INFECTION

AND

IMMUNITY, OCt. 1976, p. 1077-1086

Vol. 14, No. 4 Printed in U.S.A.

Copyright C 1976 American Society for Microbiology

Large-Scale Purification and Characterization of the Exotoxin of Pseudomonas aeruginosa STEPHEN H. LEPPLA United States Army Pathology Division, Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701

Received for publication 26 April 1976

The exotoxin (PE) of Pseudomonas aeruginosa was purified from 50-liter cultures by a simple three-step procedure, yielding 135 mg of essentially homogeneous protein. In Ouchterlony gel diffusion, PE produces a single line which does not interact with a diphtheria toxin-antitoxin precipitin line. The protein has a molecular weight of 66,000, an isoelectric point of 5.1, N-terminal arginine, and four disulfide bridges. The amino acid composition shows no apparent similarity to that of diphtheria toxin. The median lethal dose of this PE preparation in mice weighing 20 g is 0.1 ,g. The median lethal dose in 350-g rats is 20 ,ug. The cytotoxicity of PE for mouse L929 fibroblasts is completely neutralized by small amounts of specific pony antitoxin. The exotoxin possesses adenosine diphosphate-ribosylation activity. Both cytotoxic and adenosine diphosphate-ribosylation activities are shown to be properties of the intact 66,000-dalton protein. Infection by Pseudomonas aeruginosa is a frequent and serious complication in debilitated patients, particularly those whose phagocytic functions are impaired (29). The cause(s) of the special virulence of Pseudomonas in these situations is unknown. However, one possible source of the pathogenicity of this bacterium is a potent Pseudomonas exotoxin (PE) described by Liu (14). This protein, which was named exotoxin A, was first recognized by its lethality for mice. Subsequent studies showed PE to be cytopathic for a number of cultured cell lines and to inhibit amino acid incorporation and ribonucleic acid synthesis (26). Recently, Iglewski and Kabat (9) presented evidence that PE, like diphtheria exotoxin (DE), catalyzes transfer of the adenosine diphosphate (ADP) ribose portion of nicotinamide adenine dinucleotide to mammalian elongation factor 2 (EF-2). The "ADP-ribosylation" (4) of EF-2 renders it unable to cause translocation of ribosomes along messenger ribonucleic acid during protein synthesis. A study of the role of the exotoxin in infections showed that passive immunization with pony antitoxin protected mice from a lethal challenge with P. aeruginosa (16). This result, and the analogy to diphtheria, suggests that PE may be an important virulence factor and that passive or active immunization against PE might aid in control of human infections. Further studies of the role of this exotoxin in pathogenesis would be aided by the availability

of substantial amounts of purified PE and greater knowledge of its chemical properties. Methods have been described for production and purification of PE, and a few of its chemical properties were reported (3, 17). However, only modest amounts of protein were obtained, and little evidence of the purity of the preparations was provided. Reported here are an improved method for the large-scale purification of PE and some properties of the purified exotoxin. MATERIALS AND METHODS

Bacterial strain. P. aeruginosa strain PA103 was obtained from P. V. Liu. This strain produces very low levels of extracellular protease. Biochemicals. Diphtheria toxin and diphtheria antitoxin were gifts of A. M. Pappenheimer, Jr. Antitoxin to purified PE was prepared in a pony, using the immunization schedule of Liu and Hsieh (16). Nicotinamide adenine dinucleotide labeled uniformly with 14C in the adenosine moiety at a specific activity of 271 MCi/,umol and [1-_4C]iodoacetamide, 60 ACi/,Amol, were purchased from Amer-

sham/Searle (Arlington Heights, Ill).

Fermentation conditions. P. aeruginosa was grown using minor modifications of the culture conditions of Liu (15). The medium was the dialyzable fraction of Trypticase soy broth (Baltimore Biological Laboratories, Cockeysville, Md.) supplemented by addition of 50% (wt/vol) sterile glycerol and 2.0 M monosodium glutamate (filter sterilized) to final concentrations of 1% and 0.05 M, respectively. An inoculum of 100 ml was grown to late log phase in shake flasks and added to 50 liters of medium in a 70-liter fermenter (Fermentation Design, New 1077

1078

INFECT. IMMUN.

