Toxkon, Vol. 17, pp. 467-473.
0041-0101/79!0901-0467302 .00/0
© Pergamon Prass Ltd . 1979 . Printed in Endland .
PURIFICATION OF A CYTOTOXIC PROTEIN FROM PSEUDOMONAS AERUGINOSA F. LuTz Institute of Pharmacology and Toxicology, Justus-Liebig-University, Frankfurter Strasse 107, D-6300 Giessen, West Germany (Accepted jor publication 6 February 1979)
F. Lurz . Purification of a cytotoxic protein from Pseudomonds aeruginosa. Tozicon 17, 46775, 1979 .-A method for preparation of the cytotoxin from Pseudomonds aeruginosa, strain 158, is presented. The procedure described by $CfiARMANN (1976b) was improved by a precipitation step with ammonium sulphate and by subsequent chromatography of the dissolved precipitate on acrylamide-agarose. The recovery of purified toxin was 1 mg from 100 mg protein in supernatant (autolysate). The purity of the preparations was tested by sodium dodecyl sulphate gel electrophoresis, immunodiffusion and enzymatic methods for the presence of other known pseudomonal components. The molecular weight of the toxin was estimated by sodium dodecyl sulphate gel electrophoresis (25,100) and by gel chromatography (22,300-22,800). Amino acid analysis revealed a ratio of acidic to basic amino acids of 4 :1 . Cysteine was absent . The protein contains neither carbohydrate nor lipids . By disc electrophoresis the toxin separated into 4-6 fractions with the same toxicity and with iscelectric points between 5~0 and 6~4. In a slide adhesion test with human granulocytes the En, 0 0 of cell swelling was 91tg/ml. The Eoso for growth inibition of chicken embryo fibroblasts was 11 ltg/ml . At 44 Itg/ml the toxin liberates half of the hemoglobin in rabbit red cells, but did not hemolyse erythrocytes of sheep, horse and cow. The Lasso on mice was 25 rag/g body weight . In postmortem histological examination of mice, fatty liver necrosis was observed . INTRODUCTION
(1976a and b) described a toxin from Pseudomonas aeruginosa with a molecular weight of nearly 30,000. Various batches of the toxin prepared by the original method showed considerable differences in their activity (FRIMMER et a1.,1976b). Impurities (possibly proteases) might have affected the reproducibility of the preparations . In this paper an improved procedure with good reproducibility is described . Additional information about the chemical composition and the biological effects of the toxin is given. SCHABMANN
MATERIALS AND METHODS
Bacteria and growth conditions Pseudomonds aeruginosa, strain 158', was used for the preparation of the toxin. The organism was
originally isolated from bovine milk. The bacteria were stored on tryptic soy agar (Petri dishes) at 4°C after growth for 24 hr at 30°C. For cultivation 1-liter flasks containing 3~0 g of tryptic soy broth and 0~5 g of glucose in 100 ml were inoculated with stored bacteria and incubated for 24 hr at 30°C on a gyratory shaker operating at 180 c/min. About 0~6 ml of this incubation mixture was transferred into 1-liter flasks containing l00 ml of medium and incubated anew for 22 hr . To prepare the medium, flasks containing 100 ml of Cryptic soy broth were presterilized for 15 min at 121°C and 0~5 g of glucose was added. Finally the flasks were autoclaved three times for 30 min at 100°C. Purification of the Pseudomonds aeruginosa toxin (Fig . 1)
Bacteria cultures were centrifuged at 14,000 g for 60 min. The sediment was washed and bacteria resuspended in phosphate buffered saline (10 ml/300 ml culture). The autolysis of the bacteria was performed at 37°C for about 52 hr, rotating at 60 rev/min. The suspension was cooled overnight to 4°C ; this temperature was used for all further isolation steps. The autolysate was centrifuged at 24,000 g for 60 min. To the supernatant a saturated ammonilun sulphate solution was added slowly, with gentle stirring, to a final wncentration of Oß5 M. After 4 hr the precipitate was removed by centrifugation . Secondly, saturated ammonium 'The strain was a gift from Dr. West Germany.
