were incubated at 370C for 1 h with twofold dilutions of toxin in an equal volume of buffer before addition of toxin-coated sheep erythrocytes. The highest dilution of toxin that inhibited hemagglutination completely was designated as the end point 66
VOL. 19, 1978
67
PURIFICATION OF P. AERUGINOSA EXOTOXIN
and was related to a toxin standard of known concentration. Mouse lethality. The LD50 of toxin for 20-g mice was determined as previously described (2). Cytotoxicity. Cytotoxicity was measured with microcultures containing 200, rather than 1,000, L cells, and cytotoxic end points, defined as the highest toxin dilution causing 290% destruction of cell monolayers, were read visually at 72 h (10, 19). Polyacrylamide gel electrophoresis. Toxin preparations were assayed for purity by SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels according to the method of Weber and Osborn (22) and by disc electrophoresis by the method of Davis (7). Assay for adenosine diphosphate ribosyl transferase. Adenosine diphosphate ribosyl transferase activity was measured by the procedure of Collier and Kandel (6) as employed by Iglewski and Kabat (9), with wheat germ as the source of elongation factor 2 (5). The assay mixture contained 50 itl of wheat germ extract, 15 p1 of nicoimide [4C]adenine dinucleotide (280 mCi/mmol; Amersham/Searle, Arlington Heights, Ill.), 10 p1 of toxin, and 50 p1 of 125 mM tris(hydroxymethyl)aminomethane buffer (pH 8.2) containing 0.2 mM ethylenediaminetetraacetic acid and 100 mM dithiothreitol. Toxin was activated with 4 M urea plus 1% dithiothreitol for 15 min at 23°C.
Reactions were initiated by the addition of the nicotinamide adenine dinucleotide (diluted 1:1 with water), the reaction mixtures were incubated for 5 min at 23°C, and the reactions were stopped with 10% trichloroacetic acid. The precipitates were collected, washed, and counted (9).
RESULTS
AC. Treatment of CNBr-activated Sepharose 4B with AT-IgG in a concentration of 1 to 5 mg of protein per ml of beads resulted in complete coupling of the immunoglobulin. Columns measuring 1.6 by 25 cm and 2.5 by 40 cm with bed volumes of 40 and 200 ml, respectively, were made from the AT-IgG-Sepharose. The smaller columns contained Sepharose treated with AT-IgG at either high or low concentrations; the larger columns contained Sepharose treated with the lower concentration of IgG. The toxin concentration of culture supernatants applied to columns varied from 4 to 70 ,ug/ml, based on HI assay. This represented 0.5 to 2% of the total protein, as measured by the Lowry assay (14). Sample volumes varied from
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THIOCYANATE
8
,10
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20
445 40
35
FRACTION NUMBER (6 ml)
FIG. 1. AC of crude culture supernatant from P. aeruginosa PA 103. Approximately 624 mg of protein in a volume of 100 ml, dialyzed against 0.05 Mphosphate buffer (pH 7.0; equilibration buffer), was applied to a column (1.6 by 25 cm) consisting of CNBr-activated Sepharose 4B to which AT-IgG had been coupled in a ratio of 1 mg of IgG per ml of Sepharose. The column was eluted, successively, with 200 ml of equilibration buffer and 40 ml of 3 M NaSCN, and 5-ml fractions were tested for absorbance at 280 nm and for toxin content by HI. Cross-hatched bars indicate HI titers of fractions. TABLE 1. Purification of P. aeruginosa exotoxin by AC LD5o per % RecovFold purifiTotal poen Total Purification stage
Crude culture supernatant .. AC ................................... a Based on Lowry assay. b Toxin administered intravenously.
Tog)tal (,zg)~
mouseb
624,000 1,763
32.5 0.118
(,)a,
LD5o 19,200 14,940
ery
cation
78
275
68
_
TAYLOR AND POLLACK
INFECT. IMMUN.