LEPPLA

Brunswick, N.J.). The culture was aerated at 24 liters/min, stirred at 400 rpm, and maintained at 32°C. Foaming was controlled by automatic addition of Antifoam 60 (Hartwick, Trenton, N.J.). The pH, which was not regulated, rose from an initial value of 7.4 to a final value of 8.2. After 18 h of growth, the culture was cooled to 5 to 10°C; bacteria were removed by centrifugation with a continuous-flow rotor (Lourdes, Old Bethpage, N.Y.). Exotoxin purification. All purification steps were performed at 5°C. The bacteria-free culture supernatant was diluted to 200 liters with deionized water to decrease the ionic strength. Two liters of diethylaminoethyl (DEAE)-cellulose (Cellex-D, 0.9 meq/g, Bio-Rad Laboratories, Richmond, Calif.) was added, and the suspension was stirred gently for 1 h. The DEAE-cellulose was then allowed to settle, and the supernatant was discarded. The DEAE-cellulose was transferred to a 4.8- by 120-cm column, which was eluted with 3-liter portions of 0.01, 0.05, and 0.10 M NaCl in buffer A [0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 8.1]. Exotoxin was precipitated from the 0.10 M NaCl eluate by addition of solid (NH4)2SO4 to 70% saturation. The precipitate was redissolved and dialyzed against buffer A and then applied to a 1.6by 35-cm (70 ml) column of DEAE-cellulose (Whatman DE-52, Reeve Angel, Clifton, N.J.) equilibrated in buffer A. The column was eluted by a linear gradient of NaCl, 0.01 to 0.33 M, in buffer A (total volume, 1,500 ml). The exotoxin-containing fractions, emerging at 0.12 to 0.14 M NaCl, were adjusted to pH 7.2 with 1.0 M HCl and pumped directly onto a 1.6- by 35-cm (70 ml) column of hydroxylapatite (Bio-Rad Laboratories) equilibrated in 0.005 M sodium phosphate, 0.05 M NaCl, pH 7.0. The column was eluted by a linear gradient of sodium phosphate, 0.005 to 0.10 M in 0.05 M NaCl pH 7.0 (total volume, 1,000 ml). The protein peak emerging at 0.04 to 0.06 M sodium phosphate was concentrated by precipitation with ammonium sulfate, dialyzed against buffer A, and frozen at -700C in small portions. Analytical methods. Protein was measured by a procedure in which samples are precipitated on paper strips and stained with xylene brilliant cyanin G (2). The E21% of PE was determined by measuring nitrogen with a micro-Kjeldahl technique and assuming a nitrogen content of 16%. Neutral sugars were measured with the anthrone reagent (27). Analyses of carbohydrate content by gas chromatography were made by using a modification of the method of Bhatti et al. (1). Amino acid analyses were performed by a combination of methods currently in use in this laboratory (11). N-terminal amino acids were determined by a dansyl chloride method (30). Sulfhydryl groups and disulfides were measured with [14C]iodoacetamide, using modifications of the method of Steinert (28). The ['4C]iodoacetamide was diluted approximately 35fold with nonradioactive iodoacetamide, and the specific activity was determined by alkylation of reduced ribonuclease to be 2.0

x

106 cpm/,umol. For

disulfide determinations, the proteins were first reduced by incubation for 2 h at 1 to 2 mg/ml in buffer

B (9 M urea, 0.05 M Tris-hydrochloride, 0.5 mM ethylenediaminetetraacetic acid [EDTA], pH 8.3) containing 3 mM dithiothreitol. The solutions were then diluted to 200 yg/ml with buffer B containing 1 mM dithiothreitol; ['4C]iodoacetamide was added to a concentration of 2 mM. Samples of 50 gl were removed at intervals, mixed with 20 ,l of 0.10 M cysteine hydrochloride, and transferred to paper disks (Schleicher and Schuell no. 740-E, Keene, N.H.), which were washed as described by Mans and Novelli (20). Experiments using higher concentrations of dithiothreitol or iodoacetamide showed that the conditions described here led to complete reduction and alkylation of ribonuclease and PE. Radioactivity of the disks was measured by liquid scintillation counting in 2.0 ml of Liquifluor-toluene (New England Nuclear Corp., Boston, Mass.). Radial immunodiffusion according- to Mancini et al. (19) was performed in 1.0-mm-thick layers of Noble agar (Difco Laboratories, Detroit, Mich.) containing 7 ,l of pony anti-PE serum per cm2. Rings were measured after 24 h of incubation at 23°C. Double immunodiffusion was done according to the method of Ouchterlony (25). Polyacrylamide gel electrophoresis. Electrophoresis in 8 and 5% polyacrylamide gels containing sodium dodecyl sulfate (SDS) was by a modification of the procedure of Hosada and Cone (8). The gels and lower reservoir contained 0.05 M Tris acetate, 0.1% SDS, and 2 mM EDTA, pH 7.7. The upper reservoir contained the same buffer diluted fivefold. Samples of 40 gl were mixed with 10 ,ul of denaturant (3% SDS, 1% 2-mercaptoethanol, 50% glycerol, 0.05 M Tris acetate, 2 mM EDTA, pH 7.7) and, unless otherwise stated, were heated at 90°C for 3 min. Electrophoresis was performed at 3 mA/tube for 2.5 h at 23°C. Gels were either cut into 2-mm sections or stained with Coomassie brilliant blue. Isoelectric focusing was performed on polyacrylamide gel slabs containing pH 3 to 10 ampholytes (PAG plate, LKB Instruments, Inc., Rockville, Md.), using the instructions provided. Sample application strips were removed after 30 min, and electrophoresis was continued for an additional 60 min. For determination of the isoelectric point (pI) of PE, the pH at intervals on the gel was measured with a surface electrode (Ingold Electrodes, Inc., Lexington, Mass.) prior to staining with Coomassie brilliant blue. Assay of ADP-ribosylation activity. For assay of ADP-ribosylation activity, the general procedure of Collier and Kandel was followed (5). A 35 to 50% ammonium sulfate fraction of rabbit reticulocyte lysate was used as a source of EF-2. Rabbit reticulocyte lysate was prepared by F. B. Abeles. Assays (100-,l total volume) contained about 10 pmol of rabbit reticulocyte EF-2, 37 pmol of ['4C]nicotinamide adenine dinucleotide (0.01 ,uCi), 2 to 200 ng of PE, and 10 Al of a 0.50 M Tris-hydrochloride, 1 mM EDTA, 0.40 M dithiothreitol, pH 8.2, buffer. After incubation for 60 min at 23°C, samples (90 ,l) were placed on paper disks, which were processed as described above for sulfhydryl determinations. For "activation" of PE, 10-gl samples at a,pproximately 100 Ag/ml were mixed with 10 ,l of 10 M