SCHABMANN,
Institute of Bacteriology, Justus-Liebig-University Giessen 467
468
F. LUTZ
sulphate solution was added to a final concentration of 1 ~1 M. After 40 hr the precipitate was extracted with phosphate buffered saline (0~5 ml/300 ml culture) for 10 hr under gentle stimng . This step was repeated. The pooled extracts were further purified by chromatography on an Ultrogel AcA 54 column (Fig . 3) . The leucotoxic fraction (V~/Vo ~" 184) was concentrated on Aquacide (containing equal parts of A-I and A-In and rechromatographed . After dehydration and dialysis against phosphate buffered saline for 6 hr the purified toxin was stored at -30°C. Bramer Phosphate buffered saline, pH 7~4, wntained 0137 M NaCI, 268 mM KCI, 0~5 mM MgClx, 147 mM KH,PO, and 39ß mM Na,HPO,. Cell toxicity Leucotoxic activity was tested on human granulocytes aaording to the method of ScxnxMnrnl(1976a) . The leucotoxic units were referred to a volume of 20 ~1. To examine cell growth, 2 x 106 fibroblasts' from 11-day old chick embryos were incubated with increasing toxin concentrations in 5 ml of Eagle's medium (containing lactalbumin and yeast instead of amino acids). The incubation was performed in 60 mm diameter Petri dishes at 37°C in an atmosphere of air and 10 ~ carbon dioxide. After 48 hr culture supernatant fluids were decanted and the monolayers were washed with phosphate buffered saline to remove nonadherent cells. The adherent cells were detached with versene buffer containing 025 ~ bovine trypsin and aliquots counted in a Cooper Electronics Model ZF cell counter. Hemolytic activity was determined as described by BexxHeinn?R and ScxwnxTZ (1963) with erythrocytes from rabbit, sheep, horse, cow and man. Blood was collected aseptically in an equal amount of Alsever's solution and stored at 4°C for no longer than 1 week .
Biochemical assays Protein was determined by the method of LOWRY et al ., (1951) with bovine serum albumin as the standard. Amino acid analyses were performed with a Biotronic amino acid analyzer LC 6000 after hydrolysis at 110°C with 6 N HCl in vacuo for 24 and 72 hr and drying of hydrolysates under reduced pressure for 3 hr . The trypotophan content was determined by the spectrophotometric method of EnEr xocx (1967) . Total carbohydrate was determined using the tryptophan reaction (BADIN et al., 1953), hexose was determined by means of the anthrone reaction (Sarrr and MELVIN, 1953) and hexosamine was determined using the Elson-Morgan reaction (WINZLER, 195 . Interference due to nonspecific absorption in the hexosamine determination was eliminated by dichromatic readings at 450 nm and 530 nm . The standards were chicken ovalbumin and glucose/galactosamine. Sialic acid was determined (1) by the thiobarbituric acid method (WARREN, 1959) with fibrinogen as standard and (2) by testing the electrophoretic mobility of the toxin before and after incubation with neuraminidase from v?brio comma (EC 3.2.1 .18) (15 :1, w/w), for 40 min. For the carbohydrate analyses the toxin was purified by chromatography on acrylamide gel (Biogel P 30) and concentrated on a Sartorius cellulose-nitrate filter . For determination of lipids, buffer solutions containing 05-1 mg toxin were lyophilized and extracted with 100 ~1 chloroform-methanol (2 :1, v/v) . The extracts were developed on l2 ~m layers of silica gel 60 in a solvent system composed of chloroform-methanol-water (65:25 :4 or 16~25 :8~75 :2, v/v) at room temperature . After drying, spots were made visible by ultraviolet light, exposure to iodine vapours, and spraying with naphthol-sulfuric acid/sulfuric acid . Reference substances were spingomyelin and rra-cephalin in quantities of 1 fig. Protease (elastase) activity was assayed according to the procedure of ScHARMANN and BALICE (1974) with casein (Hammerstein) as substrate. The method was modified as follows: (1) Phosphate buffer, pH 8, was used instead of Tris-2. (2) The incubation temperature was increased to 37°C. (3) The linearity of casein cleavage allowed an increase of incubation time to several hours instead of a few min. Further, purified preparations were examined for the presence of deoxyribonucleate 5'-oligonucleotidohydrolase (EC 3.1 .4.5) (BERGMEYER et al., 1974), ribonucleate 3'-pyrimidino-oligonucleotidohydrolase (EC 3.1 .4.22) (ANSCNSeN et al., 1954), triacylglycerol acylhydrolase (EC 3.1 .1 .3) (ScHnanr et al ., 1974), lecithirrase with and without Cat+ (MARINE'1TI, 1965), and orthophosphoricmonoester phosphohydrolase (EC 3.1 .3 .1) (CoxN and HmnscH( 1960). For these methods toxin concentrations of approx . 