Vol. 24, No. 2
INFECTION AND IMMUNITY, May 1979, p. 394-398 0019-9567/79/05-0394/05$02.00/0
Inhibitory Effect of Pseudomonas aeruginosa on the Phagacytic and Killing Activities of Rabbit Polymorphonuclear Leukocytes: Purification and Characterization of an Inhibitor of Polymorphonuclear Leukocyte Function SHIGEO NONOYAMA,' HITOSHI KOJO,' YASUHIRO MINE,' MINORU NISHIDA,' * SACHIKO GOTO,2 AND SHOGO KUWAHARA2 Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan,' and Department of Microbiology, Toho Univerisity School of Medicine, Tokyo, Japan2
Received for publication
A clinically isolated strain of polymorphonuclear leukocyte (PMN)-resistant Pseudomonas was found to produce an extracellular substance (PMN inhibitor) that inhibits the phagocytic and killing activities of PMN. The PMN inhibitor was purified from culture filtrates by precipitation with (NH4)2SO4 and chromatography on phosphocellulose, diethylaminoethyl-cellulose, and Sephadex G-100. The preparation was homogenous as judged by disc gel electrophoresis. The purified PMN inhibitor appeared to be a protein with a molecular weight of approximately 65,000 that was inactivated by heating and by exposure to a proteolytic enzyme. It has been demonstrated that extracellular substances of Pseudomonas aeruginosa such as protease, elastase, collagenase, lecithinase, hemolysin, exotoxin, and enterotoxin play an important role in the pathogenicity of P. aeruginosa infections (4, 9, 10, 11, 13, 16, 29). Phagocytosis plays an important role in host defense against bacterial infection. During an investigation of the interactions of rabbit polymorphonuclear leukocytes (PMN) and P. aeruginosa, we encountered two different types of strains (18). These are resistant or susceptible to the phagocytic and killing activities of PMN, and the strains are found to be virulent and avirulent, respectively, as determined by the lethal dose values for mouse infection. In our preparatory study, of 15 clinically isolated P. aeruginosa tested, PMN-resistant strains are detected at comparatively high frequency (60%). PMN-resistant strains are found to produce a high-molecular-weight substance which inhibits the phagocytic and killing activities of the PMN. The present paper describes the purification and biochemical properties of the PMN inhibitor isolated from culture filtrates of PMN-resistant Pseudomonas. MATERIALS AND METHODS Organisms. P. aeruginosa strain 7005 and Escherichia coli strain 303 were used which were originally isolated from clinical sources. P. aeruginosa strain
7005 was a virulent, PMN-resistant strain producing protease, elastase, melanin, pyocyanine, and fluorescein. E. coli strain 303 was a PMN-sensitive strain. Purification of the PMN inhibitor. P. aeruginosa strain 7005 was cultivated in Trypticase soy broth (Baltimore Biological Laboratory) at 370C for 20 h to stationary phase. Cells were removed by centrifuging at 16,400 x g for 30 min, and the resulting supernatant fluid was passed through a filter (Millipore Corp.; pore size, 0.45 ,um). A 1.5-liter amount of the culture supernatant was precipitated with (NH4)2SO4 to 90% saturation. The precipitate was collected by centrifuging at 16,400 x g for 30 min and was dissolved in 100 ml of 0.01 M tris(hydroxymethyl)aminomethane (Tris) buffer containing 5% glycerol (Ishizu Pharmaceuticals, Ltd., Osaka, Japan) (pH 7.0) and dialyzed against the same buffer at 4°C for 20 h. The dialysate (148 ml) was applied to a column (1.0 by 10 cm) of phosphocellulose and then to diethylaminoethyl (DEAE)-cellulose (1.0 by 10 cm) equilibrated with the same buffer. In DEAE-cellulose chromatography a linear gradient of 0 to 0.8 M KC1 in the same buffer was used. The fractions from DEAE chromatography containing the PMN inhibitor (30 ml) were concentrated to 2 ml, and the solution was applied to a column (2.5 by 86 cm) of Sephadex G-100 equilibrated with the same buffer and eluted with the same buffer. Preparation of protease and elastase. The culture filtrate was precipitated with (NH4)2SO4, and the resulting precipitate was dissolved in 0.01 M Tris buffer (pH 7.0) and dialyzed against the same buffer. The dialysate was applied directly to a column of DEAE-cellulose, omitting the phosphocellulose chromatography step. Elution was effected under the same
394
VOL. 24, 1979
PURIFICATION OF P. AERUGINOSA EXOTOXIN
395
conditions used for purification of the PMN inhibitor. Polyacrylamide gel disc electrophoresis. Polyacrylamide gel disc electrophoresis was carried out as described by Davis (5). The PMN inhibitor was mixed with the sample gel and placed in top of the gel. Electrophoresis was performed at a constant current of 2 mA/tube for about 5 h at 200C. The gel was stained with amino black and then destained. Determination of molecular weight. The molecular weight of the PMN inhibitor was estimated by gel filtration on Sephadex G-100 column chromatography (1), which was equilibrated with 0.01 M Tris buffer containing 0.5 M KCI (pH 7.0). The molecular weight of the PMN inhibitor was compared with proteins of known molecular weights: rabbit serum albumin (69,000 molecular weight), human serum albumin (60,000 molecular weight), pyruvate kinase (57,000 molecular weight), leucin aminopeptidase (53,000 molecular weight), and egg albumin (45,000 molecular weight). Moreover, the molecular weight was determined essentially by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Weber (30). Slab gels were prepared by the method of Studier (27) and stained with Coomassie brilliant blue. Assay of protease and elastase activities. Protease activity was measured by the casein digestion method essentially as described by Johnson (7) and Morihara (17). Elastase activity was assayed by the method described by Sbarra (21). Assay of the PMN inhibitor. PMN were obtained by the method previously described (18, 19). PMN inhibitor activity was assayed by measuring the inhibition of the phagocytic and killing activities of PMN against E. coli strain 303. PMN (8 x 106 cells per ml) were shaken with E. coli strain 303 (106 colony-forming units [CFU] per ml) at 370 for 4 h. The mixture was centrifuged at 2,100 x g for 20 min to sediment broth PMN and E. coli. PMN were then selectively lysed by adding sterile distilled water, and surviving E. coli were determined by plating (18).