50 to 200 ml, and the toxin applied to columns varied from 0.2 to 4.2 mg, based on HI assay. The volume of supernatant applied to columns did not appear to be critical. However, when the total protein present in the sample exceeded 20 mg/ml of Sepharose, in the case of columns made with 1 mg of AT-IgG per ml, or 30 to 40
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_-
FIG. 3. Immunodiffusion analysis ofP. aeruginosa exotoxin (L) purified by the method of Leppla (11) with additional purification by preparative polyacrylamide gel electrophoresis and Sephadex G-150 chromatography (3) and of exotoxin (AC) purified by AC. Center well contains rabbit antiserum (AT) to AC-purified toxin, and remaining wells contain crude PA 103 culture supernatant (S), concentrated 10-fold.
mg/ml, in the case of columns made with 5 mg AT-IgG per ml, the toxin peak became contaminated with at least one other protein, as determined by SDS gels. Elution of columns with equilibration buffer resulted in a large protein peak (Fig. 1). No toxin was detectable by HI assay in this peak, either in the case of small columns made with 5 mg of AT-IgG per ml, to which as much as 2.8 mg of toxin had been applied, or in the case of large columns, to which as much as 4.2 mg of toxin had been applied. It thus appeared that the toxin capacity of small and large columns was at least 2.8 and 4.2 mg, respectively. After complete elution of unbound protein, application of 3 M NaSCN in a volume of 40 ml for small columns and 200 ml for large columns resulted in a second sharp peak (Fig. 1), which yielded 60 to 80% of the applied toxin (Table 1). In some instances, low levels of toxin FIG. 2. 10%o SDS-polyacrylamide gel electrophore- detectable by HI appeared in a number of fracsis of P. aeruginosa exotoxin prepared by different tions after the 3 M NaSCN-eluted peak. This methods. (A) Crude PA 103 culture supernatant con- trailing may have been responsible for our inacentrated 50-fold; 25-,ul sample containing 50 jig of bility to recover all of the toxin applied to colprotein. (B) Exotoxin purified by AC; 100-,il sample umns. Repeated NaSCN elutions yielded no adcontaining 15 jig ofprotein. (C) Exotoxin purified by the method ofLeppla (11) with additionalpurification ditional detectable toxin. After elution of toxin, columns were washed by preparative polyacrylamide gel electrophoresis and Sephadex G-150 chromatography (3); 10-jul sam- with two to three times the void volume of equilibration buffer and rerun as many as six ple containing 14 jig ofprotein.
VOL. 19, 1978
69 Data from a representative experiment (Table 2) demonstrate the similarity of activatable enzymatic activity of toxin purified by AC and by Leppla's method (11).
PURIFICATION OF P. AERUGINOSA EXOTOXIN
times without apparent loss of efficiency. Purity. Toxin purified by AC migrated
on
10% SDS-polyacrylamide gels as a single band, which was identical to that of highly purified toxin prepared by combination of other methods (3, 11; Fig. 2). On disc gels, some toxin preparations migrated as a single band while others showed a faint, faster-migrating band that corresponded to one of several bands of the LepplaCallahan toxin (3, 11). Antigenic and immunogenic analysis indicated that AC-prepared toxin was highly purified (Fig. 3, 4). Toxicity. The LD50 in 20-g mice of toxin purified by AC was consistently between 50 and 160 ng, comparable to the LDso reported for other purified toxins (3, 11, 13). The cytotoxicity end point for L cells was 100 to 200 pg of toxin per 200 cells, similar to purified toxin preparations produced by Callahan and by Leppla (3, 11). Both mouse lethality and cytotoxicity were neutralized with rabbit antitoxin serum in amounts comparable to those required for neutralization of these activities by Callahan's toxin. Enzymatic activity. Several AC-purified toxin preparations were examined for adenosine diphosphate ribosyl transferase activity before and after activation with dithiothreitol and urea.