50-800 ~g/ml in phosphate buffered saline or in 015 M Tris-2, pH 7~4, were tested using incubation periods up to 2 hr. Immunodiffusion
Diffusion precipitation reactions with anti-exotoxin A from sheept as well as Staphylococcus anti-a hemolysin (Wellcome) were performed using micro slides precoated with 1 ~ agarose in phosphate buffered saline . The reaction was allowed to proceed at room temperature for 24 hr in a humidified chamber. 'The fibroblasts were kindly provided by the Institute of Virology, Justus-Liebig-University, Giessen, West Germany. tAnti~acotoxin A waskindly provided by Mrs. BARBARA H. IctEwsxr, Health Sciences Center, University of Oregon, Portland, OR, U.S .A . We could also test the reaction with pony anti-exotoxin A, provided by Mr. S. H. LEeera, U.S . Army Medical Research Institute of Infectious Diseases, Fort Derrick, Frederick, Maryland, U.S .A .
Pseudfomotws aerugirrosa Tozin
469
Molecular sieve chromatography
Separation of the cytotoxin was attempted with DEAF-cellulose (2~5 x 4 cm column) as well as Sephadex, Sepharose, Biogel and Ultrogel (most column dimensions were 1~4 x 180 cm .) For elution on DEAE the gradient contained NaCI up to 1 M. Each fraction was analyzed for protein, elastase activity and leucotoxic activity. Electrophoreses
For sodium dodecyl sulphate polyacrylamide gel electrophoresis the method of LAMMLd was used with a modification as described earlier (GL03.4MANN and Lvrz, 1970). Disc-electrophoresis in polyacrylamide was carried out as described by Dnvts (1964) . The staining was performed according to the method of FntRanxKs et al., (1971) with the exception that methanol was used instead of isopropanol . Isoelectric focusing
Tscelectric focusing was performed in vertical slabs (12 x 14 x 0~3 cm) at room temperature (O'F~xRei.L ., 1975). The aqueous gel solution contained 4~7 ~ (w/v) acrylamide, 0~2~ (w/v) bisacrylamide, 001 ~ (w/v) ammonium persulphate, 0'07 ~ (v/v) N,N,N',N'-tetramethylenediamine, 2~ (v/v) ampholine (pH 3-10,4-6, 6-8 = 1 :5 :5) and 9~2 M urea. The overlay solution contained 4 M urea and 1 ~ (v/v) ampholine. After 4 hr the overlay solution was decanted and 100 ~1 of sample containing 9~5 M urea was applied followed by 100 ~1 of overlay solution . The current was initially 25 mA and was changed to 5 mA after 14 hr . After focusing, the gels were washed with 10% trichloroacetic acid (w/v) for one day to remove the ampholine and then stained as previously . The pH values were estimated after elution of the bands with distilled water. RESULTS
Pur~cation of the Pseudomonas aeruginosa toxin
The purification procedure is outlined in Fig. 1 . The supernatant fluid of the bacterial autolysate revealed about 25 protein bands when subjected to sodium dodecyl sulphate gel electrophoresis (Fig. 2). Storage and addition of low ammonium sulphate concentrations (up to 035 M) removed about one tenth of the protein content in the supernatant Fig .l : Purification scheme of the Pseudomonas aeruginosa toxin
Culture Autolyais of bacteria in phosphate buffered saline at 37 ° C for 52 hr Autolysate supernatant Ammonium sulphate fractionation (0 .35 M for 4 hr and 1 .1 M for 40 hr) 1 .1 M ammonium sulphate precipitate Repeated suspension in phosphate buffered saline Saline soluble fraction Chramatography on Ultrogel AcA 54 Leucotoxic fraction (V e /VO +K 1 .84) Rechroanatography on Ultrogel AcA 54 Dialysis against saline Purified toxin