trates by precipitation with (NH4)2SO4 and chromatography on phosphocellulose, DEAE-cellulose, and Sephadex G-100. Figure 1 shows the elution pattern of the PMN inhibitor from the phosphocellulose column. The PMN inhibitor was present in the passage through fractions with 0.01 M Tris buffer containing 5% glycerol (pH 7.0) as eluent. The fractions containing the PMN inhibitor from phosphocellulose chromatography were applied to the column of DEAE-cellulose (Fig. 2). Two major peaks of 280-nm absorbing material were eluted with KCI at about 0.2 and 0.3 M. PMNinhibiting activity was associated with the 0.4 M KCI eluate. Further purification of the PMN inhibitor was carried out by column chromatography on Sephadex G-100 (Fig. 3). The major peak of ultraviolet-absorbing material was found in tube no. 15 in the void volume, and a small peak was found in tube no. 20. The activity of the PMN inhibitor was associated with the latter peak. Polyacrylamide gel electrophoresis of the PMN inhibitor obtained by gel filtration on Sephadex G-100 showed a single band which corresponds to the fastest moving band among the bands obtained the original crude culture filtrates. Separation of the PMN inhibitor from protease and elastase. The elution patterns on the PMN inhibitor, protease, and elastase by ion-exchange chromatography on DEAE-cellulose are shown in Fig. 4. Ten peaks of 280-nm absorbing material were obtained, of which five fractions had different biological activities. Fractions 1 and 2, which passed through the column with Tris buffer, showed only proteolytic and elastolytic activiRESULTS ties, respectively. Fractions 3 and 4, which were Purification of the PMN inhibitor. The obtained by gradient elution, showed only proPMN inhibitor was purified from culture fil- teolytic activity, and fraction 5, eluted with 0.4 INHIBITORY ACTIVITY
1. 0
0-0 UV ABSORBANCE(A 280) 0
0
>4
co Co
H
B
0
H
8 Ed-4
H
w
u z
H
7X w
9c
az
0:
0
En us
H
4
6 H-
-4
0.5
9 z
0 U
z H
0
10
20
30
5s
[0 X
FRACTION NUMBER
FIG. 1. Phosphocellulose column chromatography of the PMN inhibitor. The dialysis residue (148 ml) was applied to an 8-ml bed volume of phosphocellulose in 0.01 M Tris buffer containing 5% glycerol (pH 7.0). The ionic strength increased linearly in the elation buffer system; 0 to 1.0 M KCl. Fractions (10 ml) were collected and assayed for PMN inhibitor. The arrow indicates where gradient elation was initiated.
396
INFECT. IMMUN.
NONOYAMA ET AL. z
INHIBITORY ACTIVITY 0-0 UV ABSORBANCE(A 280)
0
co u
>4 H
H
8 :> H
1.5
U
0.8 z 0 H Ez
7 ,S, U
z;1.0
o
H
0
H
E
-
tU
o H 4 6 m
0.5
I
0
H z
CD
0.4 U z 0
u
H .
10 20 30 FRACTION NUMBER
0
0
40
FIG. 2. DEAE-cellulose column chromatography of the PMN inhibitor. The PMN inhibitor (60 ml) eluted from phosphocellulose was placed on a cellulose column (1.0 by 10 cm) equilibrated with 0.01 M Tris buffer containing 5% glycerol (pH 7.0). The ionic strength increased linearly as indicated. Fractions (8 ml) were collected and assayed for the PMN inhibitor. The arrow indicates where gradient elution was initiated.
0-O
UV ABSORBANCE (A 280) 0
E-4
> 5 H
).
2:S c~J
5
cD D
z
m
W.1E
H
U4 EH, Ho H
).1
CO
-
z
H
0
10
20
30
FRACTION NUMBER
FIG. 3. Sephadex G-l OO gel filtration on the PMN inhibitor. The PMN inhibitor (2 ml), which was purified through the step of DEAE-cellulose chromatography and concentrated from 30 ml of fractions, was chromatographed on a Sephadex G-100 column (2.5 by 86 cm) in 0.01 M Tris buffer containing 5% glycerol (pH 7.0). The flow rate was 15 ml/h, and fractions of 10 ml each were collected. 3 C..
0.8
.0.8
.6
u
-
C,
01-
:>E-4
0 I: H
H
Ez
>4
U
H
H 0. 4
9c
4
W
o U H 0 U
0
0.2
9
zb zZ
E-4 0
U) uz
H
0.4
H
B
o aZ:
z
E-i
4
0
Pr
C.)_
E4
0.6 CN
C.)