DISCUSSION The advantage of our pseudomonas exotoxin AC purification procedure is that it involves only one step. The effectiveness of the procedure is underlined by the fact that exotoxin represented less than 1% of the total protein present in supernatants (Table 1), and that, based on LD50 for mice, a 200- to 300-fold purification was achieved. The preparation and use of affinity columns was relatively simple, and they could be reused. AC toxin preparations showed a single protein band on 10% SDS gels with samples containing up to 25 ,ug of protein, whereas disc gels showed either a single band or one major band and one very faint, faster-migrating band. This latter finding was variable, and it is not known whether the second band represented a breakdown product of toxin or a second distinct protein. Rabbit antiserum to AC-purified toxin yielded one precipitin line by immunodiffusion when run against purified toxin or crude supernatant, whereas antiserum against crude material gave one line to toxin. Liu indicated that antisera against his purified toxin contained several antibodies directed against nontoxic antigens (13). Callahan's most highly purified material yielded a few faint bands in addition to the main one on polyacrylamide gels (3), as did that of Leppla (11; S. Leppla, personal communication). AC purification yielded toxin with consistent mouse lethality. Although the LD5o's of 62 to 83 ng, 100 ng, and 125 ng reported by Callahan (3), Leppla (11), and Liu (13), respectively, compared well with our range of 50 to 160 ng, subsequent experience with the methods of Callahan and Leppla has yielded toxin with LD50 as high as 1 jig or more (R. DeBell and S. Leppla, personal communication). The adenosine diphosphate ribosyl transferTABLE 2. Comparison of adenosine diphosphate ribosyl transferase activity ofpurified P. aeruginosa exotoxin Purification
method'
FIG. 4. Immunodiffusion analysis of P. aeruginosa exotoxin reacted with rabbit antiserum (AS) to crude PA 103 culture supernatant (S). Wells labeled AC contain exotoxin purified by AC, and wells designated L contain exotoxin purified by the method of Leppla (11) with additional purification by preparative polyacrylamide gel electrophoresis and Sephadex G-150 chromatography (3).
Unactivated
Activated
activateo activated/
unactivated 8.7 AC ........... 1,053 9,132 4.5 9,896 Leppla method ... 2,203 aAfter subtraction of background (201 counts per
minute). bConcentration of toxin in reaction .tg/mI (11).
mixture,
7.4
70
TAYLOR AND POLLACK
ase activity of pseudomonas exotoxin originally reported by Iglewski and Kabat (9) is present in our AC-purified material. Although systematic dose response studies were not done, it appears that the enzymatic activity of our toxin is roughly comparable to that of the toxin prepared by Leppla (Table 20. The marked increase of activity upon activation of our toxin with dithiothreitol and urea suggests it is relatively intact. ACKNOWLEDGMENTS We thank Robert DeBell, Stephen H. Leppla, and L. T. Callahan HI for graciously contributing purified exotoxin, Robert DeBell for performing enzyme assays, and Emilio Weiss and John B. Robbins for reviewing the manuscript. Special thanks go to Richard F. Taylor and Richard Wistar for valuable advice and shared equipment. This investigation was supported by the Naval Medical Research and Development Command, Department of the Navy, Research Work Unit no. MR0412001.0423.
LITERATURE CITED 1. Bjorn, M. J., M. L Vasil, J. C. Sadoff, and B. H. Iglewski. 1977. Incidence of exotoxin production by Pseudomonas species. Infect. Immun. 16:362-366. 2. Callahan, L. T. III. 1974. Purification and characterization of Pseudomonas aeruginosa exotoxin. Infect. Immun. 9:113-118. 3. Callahan, L T. mI. 1976. Pseudomonas aeruginosa exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of a highly specific antitoxin serum. Infect. Immun. 14:55-61. 4. Campbell, D. H., J. S. Garvey, N. E. Cremer, and D. H. Sussdorf. 1970. Lsolation of rabbit antibodies and their subunits, p. 189-191. In Methods in immunology, 2nd ed. W. A. Benjamin, Inc., New York. 5. Chung, D. W., and R. J. Collier. 1977. Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect. Immun. 16:832-841. 6. Collier, R. J., and J. Kandel. 1971. Structure and activity of diphtheria toxin. I. Thiol-dependent dissociation of a fraction of toxin into enzymatically active and inactive fragments. J. Biol. Chem. 246:1496-1503. 7. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427.