PSEUDOMONAS EXOTOXIN

VOL. 14, 1976

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urea, 100 mM dithiothreitol, 0.03 M EDTA, 0.05 M Tris-hydrochloride, pH 8.1. After 5 min at 23°C, the samples were diluted with 1.5 ml of buffer A and samples were removed for assay. Cell cytotoxicity tests. Tests of the cytotoxic activity of exotoxin were performed in mouse L929 cell cultures using a method developed at this Institute by J. L. Middlebrook and R. Dorland (manuscript in preparation). Mixtures containing 100 1.l of serum (diluted 1:10, 1:30, 1:90, 1:270, etc.) and 2.5 .lI of PE (5 or 50 yg/ml) were incubated for 1 h and then added to wells of 24-well tissue culture plates, which had been seeded 24 h previously with 1.0 ml of medium 199 (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 2.2 g of bicarbonate per liter, 0.1 g of glutamine per liter, 100 U of penicillin per ml, 100 Ag of streptomycin per ml, and

borious. Furthermore, difficulty was often experienced in redissolving the precipitate obtained in the initial zinc acetate step described by Liu et al. (17). Jackson and Matsueda (10) reported that the same problem frequently occurs when zinc acetate is used to precipitate a myxobacter protease. For these reasons other protocols more suitable for large-scale purifications were tested. The low pl (5.0) of the protein (3) suggested that it could be adsorbed from culture supernatants by anion-exchange resins. A protocol starting with adsorption to DEAEcellulose proved successful. Analysis of such a purification is shown in Table 1. Detection and quantitation of PE was conveniently achieved 10% (vol/vol) fetal calf serum and containing 5 x 104 during the purification by radial immunodiffusion in agar gels containing antitoxin. In the mouse L929 fibroblast cells/ml. After incubation for 48 h, the monolayers were washed with a balanced preparation described in Table 1 the toxin-consalt solution to remove disattached cells. Remaining taining fractions obtained at each step were cells were solubilized by addition of 0.5 ml of 0.1 M also assayed for carbohydrate, protein, proteoNaOH per well. Portions were assayed for protein by lytic activity, and ADP-ribosylation activity. an automated Lowry procedure. The culture supernatant contained 5 to 10 ,ug of PE per ml, equal to that reported for cultures RESULTS grown in shake flasks (17). This constitutes 5 to Preparation of exotoxin. PE was produced 10% of the protein secreted by the bacterium. A by growing strain PA103 in a 70-liter fermenter previous report that culture supernatants conusing the medium developed by Liu (15). Ini- tain 30 mg of protein per ml (17) apparently was tially, several 50-liter culture supernatants based on a protein assay that responded to varwere processed by combining the purification ious low-molecular-weight materials. Adsorpprocedure of Liu et al. (17) with chromatogra- tion and elution of PE from DEAE-cellulose phy on hydroxylapatite, which was shown by achieved a 25-fold reduction in volume and a 6Callahan (3) to be an effective purification step. fold increase in specific activity. ChromatograThis protocol produced good yields of pure PE, phy on a DEAE-cellulose column eluted with a portions of which were used to prepare a high- linear gradient of NaCl produced two peaks of titer pony antitoxin, by using the immuniza- ultraviolet-absorbing material. Radial immution schedule of Liu and Hsieh (16). However, nodiffusion showed all the toxin to be in the this combination of steps included repeated cen- first peak, eluted at 0.12 to 0.14 M NaCl. The toxin-containing peak could be applied trifugation of large volumes, which proved laTABLE 1. Purification of P. aeruginosa exotoxin Purification Total vol (ml) stage stage

Total protein' (mg) telna

Total toxin by radial diffu-

~~~siona (mg)

Total toxin Total carbohySp actc drates (mg) tion" (mg)drts(g

by ribosyla-

6 350e 28,000 Culture 5,500