470 0~
F . LUTZ
n
-Ovalbumin dimer -Bovine serum albumin -Ovalbumin
0,6 0
0,8 -{
Trypsin _ Lottoglobulin =Myoglobin from horse Dye front
1,0 ~
1 FIG .
2.
2
3
~
5
6
7
8
9
SODIUM DODECYL SULPHATE POLYACRYLAMmE GEL ELECTROPHORESIS OF PREPARATIONS OBTAINED DURING PURIFICATION OF THE Pseudomonas aeruginosa Toxnv .
Electrophoresis was performed using 10 % acrylamide and 0~1 ~ sodium dodecyl sulphate . The samples were heat treated (95°C) for 3 min in sample buffer prior to electrophoresis . The gels were electrophoresed at 3 mA/gel for 30 min and then at 6 mA/gel for 60 min. Bovine serum albumin (68,000), ovalbumin (45,000), trypsin from bovine pancreas (23,800) and ß-lactoglobulin (18,200)/myoglobulin from horse (17,800) were used as molecular weight (Mr) markers . (1) P . aeruginosa ; (2) Supernatant fluid of bacterial autolysate ; (3) Precipitate of supernatant autolysate after cooling overnight ; (4) Precipitate of supernatant autolysate after fractionation with 035 M ammonium sulphate ; (5) Precipitate of supernatant autolysate after fractionation between 035 and 1 ~ I M ammonium sulphate ; (6) Phosphate buffered saline soluble fraction of the 035-1~1 M ammonium sulphate precipitate (Ist extraction) ; (7) Phosphate buffered saline soluble fraction of the 0~3~1 ~ 1 M ammonium sulphate precipitate (2nd extraction) ; (8) Phosphate buffered saline insoluble fraction of the 035-1 ~ 1 M ammonium sulphate pecipitate ; (9) Purified toxin after rechromatography on Ultrogel AcA 54 . autolysate fluid with negligible leucotoxic activity . Fractionation between 035 and l~l M ammonium sulphate concentration precipitated 6 % of the total protein and nearly all of the leucotoxic activity . An increased solubility of the toxin after precipitation, a decrease to 1/5 of the protease impurities and a decrease of the concentration of precipitant from 1~4 to 1~1 M was observed when the ammonium sulphate precipitation was prolonged to 40 hr . The soluble protein material recovered from the l~l M ammonium sulphate precipitate showed 8-12 Coomassie blue stainable bands in sodium dodecyl sulphate gel electrophoresis . The material was chromatographed on DEAE-cellulose, Sephadex, Sephacryl, Biogel or acrylamide-agarose (Ultrogel) columns . None of these methods was completely satisfactory, although Ultrogel-AcA 54 columns gave the best separation profile. A typical example is shown in Fig. 3 . The leucotoxic activity constituting the main peak is overlapped both by a preceding protein fraction and by the following elastase . After rechromatography the leucotoxic activity was uniform as shown by sodium dodecyl sulphate gel electrophoresis. The results of the purification scheme are summarized in Table 1 . Approximately 3~5 mg of the pure toxin were obtained from a preparation batch of 330 mg protein . In the purified toxin preparations, protease (elastase), ribonuclease, deoxyribonuclease, lipase, lecithinase and alkaline phosphatase activities were not detected . The purified toxin showed no precipitation line in gel-diffusion with anti-exotoxin A and staphylococcus anti-a hemolysin .
471
Pseudomonas aeruginosa Toxin Molecular weight of reference proteins 21000 12 300 68 000 45000 -1
ry, r,rd~ r~ i ~~ r-g Fractions
Leucotoxic activity
Protein (mg/ml) 0.3
.1.~1 / m.l .l.. . . ..