8. Holmes, R. K., and R. B. Perlow. 1975. Quantitative
INFECT. IMMUN. assay of diphtherial toxin and of immunologically crossreacting proteins by reversed passive hemagglutination. Infect. Immun. 12:1392-1400. 9. Iglewski, B. H., and D. Kabat. 1975. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin. Proc. Natl. Acad. Sci. U.S.A. 72:2284-2288. 10. Knudsen, R. C., L. T. Callahan Im, A. Ahmed, and K. W. Sell. 1974. Use of microculture plates and the multiple automated sample harvester for in vitro microassay ofbacterial toxins. Appl. Microbiol. 28:326-327. 11. Leppla, S. H. 1976. Large-scale purification and characterization of the exotoxin of Pseudomonas aeruginosa. Infect. Immun. 14:1077-1086. 12. Liu, P. V. 1966. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. III. Identity of the lethal toxins produced in vitro and in vivo. J. Infect. Dis. 116:481 489. 13. Liu, P. V., S. Yoshii, and H. Hsieh. 1973. Exotoxins of Pseudomonas aeruginosa. II. Concentration, purification, and characterization of exotoxin A. J. Infect. Dis. 128:514-519. 14. 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. 15. March, S. C., I. Parikh, and P. Cuatrecasas. 1974. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem. 60:149-152. 16. Pavlovskis, 0. R., and F. B. Gordon. 1972. Pseudomonas aeruginosa exotoxin: effect on cell cultures. J. Infect. Dis. 125:631-636. 17. Pavlovskis, 0. R., M. Pollack, L T. Callahan III, and B. H. Iglewski. 1977. Passive protection by antitoxin in experimental Pseudomonas aeruginosa burn infections. Infect. Immun. 18:596-602. 18. Pavlovskis, 0. R., and A. H. Shackelford. 1974. Pseudomonas aeruginosa exotoxin in mice: localization and effect on protein synthesis. Infect. Immun. 9:540-546. 19. Poliack, M., L T. Callahan HI, and N. S. Taylor. 1976. Neutralizing antibody to Pseudomonas aeruginosa exotoxin in human sera: evidence for in vivo toxin production during infections. Infect. Immun. 14:942-947. 20. Pollack, M., and N. S. Taylor. 1977. Serum antibody to Pseudomonas aeruginosa exotoxin measured by a passive hemagglutination assay. J. Clin. Microbiol. 6:58-61. 21. Pollack, M., N. S. Taylor, and L. T. Callahan III. 1977. Exotoxin production by clinical isolates of Pseudomonas aeruginosa. Infect. Immun. 15:776-7K0. 22. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.
INFECTION AND IMMUNITY, Jan. 1978, p. 66-70 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Purification of Pseudomonas aeruginosa Exotoxin by Affinity Chromatography NANCY S. TAYLOR AND MATTHEW POLLACK* Department of Microbiology, Naval Medical Research Institute, Bethesda, Maryland 20014 Received for publication 1 September 1977
Pseudomonas aeruginosa exotoxin A was purified by affinity chromatography from culture supernatants by elution of toxin from antitoxin immunoglobulin GSepharose 4B with 3 M NaSCN. The purity, toxicity, and enzymatic activity of exotoxin obtained were comparable to those of toxin purified by previously reported multiple-step procedures. Exotoxin A, produced by most clinical strains of Pseudomonas aeruginosa (1, 21), is highly toxic for animals and tissue cultures and inhibits protein synthesis in vitro (16) and in vivo (18) by the enzymatic addition of adenosine 5'-diphosphate ribose to elongation factor 2 (9). Exotoxin A has been purified from culture supernatants of a nonproteolytic P. aeruginosa strain, PA 103, by several physicochemical methods (2, 3, 11, 13). These procedures are complex and yield toxin with variable purity and potency. We describe a fast and simple one-step procedure for P. aeruginosa exotoxin purification utilizing affinity chromatography (AC) with good recovery of highly pure, toxic, and enzymatically active material.