FRACTION NUMBER
FIG. 4. Preparation of protease and elastase with a column of DEAE-cellulose. The dialysis residue (20 ml) was placed on a cellulose column (1.0 by 10 cm) equilibrated with 0.01 M Tris buffer containing 5% glycerol (pH 7.0). The ionic strength increased linearly in the elution buffer system; 0 to 0.8 M KCl. Fractions (6 ml) were collected and assayed for proteolytic enzyme, elastolytic enzyme, and PMN inhibitor. The arrow indicates where gradient elution was initiated.
VOL. 24, 1979
PURIFICATION OF P. AERUGINOSA EXOTOXIN
M KCl, showed the activity of the PMN inhibitor. This indicated that the PMN inhibitor was a separate extracellular product from protease and elastase. Biochemical properties ofthe PMN inhibitor. In these experiments, the 30 ml of the PMN inhibitor which was separated from 1.5 liters of culture filtrate and purified by gel filtration on Sephadex G-100 column chromatography was used. This purified PMN inhibitor had an absorbance at 280 nm of 0.035. In the absence of the PMN inhibitor, the viable cell counts of E. coli strain 303 decreased from 106 to 6 x 102 CFU/ml after 4 h of incubation in the assay for phagocytosis and killing. In the presence of PMN inhibitor, the viable cell count of E. coli strain 303 did not decrease (3 x 105 CFU/ml). This PMN inhibitor was used at the original concentration yielded by the purification procedure and was referred to as 35 Am0/mi. The biochemical properties of the PMN inhibitor are as follows. The molecular weight of the PMN inhibitor was estimated to be about 65,000 by gel filtration on Sephadex G-100. The PMN inhibitor preparation contained two protein components of molecular weights of about 66,000 and 9,000 when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The effect of heat treatment on the stability of the PMN inhibitor was tested. The PMN inhibitor was treated at four different temperatures (37, 50, 70, 10000) for 30 min (pH 7.4). The activity of the PMN inhibitor was lost rapidly as temperature increased. After exposure of the PMN inhibitor solution to five different pH values at 370C for 2 h, the pH's of the solutions were adjusted to 7.2 with 0.1 N NaOH or 0.1 N HCl, and the remaining activity of the PMN inhibitor was measured. Treatment of the PMN inhibitor at pH values from 5 to 9 had little effect on its activity, but exposure to pH 3 and 11 caused loss of activity. The effect of proteolytic enzyme on the stability of the PMN inhibitor was examined (Fig. 5); the protease used (51 U) was separated by chromatography on DEAE-cellulose (fraction 3). Under conditions of 370C and pH 7.4, the PMN inhibitor rapidly lost its ability to inhibit PMN phagocytosis and killing activities.
DISCUSSION Various investigations on lethal toxicity and cytotoxicity of extracellular substances produced from P. aeruginosa have been carried out to study the pathogenicity of P. aeruginosa (4, 8, 16, 28, 29). Liu (12, 13, 15), Callahan (2, 3), and Pavlovskis (20) extracted an exotoxin (PA toxin) from a culture filtrate of a non-proteolytic
397
sI EA
:m
z H
P4
0
-
1
2
3
TIME (HOUR)
FIG. 5. Effect ofproteolytic enzyme on the stability of the PMN inhibitor. Protease (51 U) was added to 4 ml of PMN inhibitor (35 Am/ml) at 37° C, pH 7.4. The residual activity of the PMN inhibitor was measured through inhibition of the phagocytic and killing activities of PMN against E. coli strain 303 and was expressed as the number (CFU) pf E. coli strain 303 surviving after exposure to PMN.
strain of P. aeruginosa and purified it as a heatlabile protein with a molecular weight of 50,000 to 54,000. Iglewski and Kabat (6) describe an enzymatic activity of this exotoxin which appears to be similar to nicotinamide adenine dinucleotide-dependent adenosine diphosphate-ribosyltransferase activity. Furthermore, Scharman (24-26) found a toxic substance, leukocidin, which destroyed PMN from various kinds of animals, but not erythrocytes, in cultures of P. aeruginosa. The purified leukocidin has a molecular weight of 27,000 and the ultraviolet light absorbency of protein, and is thermolabile and inactivated by pronase, but not by several other proteases. In the previous study, we demonstrated that there is a close relationship between the virulence to mice and the susceptibility of clinical isolates of P. aeruginosa to the phagocytic and killing activities of PMN (18). Furthermore, we find that virulent strains of P. aeruginosa produce an extracellular substance (the PMN inhibitor) that inhibits the phagocytic and killing activities of PMN (18). In these studies on the purification of the PMN inhibitor, the PMN inhibitor was eluted in a different fraction from protease and elastase by DEAE-cellulose chromatography. The PMN inhibitor had the ability to inhibit phagocytic and killing activities of PMN. On the other hand, protease and elastase had no such ability. In addition, the PMN inhibitor had the properties of a protein being inactivated by heat and proteolytic enzyme treatment and had a molecular weight of 65,000.
398
NONOYAMA ET AL.