-6 400 -1 600
0 .2-
- 400 - 100 - 0
0~
FIG. 3. FIRST COLUMN GEL FILTRATION OF PARTLY PURIFIED PSelfdOilIOpaS aerfIgiROSa TOXIN AFTER PRECIPITATION WITH O'3S-I'1 M AMMONIUM SULPHATE ON ACRYLAMIDE-AGAROSE . Max. 8 ml were applied to an Ultrogel AcA 54 column (2 "5 x 130 cm) equilibrated with phos-
phate buffered saline. The flow rate was 12 ml/hr (3 ml tubes) . Efliuent was analyzed for protein ( ), leucotoxic activity (. . . . . . . . ) and elastase (---) . Bovine serum albumin (68,000), ovalbumin (45,000), trypsin inhibitor from soybean (21,000) and cytochrome c from equine heart (12,300) were used as reference proteins. Tubes of fraction d were combined, dehydrated and rechromatographed. TABLE 1 . PURIFICATION OF TOXIN FROM
Stage
Protein Volume Conc . Total Ono (mg/rnl) (rug)
Srlpernatant fluid~of bacterial autolysate 26 Dissolved amonium sulphate precipitate 1st extract' 1 "6 2nd extract l'6 1 st Ultrogel AcA S4 filtration pooled fraction d$ 4S concentrated fraction d§ 32 2nd Ultrogel AcA S4 filtration pooled fraction d 47 concentrated and dialyzed fraction dll 4'6
12'7
Pseudomonas aeruginosa
Yield ( ~)
330
Activity (U/ml)
Leucotoxic activity Total Yield Spec . act. (U) (~) (U/mg)
3200
83,300
250
18,800 21,900
23 26
3300
3800
4'1 3'7
6"6 S'9
2~0 l'8
11,800 13,700
0~12 I "4
5"4 4v
l'6 l'4
44(1 5400
20,000 17,300
24 21
0~09
4'2
1"3
320
14,900
18
0'S
3'S
l'1
3000
13,700
16
aôiaa
3900
'First extract means the soluble part of 0'35-1'1 M ammonium sulphate precipitate during first uptake in phosphate buffered saline for 10 hr. tSecond extract means the soluble part of 0'35-1'1 M ammonium sulphate precipitate during second uptake in phosphate buffered saline for 10 hr . $Pooled fraction d is the leucotoxic fraction of the eluate of gel filtration on acrylamide-agarose (as shown in Fig. 3) . §Concentrated fraction d is the pooled fraction d after dehydration of Aquacide. CConcentrated and dialyzed fraction d is the leucotoxic fraction from the second gel filtration on acrylamidc,agarose after dehydration on Aquacide and dialysis against phosphate buffered saline .
47 2
F. LUTZ
Characterization of the Pseudomonas aeruginosa toxin
Molecular weight determinations : Ultrogel AcA 54 gel filtration gave an average VelVo of 184 (13 experiments) which corresponds to an apparent molecular weight of 22,800 . Biogel P 30 gel filtration gave an average VelVo of 147, which corresponds to an apparent molecular weight of 22,300 (nine experiments) . Sodium dodecyl sulphate gel electrophoresis of the denatured toxin showed an apparent molecular weight of 25,100 (eight experiments) . Chemical composition: Table 2 shows the results of studies on the composition of the toxin. The molecular weight calculated from the amino acid content is 23,100. The ratio of acidic to basic amino acids is 49 :12. The prevalence of acidic residues is in agreement with its behavior in isoelectric focusing. The percentage of hydrophobic amino acids is low and only traces of cysteine were found . The content of carbohydrate was very small and no lipids were detected . Surface charges: upon disc-electrophoresis on polyacrylamide gel without sodium dodecyl sulphate, SCHARMANN (19766) found five bands of `leucocidin' . In our experiments the number of protein bands was from 4 to 6 depending on the preparation batch (not T~,aLE
2.