and to crude culture supernatant (19). Preparation of AT-IgG. To prepare antitoxin immunoglobulin G (AT-IgG), sheep antitoxin serum was absorbed with Formalin-killed whole cells of strain PA 103 to eliminate antibodies to cellular antigens (17); this absorption did not affect the antitoxin titer of the serum. The absorbed serum was then subjected to three successive precipitations with 50, 40, and 33% solutions of saturated ammonium sulfate (4). The final precipitate was redissolved in normal saline, dialyzed against 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5) adjusted to a conductivity of 8,000 Q with 17% NaCl, and applied to a diethylaminoethyl-cellulose column (1 by 20 cm), and the IgG was eluted with the same buffer. AC. Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) was activated with cyanogen bromide (CNBr) (15). Activated Sepharose was reacted with purified AT-IgG in the proportion of 1 or 5 mg of AT-IgG per ml of Sepharose (8). The AT-IgGSepharose was suspended in equilibration buffer, and columns were poured and equilibrated with several volumes of buffer. Culture supernatant, dialyzed against equilibration buffer, was applied to the ATIgG-Sepharose columns, and equilibration buffer was added until no more material with an absorbance of 280 nm was detected in the elutate. Columns were then eluted with 3 M sodium thiocyanate (NaSCN), followed by extensive washing with equilibration buffer. Protein-containing fractions eluted with the NaSCN were dialyzed as soon as possible against equilibration buffer without azide and assayed for toxin. All of the above procedures were perfonned at
MATERIALS AND METHODS Culture preparation. The toxigenic, nonproteolytic P. aeruginosa strain PA 103 (12) was donated by P. V. Liu, University of Louisville, Louisville, Ky. Cultures were grown without nitrilotriacetic acid (21). After centrifugation at 10,000 rpm for 30 min, culture supernatants were filter sterilized, concentrated with a PM-30 membrane (Amicon Corp., Lexington, Mass.), and dialyzed overnight against several changes of 0.05 M phosphate buffer (pH 7.0) with 0.02% sodium azide. All procedures were performed at 4°C. Antitoxin serum. Sheep antitoxin serum was prepared against toxin lot 11475 (2, 3, 20). This toxin preparation showed one major and several minor protein bands after electrophoresis in 10% sodium dodecyl sulfate (SDS) gels and had a 50% lethal dose (LD5o) of 62 ng for 20-g mice (3). The sheep antitoxin serum yielded a single precipitin line to purified toxin (100 ,ug/ml) and PA 103 culture filtrate by immunodiffusion, and demonstrated a passive hemagglutination titer of 1:32,000 (20). One milliliter of the antitoxin serum neutralized 768 ,ug of purified toxin, based on mouse lethality (2), or 640 Mug, based on cytotoxicity for L cells (10, 19), and it reacted with 1,088 Mg in a hemagglutination inhibition (HI) test (see below). Rabbit antisera were prepared to AC-purified toxin
40C.
Protein measurement. Protein concentrations were determined by the method of Lowry et al. (14),
using Dade Lab-trol (American Hospital Supply Corp., Miami, Fla.) as the standard. HI assay for exotoxin. A microtiter hemagglutination assay for P. aeruginosa exotoxin antibody (21) was used to measure toxin by HI. Four hemagglutinating units of sheep antitoxin serum in 25 p1 of Veronal buffer were incubated at 370C for 1 h with twofold dilutions of toxin in an equal volume of buffer before addition of toxin-coated sheep erythrocytes. The highest dilution of toxin that inhibited hemagglutination completely was designated as the end point 66
VOL. 19, 1978
67
PURIFICATION OF P. AERUGINOSA EXOTOXIN
and was related to a toxin standard of known concentration. Mouse lethality. The LD50 of toxin for 20-g mice was determined as previously described (2). Cytotoxicity. Cytotoxicity was measured with microcultures containing 200, rather than 1,000, L cells, and cytotoxic end points, defined as the highest toxin dilution causing 290% destruction of cell monolayers, were read visually at 72 h (10, 19). Polyacrylamide gel electrophoresis. Toxin preparations were assayed for purity by SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels according to the method of Weber and Osborn (22) and by disc electrophoresis by the method of Davis (7). Assay for adenosine diphosphate ribosyl transferase. Adenosine diphosphate ribosyl transferase activity was measured by the procedure of Collier and Kandel (6) as employed by Iglewski and Kabat (9), with wheat germ as the source of elongation factor 2 (5). The assay mixture contained 50 itl of wheat germ extract, 15 p1 of nicoimide [4C]adenine dinucleotide (280 mCi/mmol; Amersham/Searle, Arlington Heights, Ill.), 10 p1 of toxin, and 50 p1 of 125 mM tris(hydroxymethyl)aminomethane buffer (pH 8.2) containing 0.2 mM ethylenediaminetetraacetic acid and 100 mM dithiothreitol. Toxin was activated with 4 M urea plus 1% dithiothreitol for 15 min at 23°C.
Reactions were initiated by the addition of the nicotinamide adenine dinucleotide (diluted 1:1 with water), the reaction mixtures were incubated for 5 min at 23°C, and the reactions were stopped with 10% trichloroacetic acid. The precipitates were collected, washed, and counted (9).