Further studies will be required to clarify whether the PMN inhibitor described here is the same substance as PA toxin or leukocidin. However, the PMN inhibitor seems to be a different substance from PA toxin and leukocidin in that PA toxin (21) does not affect ingestion and killing of bacteria by PMN and leukocidin (22, 23) has a molecular weight of 27,000. Although the role of the PMN inhibitor of P. aeruginosa in vivo is not fully studied in the present, it is probable that the PMN inhibitor may play an important role in early stage of P. aeruginosa infection in humans in that the PMN inhibitor inhibits the phagocytic and killing activities of PMN. LITERATURE CMD 1. Andrews, P. 1964. Estimation of the molecular weight of proteins by Sephadex gel-filtration. Biochem. J. 91: 222-233. 2. Callahan, L. T., m. 1974. Purification and characterization of Pseudomonas aeruginosa exotoxin. Infect. Immun. 9:113-118. 3. Callahan, L T., m. 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. Carney, S. A., and R. J. Jones. 1968. Biological and immunochemical properties of culture filtrates of virulent and avirulent strains of Pseudomonas aeruginosa. Br. J. Exp. Pathol. 49:395-410. 5. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum protein. Ann. N.Y. Acad. Sci. 121:404-427. 6. 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. 7. Johnson, G. G., J. M. Morris, and R. S. Berk. 1967. The extracellular protease from Pseudomonas aeruginosa exhibiting elastase activity. Can. J. Microbiol. 13: 711-719. 8. Kusama, H., and R. H. Suss. 1972. Vascular permeability factor of Pseudomonas aeruginosa. Infect. Immun. 5:363-369. 9. Liu, P. V. 1964. Factors that influence toxigenicity of Pseudomonas aeruginosa. J. Bacteriol. 88:1421-1427. 10. Liu, P. V. 1966. The roles of various fractions ofPseudomonas aeruginosa in its pathogenesis. III. Identity of the lethal toxins produced in vitro and in vivo. J. Infect. Dis. 116:481-489. 11. Liu, P. V. 1966. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. II. Effects of lecithinase and protease. J. Infect. Dis. 116:112-116. 12. Liu, P. V. 1973. Exotoxins of Pseudomonas aeruginosa. I. Factors that influence the production of exotoxin A.
INFECT. IMMUN. J. Infect. Dis. 128:506-513. 13. Liu, P. V. 1974. Extracellular toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130:94-99. 14. Liu, P. V., and H. Hsieh. 1973. Exotoxins of Pseudomonas aeruginosa. III. Characteristics of antitoxin A. J. Infect. Dis. 128:520-525. 15. Liu, P. V., S. Yoshii, and IL Hsieh. 1973. Exotoxins of Pseudomonas aeruginosa. H. Concentration, purification, and characterization of exotoxin A. J. Infect. Dis. 128:514-519. 16. Meinke, G., and R. S. Berk. 1970. In vivo studies with a toxic fraction of Pseudomonas aeruginosa. Proc. Soc. Exp. Biol. Med. 135:360-363. 17. Morihara, K. 1965. Production of elastase and proteinase by Pseudomonas aeruginosa. J. Bacteriol. 88:745-757. 18. Nishida, M., Y. Mine, S. Nonoyama, and S. Goto. 1975. Inhibiting effect of Pseudomonas aeruginosa on the phagocytic and killing activity of polymorphonuclear leukocytes, p. 277-281. Ist ISC-IAMS Proceedings (1974), Vol. 4. 19. Nishida, M., Y. Mine, S. Nonoyama, and Y. Yokota. 1976. Effect of antibiotics on the phagocytosis and killing of Pseudomonas aeruginosa by rabbit polymorphonuclear leukocytes. Chemotherapy 22:203-210. 20. Pavlovskis, 0. R., L. T. Callahan, and R. D. Meyer. 1974. Characterization of exotoxin of Pseudomonas aeruginosa. J. Infect. Dis. 130:100-102. 21. Sbarra, A. J., R. F. Gilfilla, and W. A. Bardawill. 1960. A plate assay for elastase. Nature (London) 188: 322-323. 22. Scharmann, W. 1976. Formation and isolation of leukocidin from Pseudomonas aeruginosa. J. Gen. Microbiol. 93:283-291. 23. Scharmann, W. 1976. Purification and characterization of leukocidin from Pseudomonas aeruginosa. J. Gen. Microbiol. 93:292-302. 24. Scharmann, W. 1976. The cytotoxic action of leukocidin from Pseudomonas aeruginosa on human polymorphonuclear leukocytes. J. Gen. Microbiol. 93:303-308. 25. Scharmann, W. 1976. Cytotoxic effects of leukocidin from Pseudomonas aeruginosa on polymorphonuclear leukocytes from cattle. Infect. Immun. 13:836-843. 26. Scharmann, W. 1976. Interaction of purified leukocidin from Pseudomonas aeruginosa with bovine polymorphonuclear leukocytes. Infect. Immun. 13:1046-1053. 27. Studier, F. W. 1972. Genetic and biochemical analysis of the simple phage genes information about basic genetic processes. Science 176:367-376. 28. Tabuchi, K., and B. B. Diena. 1974. A cytotoxicity assay of Pseudomonas aeruginosa culture filtrates by a dyeuptake test. Res. Commun. Chem. Pathol. Pharmacol. 8:353-364. 29. Tabuchi, K., H. Sata, and B. B. Diena. 1974. Pathogenic and immunological properties of culture filtrates of Pseudomonas aeruginosa. Can. J. Microbiol. 20:14171422. 30. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406-4412.