Pseudomonas aeruginosa Toxm
C~3EMICAL COMPOSITION OF
Amino acid analysis Amino acid Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Trp Total residues (Formula weight) Basic: acidic Carbohydrate analysis Sugar Total Hexose Hexosamine 5ialic acid
Molar ratios" Relative molar Probable No . of quantities residues per molecule 229 23 176 18 200 20 261 26 5~1 5 21 .8 22 129 13 0~ 1 t 0 20~ 1 20 2 1~9 9 8~7 14 14 .1 8 8~4 7 6~9 8 7~7 5~1 5 4~1 4 7~9$ 8 2l2 (23,100) 12 : 49
Content§ (ßg/100 hg protein) 014 (0-027) 007 (0-019) 0~1 (0-023) 001 (0-0~0~
"The values represent an average of three determinations . The data were corrected for hydrolytic destruction of serine and threonine and slow release of valine, leucine and isoleucine on the basis of results from 24- and 72-hr analyses. No detectable amounts of unusual amino acids were found. $Cysteine was found in one determination only as trace. $Tryptophan was determined spectrophotometrically. §The values represent an average of five determinations. The range of all values is given in parenthesis.
473
Pseudomonas aen~ginosa Toxin Number of adherent cells
6
8x10 -
6
4x10 -
2x1o 6-
° 0
hko . 4 . Exrrcr
Ô
of
1
3
10
Ng/ml
Cdncentration of cytotoxin
°
30
Pseudomonas aert~ginosa ro~m~t ox cer~, aaow~rx or c>~cxL~v FIBROHLASIS .
EI~RYO
The different symbols represent experiments with four different batches of the toxin. Each point represents one Petri dish. For further details see Materials and Methods section .
shown) . On the basis of protein content, estimated by Coomassie blue-staining of the gel slabs and by the Folin reaction after elution from the gel, the leucotoxic activity of the different bands was similar. Different isoelectric points between 5~0 and 6~4 were obtained for each of the different toxin species. Stability : The activity of the toxin during storage in phosphate buffered saline at - 30°C decreased at a rate of about 3 ~ monthly. Biological activity of the Pseudomonas
aeroginosa toxin The toxicity of the isolated protein to human granulocytes was standardized by estimating the changes in cells 1 hr after application. Average preparations needed 9 ~ 3 ~g toxin per ml (mean ~ S.D.) to swell all polymorphonuclear leucocytes in a field containing several thousand cells (12 experiments) . At 3 ~g toxin per ml most of the cells showed pseudopodia. Growth of isolated chicken embryo fibroblasts was inhibited by the toxin in a dosedependent manner (Fig. 4). Fifty per cent inhibition of cell growth was obtained at 11 f 3 ~g toxin per ml medium. For the release of 50 % of hemoglobin from rabbit erythrocytes a concentration of 44 ~ 9 ~g toxin per ml was needed (five experiments) . For human erythrocytes the concentration causing 50 ~ hemolytis was estimated to be nearly 1 mg toxin/ml. No hemolytic activity of the toxin could be detected using erythrocytes from sheep, horse or cow at concentrations up to 0~9 mg per ml. Both hemolytic and leucotoxic activity of the toxin were destroyed totally by heating to 95°C for 15-30 min at pH 7'4 or pH 3, or by treatment with three volumes of ethanol at -5°C. The Lasso to mice (Haas-NMRI weighing about 30 g) was about 25 ng/g body weight after i.v. injection of 0~1 m.l volumes of diluted toxin. Time until death was between 18 and 46 hr at Lasso and shortened to 0'S-3 hr after injection of 2 ~g per g body weight. Dishevelling offur appeared a few hr after application of lethal doses of the toxin. A fatty degeneration of the liver was seen in postmortem histological examination. DISCUSSION
The molecular weight of 25,100 in sodium dodecyl sulphate gel electrophoresis for the
474
F. LUTZ