RESULTS
AC. Treatment of CNBr-activated Sepharose 4B with AT-IgG in a concentration of 1 to 5 mg of protein per ml of beads resulted in complete coupling of the immunoglobulin. Columns measuring 1.6 by 25 cm and 2.5 by 40 cm with bed volumes of 40 and 200 ml, respectively, were made from the AT-IgG-Sepharose. The smaller columns contained Sepharose treated with AT-IgG at either high or low concentrations; the larger columns contained Sepharose treated with the lower concentration of IgG. The toxin concentration of culture supernatants applied to columns varied from 4 to 70 ,ug/ml, based on HI assay. This represented 0.5 to 2% of the total protein, as measured by the Lowry assay (14). Sample volumes varied from
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THIOCYANATE
8
,10
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20
445 40
35
FRACTION NUMBER (6 ml)
FIG. 1. AC of crude culture supernatant from P. aeruginosa PA 103. Approximately 624 mg of protein in a volume of 100 ml, dialyzed against 0.05 Mphosphate buffer (pH 7.0; equilibration buffer), was applied to a column (1.6 by 25 cm) consisting of CNBr-activated Sepharose 4B to which AT-IgG had been coupled in a ratio of 1 mg of IgG per ml of Sepharose. The column was eluted, successively, with 200 ml of equilibration buffer and 40 ml of 3 M NaSCN, and 5-ml fractions were tested for absorbance at 280 nm and for toxin content by HI. Cross-hatched bars indicate HI titers of fractions. TABLE 1. Purification of P. aeruginosa exotoxin by AC LD5o per % RecovFold purifiTotal poen Total Purification stage
Crude culture supernatant .. AC ................................... a Based on Lowry assay. b Toxin administered intravenously.
Tog)tal (,zg)~
mouseb
624,000 1,763
32.5 0.118
(,)a,
LD5o 19,200 14,940
ery
cation
78
275
68
_
TAYLOR AND POLLACK
INFECT. IMMUN.
50 to 200 ml, and the toxin applied to columns varied from 0.2 to 4.2 mg, based on HI assay. The volume of supernatant applied to columns did not appear to be critical. However, when the total protein present in the sample exceeded 20 mg/ml of Sepharose, in the case of columns made with 1 mg of AT-IgG per ml, or 30 to 40
_ Y;_u.a.
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_
_-
FIG. 3. Immunodiffusion analysis ofP. aeruginosa exotoxin (L) purified by the method of Leppla (11) with additional purification by preparative polyacrylamide gel electrophoresis and Sephadex G-150 chromatography (3) and of exotoxin (AC) purified by AC. Center well contains rabbit antiserum (AT) to AC-purified toxin, and remaining wells contain crude PA 103 culture supernatant (S), concentrated 10-fold.
mg/ml, in the case of columns made with 5 mg AT-IgG per ml, the toxin peak became contaminated with at least one other protein, as determined by SDS gels. Elution of columns with equilibration buffer resulted in a large protein peak (Fig. 1). No toxin was detectable by HI assay in this peak, either in the case of small columns made with 5 mg of AT-IgG per ml, to which as much as 2.8 mg of toxin had been applied, or in the case of large columns, to which as much as 4.2 mg of toxin had been applied. It thus appeared that the toxin capacity of small and large columns was at least 2.8 and 4.2 mg, respectively. After complete elution of unbound protein, application of 3 M NaSCN in a volume of 40 ml for small columns and 200 ml for large columns resulted in a second sharp peak (Fig. 1), which yielded 60 to 80% of the applied toxin (Table 1). In some instances, low levels of toxin FIG. 2. 10%o SDS-polyacrylamide gel electrophore- detectable by HI appeared in a number of fracsis of P. aeruginosa exotoxin prepared by different tions after the 3 M NaSCN-eluted peak. This methods. (A) Crude PA 103 culture supernatant con- trailing may have been responsible for our inacentrated 50-fold; 25-,ul sample containing 50 jig of bility to recover all of the toxin applied to colprotein. (B) Exotoxin purified by AC; 100-,il sample umns. Repeated NaSCN elutions yielded no adcontaining 15 jig ofprotein. (C) Exotoxin purified by the method ofLeppla (11) with additionalpurification ditional detectable toxin. After elution of toxin, columns were washed by preparative polyacrylamide gel electrophoresis and Sephadex G-150 chromatography (3); 10-jul sam- with two to three times the void volume of equilibration buffer and rerun as many as six ple containing 14 jig ofprotein.