INFECTION AND IMMUNITY, May 1979, p. 394-398 0019-9567/79/05-0394/05$02.00/0
Inhibitory Effect of Pseudomonas aeruginosa on the Phagacytic and Killing Activities of Rabbit Polymorphonuclear Leukocytes: Purification and Characterization of an Inhibitor of Polymorphonuclear Leukocyte Function SHIGEO NONOYAMA,' HITOSHI KOJO,' YASUHIRO MINE,' MINORU NISHIDA,' * SACHIKO GOTO,2 AND SHOGO KUWAHARA2 Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan,' and Department of Microbiology, Toho Univerisity School of Medicine, Tokyo, Japan2
Received for publication
A clinically isolated strain of polymorphonuclear leukocyte (PMN)-resistant Pseudomonas was found to produce an extracellular substance (PMN inhibitor) that inhibits the phagocytic and killing activities of PMN. The PMN inhibitor was purified from culture filtrates by precipitation with (NH4)2SO4 and chromatography on phosphocellulose, diethylaminoethyl-cellulose, and Sephadex G-100. The preparation was homogenous as judged by disc gel electrophoresis. The purified PMN inhibitor appeared to be a protein with a molecular weight of approximately 65,000 that was inactivated by heating and by exposure to a proteolytic enzyme. It has been demonstrated that extracellular substances of Pseudomonas aeruginosa such as protease, elastase, collagenase, lecithinase, hemolysin, exotoxin, and enterotoxin play an important role in the pathogenicity of P. aeruginosa infections (4, 9, 10, 11, 13, 16, 29). Phagocytosis plays an important role in host defense against bacterial infection. During an investigation of the interactions of rabbit polymorphonuclear leukocytes (PMN) and P. aeruginosa, we encountered two different types of strains (18). These are resistant or susceptible to the phagocytic and killing activities of PMN, and the strains are found to be virulent and avirulent, respectively, as determined by the lethal dose values for mouse infection. In our preparatory study, of 15 clinically isolated P. aeruginosa tested, PMN-resistant strains are detected at comparatively high frequency (60%). PMN-resistant strains are found to produce a high-molecular-weight substance which inhibits the phagocytic and killing activities of the PMN. The present paper describes the purification and biochemical properties of the PMN inhibitor isolated from culture filtrates of PMN-resistant Pseudomonas. MATERIALS AND METHODS Organisms. P. aeruginosa strain 7005 and Escherichia coli strain 303 were used which were originally isolated from clinical sources. P. aeruginosa strain
7005 was a virulent, PMN-resistant strain producing protease, elastase, melanin, pyocyanine, and fluorescein. E. coli strain 303 was a PMN-sensitive strain. Purification of the PMN inhibitor. P. aeruginosa strain 7005 was cultivated in Trypticase soy broth (Baltimore Biological Laboratory) at 370C for 20 h to stationary phase. Cells were removed by centrifuging at 16,400 x g for 30 min, and the resulting supernatant fluid was passed through a filter (Millipore Corp.; pore size, 0.45 ,um). A 1.5-liter amount of the culture supernatant was precipitated with (NH4)2SO4 to 90% saturation. The precipitate was collected by centrifuging at 16,400 x g for 30 min and was dissolved in 100 ml of 0.01 M tris(hydroxymethyl)aminomethane (Tris) buffer containing 5% glycerol (Ishizu Pharmaceuticals, Ltd., Osaka, Japan) (pH 7.0) and dialyzed against the same buffer at 4°C for 20 h. The dialysate (148 ml) was applied to a column (1.0 by 10 cm) of phosphocellulose and then to diethylaminoethyl (DEAE)-cellulose (1.0 by 10 cm) equilibrated with the same buffer. In DEAE-cellulose chromatography a linear gradient of 0 to 0.8 M KC1 in the same buffer was used. The fractions from DEAE chromatography containing the PMN inhibitor (30 ml) were concentrated to 2 ml, and the solution was applied to a column (2.5 by 86 cm) of Sephadex G-100 equilibrated with the same buffer and eluted with the same buffer. Preparation of protease and elastase. The culture filtrate was precipitated with (NH4)2SO4, and the resulting precipitate was dissolved in 0.01 M Tris buffer (pH 7.0) and dialyzed against the same buffer. The dialysate was applied directly to a column of DEAE-cellulose, omitting the phosphocellulose chromatography step. Elution was effected under the same
394
VOL. 24, 1979
PURIFICATION OF P. AERUGINOSA EXOTOXIN
395
conditions used for purification of the PMN inhibitor. Polyacrylamide gel disc electrophoresis. Polyacrylamide gel disc electrophoresis was carried out as described by Davis (5). The PMN inhibitor was mixed with the sample gel and placed in top of the gel. Electrophoresis was performed at a constant current of 2 mA/tube for about 5 h at 200C. The gel was stained with amino black and then destained. Determination of molecular weight. The molecular weight of the PMN inhibitor was estimated by gel filtration on Sephadex G-100 column chromatography (1), which was equilibrated with 0.01 M Tris buffer containing 0.5 M KCI (pH 7.0). The molecular weight of the PMN inhibitor was compared with proteins of known molecular weights: rabbit serum albumin (69,000 molecular weight), human serum albumin (60,000 molecular weight), pyruvate kinase (57,000 molecular weight), leucin aminopeptidase (53,000 molecular weight), and egg albumin (45,000 molecular weight). Moreover, the molecular weight was determined essentially by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Weber (30). Slab gels were prepared by the method of Studier (27) and stained with Coomassie brilliant blue. Assay of protease and elastase activities. Protease activity was measured by the casein digestion method essentially as described by Johnson (7) and Morihara (17). Elastase activity was assayed by the method described by Sbarra (21). Assay of the PMN inhibitor. PMN were obtained by the method previously described (18, 19). PMN inhibitor activity was assayed by measuring the inhibition of the phagocytic and killing activities of PMN against E. coli strain 303. PMN (8 x 106 cells per ml) were shaken with E. coli strain 303 (106 colony-forming units [CFU] per ml) at 370 for 4 h. The mixture was centrifuged at 2,100 x g for 20 min to sediment broth PMN and E. coli. PMN were then selectively lysed by adding sterile distilled water, and surviving E. coli were determined by plating (18).