toxin prepared by the modified purification method was slightly less than that of the toxin lSOlated by SCHARIYIANN (19766), which was estimated at 27,500. Direct comparison* by this method showed that the difference is real. The conditions under which the toxin is released from bacteria may be responsible for this difference. The fragments arising by autolysis might each contain the toxic part of the sequence but differ otherwise from each other. Therefore both molecular weight and surface charge may be variable. In our experiments the number of charged species of the Pseudomonas toxin varied from four to six depending upon preparation batch, whereas the leucotoxic activites of these fractions were the same. Sodium dodecyl sulphate gel electrophoresis had shown that isolated toxin behaved as a homogenous moiety . Nevertheless the purified preparation could be contaminated by proteins which could not be detected. Fatty liver degeneration was found in mice after application ofthe toxin . Exotoxin A acts similarly to diphtheria toxin which has been shown to cause fatty liver degeneration (SOLOTOROVSKY and .TOHNSON, 1970). Contamination of the purified toxin preparation with exotoxin A or its cleavage products is unlikely because first, the data from amino acid composition show no apparent similarities to that of exotoxin A which has an arginine aysine ratio of 3 :1 and seven halfcysteine residues per molecule (LEPPLA, 1976) . Secondly, purified toxin as well as autolysate supernatant fluid failed to react with anti-exotoxin A in gel-diffusion . A low hemolytic activity of the toxin on rabbit and human erythrocytes was found, in basic agreementwith the reSUItS OÎ SCHARMANN (19766) . This activity was neither heat stable nor stable in ethanol as shown for hemolysin of P. aeruginosa (Llu et al., 1961 ; BERK, 1964) . Activities of enzymes causing hemolytis (Liu et a1., 1961) were not detected at the purified toxin preparation . Furthermore, since the hemolytic activity increased during the preparation procedure parallel to the leucotoxic activity (i.e. 20-fold increase by ammonium sulphate precipitation of autolysate supernatant fluid, and 1 ~8-fold increase by gel filtration on acrylamide-agarose), it is probable that this hemolytic activity is associated with the toxin molecule . SCHARMANN (1976a, b) named the toxin `leucocidin' after experiments using leucocytes . Staphylococcal leucocidin is toxic only to polymorphonuclear leucocytes and macrophages of rabbit, man and mice (Woontx, 1970; BLOB&h et a1., 1973), whereas the toxin from Pseudomonas aerugittosa is toxic to all cells isolated from parenchyma, their cell lines in cultures and tumor cells except for thrombocytes (SCHARMANN, 19766 ; FRIMMER et al., 19766) . The cytopathogenic effect was similar in all tests. The toxin causes cardiovascular failure in mice and rats (FRIMMER et al., 1976a) . Pseudomonas toxin and leucocidin of Staphylococcus show, however, important differences. Staphylococcus leucocidin consists of two different proteins and is only toxic when both components are present, whereas differently charged species of Pseudomonas toxin are toxic as single fractions. Acknowledgements-This work is partly supported by the Deutsche Forschungsgemeinschaft . I am grateful for the technical assistance of Mr . K. SrUMPF and Mrs. D. ESL in performing the amino acid analysis . REFERENCES ANFIN$EN, C. B., REDFIELD, R. R., CHDATE, W, L PAGE, J. and CARROL, VV . R. (1954) Studies on the grOSS structure, cross-linkages and terminal sequences in ribonuelease . J. biol, Chem . 207, 201. BADIN, J., JACK90N, C. and SCHUBERT, M. (1953) Improved method for determination of plasma polysaccharides with tryptophan . Proc . Soc. exp, Blot . Med. 84, 288. HERGMEYER, H. U., GAWEHN, K. and Gt[nssL, M. (1974) Enzyme als biochemische Reagenticn, fn : ,Metlrnderr der enzymatischen Analyse, p. 454, (BERGNIEYER, H. U,, Ed .). Weinheim : Bergstr. Verlag Chemie . *Dr. SCHARMANN kindly provided Pseudomonas toxin.
Pseudomonas aeruginosa Toxin BERK, R. S.
559.