VOL. 19, 1978
69 Data from a representative experiment (Table 2) demonstrate the similarity of activatable enzymatic activity of toxin purified by AC and by Leppla's method (11).
PURIFICATION OF P. AERUGINOSA EXOTOXIN
times without apparent loss of efficiency. Purity. Toxin purified by AC migrated
on
10% SDS-polyacrylamide gels as a single band, which was identical to that of highly purified toxin prepared by combination of other methods (3, 11; Fig. 2). On disc gels, some toxin preparations migrated as a single band while others showed a faint, faster-migrating band that corresponded to one of several bands of the LepplaCallahan toxin (3, 11). Antigenic and immunogenic analysis indicated that AC-prepared toxin was highly purified (Fig. 3, 4). Toxicity. The LD50 in 20-g mice of toxin purified by AC was consistently between 50 and 160 ng, comparable to the LDso reported for other purified toxins (3, 11, 13). The cytotoxicity end point for L cells was 100 to 200 pg of toxin per 200 cells, similar to purified toxin preparations produced by Callahan and by Leppla (3, 11). Both mouse lethality and cytotoxicity were neutralized with rabbit antitoxin serum in amounts comparable to those required for neutralization of these activities by Callahan's toxin. Enzymatic activity. Several AC-purified toxin preparations were examined for adenosine diphosphate ribosyl transferase activity before and after activation with dithiothreitol and urea.
DISCUSSION The advantage of our pseudomonas exotoxin AC purification procedure is that it involves only one step. The effectiveness of the procedure is underlined by the fact that exotoxin represented less than 1% of the total protein present in supernatants (Table 1), and that, based on LD50 for mice, a 200- to 300-fold purification was achieved. The preparation and use of affinity columns was relatively simple, and they could be reused. AC toxin preparations showed a single protein band on 10% SDS gels with samples containing up to 25 ,ug of protein, whereas disc gels showed either a single band or one major band and one very faint, faster-migrating band. This latter finding was variable, and it is not known whether the second band represented a breakdown product of toxin or a second distinct protein. Rabbit antiserum to AC-purified toxin yielded one precipitin line by immunodiffusion when run against purified toxin or crude supernatant, whereas antiserum against crude material gave one line to toxin. Liu indicated that antisera against his purified toxin contained several antibodies directed against nontoxic antigens (13). Callahan's most highly purified material yielded a few faint bands in addition to the main one on polyacrylamide gels (3), as did that of Leppla (11; S. Leppla, personal communication). AC purification yielded toxin with consistent mouse lethality. Although the LD5o's of 62 to 83 ng, 100 ng, and 125 ng reported by Callahan (3), Leppla (11), and Liu (13), respectively, compared well with our range of 50 to 160 ng, subsequent experience with the methods of Callahan and Leppla has yielded toxin with LD50 as high as 1 jig or more (R. DeBell and S. Leppla, personal communication). The adenosine diphosphate ribosyl transferTABLE 2. Comparison of adenosine diphosphate ribosyl transferase activity ofpurified P. aeruginosa exotoxin Purification
method'
FIG. 4. Immunodiffusion analysis of P. aeruginosa exotoxin reacted with rabbit antiserum (AS) to crude PA 103 culture supernatant (S). Wells labeled AC contain exotoxin purified by AC, and wells designated L contain exotoxin purified by the method of Leppla (11) with additional purification by preparative polyacrylamide gel electrophoresis and Sephadex G-150 chromatography (3).
Unactivated
Activated
activateo activated/
unactivated 8.7 AC ........... 1,053 9,132 4.5 9,896 Leppla method ... 2,203 aAfter subtraction of background (201 counts per
minute). bConcentration of toxin in reaction .tg/mI (11).
mixture,
7.4
70
TAYLOR AND POLLACK
ase activity of pseudomonas exotoxin originally reported by Iglewski and Kabat (9) is present in our AC-purified material. Although systematic dose response studies were not done, it appears that the enzymatic activity of our toxin is roughly comparable to that of the toxin prepared by Leppla (Table 20. The marked increase of activity upon activation of our toxin with dithiothreitol and urea suggests it is relatively intact. ACKNOWLEDGMENTS We thank Robert DeBell, Stephen H. Leppla, and L. T. Callahan HI for graciously contributing purified exotoxin, Robert DeBell for performing enzyme assays, and Emilio Weiss and John B. Robbins for reviewing the manuscript. Special thanks go to Richard F. Taylor and Richard Wistar for valuable advice and shared equipment. This investigation was supported by the Naval Medical Research and Development Command, Department of the Navy, Research Work Unit no. MR0412001.0423.