trates by precipitation with (NH4)2SO4 and chromatography on phosphocellulose, DEAE-cellulose, and Sephadex G-100. Figure 1 shows the elution pattern of the PMN inhibitor from the phosphocellulose column. The PMN inhibitor was present in the passage through fractions with 0.01 M Tris buffer containing 5% glycerol (pH 7.0) as eluent. The fractions containing the PMN inhibitor from phosphocellulose chromatography were applied to the column of DEAE-cellulose (Fig. 2). Two major peaks of 280-nm absorbing material were eluted with KCI at about 0.2 and 0.3 M. PMNinhibiting activity was associated with the 0.4 M KCI eluate. Further purification of the PMN inhibitor was carried out by column chromatography on Sephadex G-100 (Fig. 3). The major peak of ultraviolet-absorbing material was found in tube no. 15 in the void volume, and a small peak was found in tube no. 20. The activity of the PMN inhibitor was associated with the latter peak. Polyacrylamide gel electrophoresis of the PMN inhibitor obtained by gel filtration on Sephadex G-100 showed a single band which corresponds to the fastest moving band among the bands obtained the original crude culture filtrates. Separation of the PMN inhibitor from protease and elastase. The elution patterns on the PMN inhibitor, protease, and elastase by ion-exchange chromatography on DEAE-cellulose are shown in Fig. 4. Ten peaks of 280-nm absorbing material were obtained, of which five fractions had different biological activities. Fractions 1 and 2, which passed through the column with Tris buffer, showed only proteolytic and elastolytic activiRESULTS ties, respectively. Fractions 3 and 4, which were Purification of the PMN inhibitor. The obtained by gradient elution, showed only proPMN inhibitor was purified from culture fil- teolytic activity, and fraction 5, eluted with 0.4 INHIBITORY ACTIVITY
1. 0
0-0 UV ABSORBANCE(A 280) 0
0
>4
co Co
H
B
0
H
8 Ed-4
H
w
u z
H
7X w
9c
az
0:
0
En us
H
4
6 H-
-4
0.5
9 z
0 U
z H
0
10
20
30
5s
[0 X
FRACTION NUMBER
FIG. 1. Phosphocellulose column chromatography of the PMN inhibitor. The dialysis residue (148 ml) was applied to an 8-ml bed volume of phosphocellulose in 0.01 M Tris buffer containing 5% glycerol (pH 7.0). The ionic strength increased linearly in the elation buffer system; 0 to 1.0 M KCl. Fractions (10 ml) were collected and assayed for PMN inhibitor. The arrow indicates where gradient elation was initiated.
396
INFECT. IMMUN.
NONOYAMA ET AL. z
INHIBITORY ACTIVITY 0-0 UV ABSORBANCE(A 280)
0
co u
>4 H
H
8 :> H
1.5
U
0.8 z 0 H Ez
7 ,S, U
z;1.0
o
H
0
H
E
-
tU
o H 4 6 m
0.5
I
0
H z
CD
0.4 U z 0
u
H .
10 20 30 FRACTION NUMBER
0
0
40
FIG. 2. DEAE-cellulose column chromatography of the PMN inhibitor. The PMN inhibitor (60 ml) eluted from phosphocellulose was placed on a cellulose column (1.0 by 10 cm) equilibrated with 0.01 M Tris buffer containing 5% glycerol (pH 7.0). The ionic strength increased linearly as indicated. Fractions (8 ml) were collected and assayed for the PMN inhibitor. The arrow indicates where gradient elution was initiated.
0-O
UV ABSORBANCE (A 280) 0
E-4
> 5 H
).
2:S c~J
5
cD D
z
m
W.1E
H
U4 EH, Ho H
).1
CO
-
z
H
0
10
20
30
FRACTION NUMBER
FIG. 3. Sephadex G-l OO gel filtration on the PMN inhibitor. The PMN inhibitor (2 ml), which was purified through the step of DEAE-cellulose chromatography and concentrated from 30 ml of fractions, was chromatographed on a Sephadex G-100 column (2.5 by 86 cm) in 0.01 M Tris buffer containing 5% glycerol (pH 7.0). The flow rate was 15 ml/h, and fractions of 10 ml each were collected. 3 C..