47 5
(1964) Partial purification of the extracellular hemolysin of Pseudomonas aeruginosa. J. Bact . 88,
A. W. and SCHWARTZ, L. L. (1963) Isolation and composition of Staphylococeus alpha toxin. J. gen. Microbiol. 30, 455. BLOBEL, H., SCHAEG, W. and SosoLL, H. (1973) Current status of staphylococcal leucocidin research . Contributions to Microbiol. Immunology 1, 359. CoxN, Z. A. and HIRSCH, J . G. (1960) The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. J. exp. Med. 112, 983. DAMS, B. J. (]964) Disc electrophoresis-II . Method and application to human serum proteins . Ann, N.Y . Acad . Sct. 121, 404. EDELHOCH, H. (1967) Spectroscopic determination of tryptophan and tyrosine in proteins . Biochemistry 6, 1948 . FAIRBANKS, G ., STECK, T. L. and WALLACH, D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane . Biochemistry 10, 2606. FRIMMER, M., NEUHOF, H., SCHARMANN, W. and ScruscrtKe, B. (1976x) Cardiovascular reactions induced by leucocidin from Pseudomonas aeruginosa . Naurryre-Schmiedebergs Arch. exp. Path. Pharmak. 294, 85. FRIMMER, M., HOMANN, J., PETZINCER, E., RUFEGER, U. and SCHARMANN, W. (]976b) Comparative studies on isolated rat hepatocytes and AS-30D hepatoma cells with leucocidin from Pseudomonas aeruginosa . Narrnyn-Schmiedebergs Arch. exp. Path . Pharmak. 295, 63 . GLOSSMAN, H. and Llrrz, F. (1970) Molecular weights of pig liver cell membrane proteins . Hoppe-Seyler's Z. physlol. Chem . 351, 1583 . LEPPLA, S. H. (1976) Large-scale purification and characterization of the exotoxin of Pseudomonas aeruginosa. Infect . Immrrn . 14, 1077 . LIU, P. V., AsE, Y. and BATES, J. L. (1961) The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis . J. infect . Dis. 108, 218. Lowxv, O. H., ROSENBROUGH, N. J., FARR, A. L. and RANOwLL, R . J. (1951) Protein measurement with the Folin phenol reagent. J. biol . Chem . 193, 265. MARINETTI, G. V. (1965) The action of phospholipase A on lipoproteins . Biochim. biophys . Acta 98, 554. O'FARRELL, P. H. (1975) High resolution two-dimensional electrophoresis of proteins . J. biol . Chem . 250, 4007 . SCHARMANN, W. (1976x) Formation and isolation of leucocidin from Pseudomonas aeruginosa. J. gen. Microbiol. 93, 283. SCHARMANN, W. (19766) Purification and characterization of leucocidin from Pseudomonas aeruginosa. J. gen. Microbiol. 93, 292. SCHARMANN, W. and BALKE, E. (1974) Untersuchungen Iiber eine Protease (Elastase) von Pseudomonas aeruginosa . 1. Bildung and Reinigung der Protease . Hoppe-Seyler's Z. physiol. Chem . 355, 443. SCHM>DT, F. H., STORK, H. and voN DAHL, K. (1974) Lipase-photometrische Messung. In : Methoden der enzymatischen Analyse, p. 848, (BERGMEYER, H. U., Ed.). Weinheim :Bergstr . Verlag Chemie. SCOTT, T. A., JR . and MELVIN, E. H. (1953) Determination of dextran with anthrone . Analyt . Chem . 25, 1656 . SOLOTOROVSKY, M. and JOHNSON, W. (1970) Tissue culture and bacterial protein toxins. In : Microbial Toxins, Vol. 1, p. 277, (AIL, S. J., KADIS, S. and MOrrrtE, T. C., Eds.). New York : Academic Press. WARREN, L. (1959) The thiobarbituric acid assay for sialic acids. J. biol . Chem. 234, 1971 . WINZLER, R. J. (1955) Determination of serum glycoproteins. In : Methods of Biochemical Analysis, Vol. 2, p. 279, (CLICK, G., Ed .) . New York : Interscience Publications. Woonnv, A. M. (1970) Staphylococcal leucocidin . In : Microbial Toxins, Vol. 3, p. 327, (Montie, T. C., Kwnls, S., and AIL, S. J., Eds.) . New York : Academic Press.
BERNHEIMER,