LITERATURE CITED 1. Bjorn, M. J., M. L Vasil, J. C. Sadoff, and B. H. Iglewski. 1977. Incidence of exotoxin production by Pseudomonas species. Infect. Immun. 16:362-366. 2. Callahan, L. T. III. 1974. Purification and characterization of Pseudomonas aeruginosa exotoxin. Infect. Immun. 9:113-118. 3. Callahan, L T. mI. 1976. Pseudomonas aeruginosa exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of a highly specific antitoxin serum. Infect. Immun. 14:55-61. 4. Campbell, D. H., J. S. Garvey, N. E. Cremer, and D. H. Sussdorf. 1970. Lsolation of rabbit antibodies and their subunits, p. 189-191. In Methods in immunology, 2nd ed. W. A. Benjamin, Inc., New York. 5. Chung, D. W., and R. J. Collier. 1977. Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect. Immun. 16:832-841. 6. Collier, R. J., and J. Kandel. 1971. Structure and activity of diphtheria toxin. I. Thiol-dependent dissociation of a fraction of toxin into enzymatically active and inactive fragments. J. Biol. Chem. 246:1496-1503. 7. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427.
8. Holmes, R. K., and R. B. Perlow. 1975. Quantitative
INFECT. IMMUN. assay of diphtherial toxin and of immunologically crossreacting proteins by reversed passive hemagglutination. Infect. Immun. 12:1392-1400. 9. Iglewski, B. H., and D. Kabat. 1975. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin. Proc. Natl. Acad. Sci. U.S.A. 72:2284-2288. 10. Knudsen, R. C., L. T. Callahan Im, A. Ahmed, and K. W. Sell. 1974. Use of microculture plates and the multiple automated sample harvester for in vitro microassay ofbacterial toxins. Appl. Microbiol. 28:326-327. 11. Leppla, S. H. 1976. Large-scale purification and characterization of the exotoxin of Pseudomonas aeruginosa. Infect. Immun. 14:1077-1086. 12. Liu, P. V. 1966. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. III. Identity of the lethal toxins produced in vitro and in vivo. J. Infect. Dis. 116:481 489. 13. Liu, P. V., S. Yoshii, and H. Hsieh. 1973. Exotoxins of Pseudomonas aeruginosa. II. Concentration, purification, and characterization of exotoxin A. J. Infect. Dis. 128:514-519. 14. 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. 15. March, S. C., I. Parikh, and P. Cuatrecasas. 1974. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem. 60:149-152. 16. Pavlovskis, 0. R., and F. B. Gordon. 1972. Pseudomonas aeruginosa exotoxin: effect on cell cultures. J. Infect. Dis. 125:631-636. 17. Pavlovskis, 0. R., M. Pollack, L T. Callahan III, and B. H. Iglewski. 1977. Passive protection by antitoxin in experimental Pseudomonas aeruginosa burn infections. Infect. Immun. 18:596-602. 18. Pavlovskis, 0. R., and A. H. Shackelford. 1974. Pseudomonas aeruginosa exotoxin in mice: localization and effect on protein synthesis. Infect. Immun. 9:540-546. 19. Poliack, M., L T. Callahan HI, and N. S. Taylor. 1976. Neutralizing antibody to Pseudomonas aeruginosa exotoxin in human sera: evidence for in vivo toxin production during infections. Infect. Immun. 14:942-947. 20. Pollack, M., and N. S. Taylor. 1977. Serum antibody to Pseudomonas aeruginosa exotoxin measured by a passive hemagglutination assay. J. Clin. Microbiol. 6:58-61. 21. Pollack, M., N. S. Taylor, and L. T. Callahan III. 1977. Exotoxin production by clinical isolates of Pseudomonas aeruginosa. Infect. Immun. 15:776-7K0. 22. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.