0.8
.0.8
.6
u
-
C,
01-
:>E-4
0 I: H
H
Ez
>4
U
H
H 0. 4
9c
4
W
o U H 0 U
0
0.2
9
zb zZ
E-4 0
U) uz
H
0.4
H
B
o aZ:
z
E-i
4
0
Pr
C.)_
E4
0.6 CN
C.)
FRACTION NUMBER
FIG. 4. Preparation of protease and elastase with a column of DEAE-cellulose. The dialysis residue (20 ml) was placed on a cellulose column (1.0 by 10 cm) equilibrated with 0.01 M Tris buffer containing 5% glycerol (pH 7.0). The ionic strength increased linearly in the elution buffer system; 0 to 0.8 M KCl. Fractions (6 ml) were collected and assayed for proteolytic enzyme, elastolytic enzyme, and PMN inhibitor. The arrow indicates where gradient elution was initiated.
VOL. 24, 1979
PURIFICATION OF P. AERUGINOSA EXOTOXIN
M KCl, showed the activity of the PMN inhibitor. This indicated that the PMN inhibitor was a separate extracellular product from protease and elastase. Biochemical properties ofthe PMN inhibitor. In these experiments, the 30 ml of the PMN inhibitor which was separated from 1.5 liters of culture filtrate and purified by gel filtration on Sephadex G-100 column chromatography was used. This purified PMN inhibitor had an absorbance at 280 nm of 0.035. In the absence of the PMN inhibitor, the viable cell counts of E. coli strain 303 decreased from 106 to 6 x 102 CFU/ml after 4 h of incubation in the assay for phagocytosis and killing. In the presence of PMN inhibitor, the viable cell count of E. coli strain 303 did not decrease (3 x 105 CFU/ml). This PMN inhibitor was used at the original concentration yielded by the purification procedure and was referred to as 35 Am0/mi. The biochemical properties of the PMN inhibitor are as follows. The molecular weight of the PMN inhibitor was estimated to be about 65,000 by gel filtration on Sephadex G-100. The PMN inhibitor preparation contained two protein components of molecular weights of about 66,000 and 9,000 when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The effect of heat treatment on the stability of the PMN inhibitor was tested. The PMN inhibitor was treated at four different temperatures (37, 50, 70, 10000) for 30 min (pH 7.4). The activity of the PMN inhibitor was lost rapidly as temperature increased. After exposure of the PMN inhibitor solution to five different pH values at 370C for 2 h, the pH's of the solutions were adjusted to 7.2 with 0.1 N NaOH or 0.1 N HCl, and the remaining activity of the PMN inhibitor was measured. Treatment of the PMN inhibitor at pH values from 5 to 9 had little effect on its activity, but exposure to pH 3 and 11 caused loss of activity. The effect of proteolytic enzyme on the stability of the PMN inhibitor was examined (Fig. 5); the protease used (51 U) was separated by chromatography on DEAE-cellulose (fraction 3). Under conditions of 370C and pH 7.4, the PMN inhibitor rapidly lost its ability to inhibit PMN phagocytosis and killing activities.
DISCUSSION Various investigations on lethal toxicity and cytotoxicity of extracellular substances produced from P. aeruginosa have been carried out to study the pathogenicity of P. aeruginosa (4, 8, 16, 28, 29). Liu (12, 13, 15), Callahan (2, 3), and Pavlovskis (20) extracted an exotoxin (PA toxin) from a culture filtrate of a non-proteolytic
397
sI EA
:m
z H
P4
0
-
1
2
3
TIME (HOUR)
FIG. 5. Effect ofproteolytic enzyme on the stability of the PMN inhibitor. Protease (51 U) was added to 4 ml of PMN inhibitor (35 Am/ml) at 37° C, pH 7.4. The residual activity of the PMN inhibitor was measured through inhibition of the phagocytic and killing activities of PMN against E. coli strain 303 and was expressed as the number (CFU) pf E. coli strain 303 surviving after exposure to PMN.
strain of P. aeruginosa and purified it as a heatlabile protein with a molecular weight of 50,000 to 54,000. Iglewski and Kabat (6) describe an enzymatic activity of this exotoxin which appears to be similar to nicotinamide adenine dinucleotide-dependent adenosine diphosphate-ribosyltransferase activity. Furthermore, Scharman (24-26) found a toxic substance, leukocidin, which destroyed PMN from various kinds of animals, but not erythrocytes, in cultures of P. aeruginosa. The purified leukocidin has a molecular weight of 27,000 and the ultraviolet light absorbency of protein, and is thermolabile and inactivated by pronase, but not by several other proteases. In the previous study, we demonstrated that there is a close relationship between the virulence to mice and the susceptibility of clinical isolates of P. aeruginosa to the phagocytic and killing activities of PMN (18). Furthermore, we find that virulent strains of P. aeruginosa produce an extracellular substance (the PMN inhibitor) that inhibits the phagocytic and killing activities of PMN (18). In these studies on the purification of the PMN inhibitor, the PMN inhibitor was eluted in a different fraction from protease and elastase by DEAE-cellulose chromatography. The PMN inhibitor had the ability to inhibit phagocytic and killing activities of PMN. On the other hand, protease and elastase had no such ability. In addition, the PMN inhibitor had the properties of a protein being inactivated by heat and proteolytic enzyme treatment and had a molecular weight of 65,000.
398
NONOYAMA ET AL.
Further studies will be required to clarify whether the PMN inhibitor described here is the same substance as PA toxin or leukocidin. However, the PMN inhibitor seems to be a different substance from PA toxin and leukocidin in that PA toxin (21) does not affect ingestion and killing of bacteria by PMN and leukocidin (22, 23) has a molecular weight of 27,000. Although the role of the PMN inhibitor of P. aeruginosa in vivo is not fully studied in the present, it is probable that the PMN inhibitor may play an important role in early stage of P. aeruginosa infection in humans in that the PMN inhibitor inhibits the phagocytic and killing activities of PMN. LITERATURE CMD 1. Andrews, P. 1964. Estimation of the molecular weight of proteins by Sephadex gel-filtration. Biochem. J. 91: 222-233. 2. Callahan, L. T., m. 1974. Purification and characterization of Pseudomonas aeruginosa exotoxin. Infect. Immun. 9:113-118. 3. Callahan, L T., m. 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. Carney, S. A., and R. J. Jones. 1968. Biological and immunochemical properties of culture filtrates of virulent and avirulent strains of Pseudomonas aeruginosa. Br. J. Exp. Pathol. 49:395-410. 5. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum protein. Ann. N.Y. Acad. Sci. 121:404-427. 6. 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. 7. Johnson, G. G., J. M. Morris, and R. S. Berk. 1967. The extracellular protease from Pseudomonas aeruginosa exhibiting elastase activity. Can. J. Microbiol. 13: 711-719. 8. Kusama, H., and R. H. Suss. 1972. Vascular permeability factor of Pseudomonas aeruginosa. Infect. Immun. 5:363-369. 9. Liu, P. V. 1964. Factors that influence toxigenicity of Pseudomonas aeruginosa. J. Bacteriol. 88:1421-1427. 10. Liu, P. V. 1966. The roles of various fractions ofPseudomonas aeruginosa in its pathogenesis. III. Identity of the lethal toxins produced in vitro and in vivo. J. Infect. Dis. 116:481-489. 11. Liu, P. V. 1966. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. II. Effects of lecithinase and protease. J. Infect. Dis. 116:112-116. 12. Liu, P. V. 1973. Exotoxins of Pseudomonas aeruginosa. I. Factors that influence the production of exotoxin A.
INFECT. IMMUN. J. Infect. Dis. 128:506-513. 13. Liu, P. V. 1974. Extracellular toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130:94-99. 14. Liu, P. V., and H. Hsieh. 1973. Exotoxins of Pseudomonas aeruginosa. III. Characteristics of antitoxin A. J. Infect. Dis. 128:520-525. 15. Liu, P. V., S. Yoshii, and IL Hsieh. 1973. Exotoxins of Pseudomonas aeruginosa. H. Concentration, purification, and characterization of exotoxin A. J. Infect. Dis. 128:514-519. 16. Meinke, G., and R. S. Berk. 1970. In vivo studies with a toxic fraction of Pseudomonas aeruginosa. Proc. Soc. Exp. Biol. Med. 135:360-363. 17. Morihara, K. 1965. Production of elastase and proteinase by Pseudomonas aeruginosa. J. Bacteriol. 88:745-757. 18. Nishida, M., Y. Mine, S. Nonoyama, and S. Goto. 1975. Inhibiting effect of Pseudomonas aeruginosa on the phagocytic and killing activity of polymorphonuclear leukocytes, p. 277-281. Ist ISC-IAMS Proceedings (1974), Vol. 4. 19. Nishida, M., Y. Mine, S. Nonoyama, and Y. Yokota. 1976. Effect of antibiotics on the phagocytosis and killing of Pseudomonas aeruginosa by rabbit polymorphonuclear leukocytes. Chemotherapy 22:203-210. 20. Pavlovskis, 0. R., L. T. Callahan, and R. D. Meyer. 1974. Characterization of exotoxin of Pseudomonas aeruginosa. J. Infect. Dis. 130:100-102. 21. Sbarra, A. J., R. F. Gilfilla, and W. A. Bardawill. 1960. A plate assay for elastase. Nature (London) 188: 322-323. 22. Scharmann, W. 1976. Formation and isolation of leukocidin from Pseudomonas aeruginosa. J. Gen. Microbiol. 93:283-291. 23. Scharmann, W. 1976. Purification and characterization of leukocidin from Pseudomonas aeruginosa. J. Gen. Microbiol. 93:292-302. 24. Scharmann, W. 1976. The cytotoxic action of leukocidin from Pseudomonas aeruginosa on human polymorphonuclear leukocytes. J. Gen. Microbiol. 93:303-308. 25. Scharmann, W. 1976. Cytotoxic effects of leukocidin from Pseudomonas aeruginosa on polymorphonuclear leukocytes from cattle. Infect. Immun. 13:836-843. 26. Scharmann, W. 1976. Interaction of purified leukocidin from Pseudomonas aeruginosa with bovine polymorphonuclear leukocytes. Infect. Immun. 13:1046-1053. 27. Studier, F. W. 1972. Genetic and biochemical analysis of the simple phage genes information about basic genetic processes. Science 176:367-376. 28. Tabuchi, K., and B. B. Diena. 1974. A cytotoxicity assay of Pseudomonas aeruginosa culture filtrates by a dyeuptake test. Res. Commun. Chem. Pathol. Pharmacol. 8:353-364. 29. Tabuchi, K., H. Sata, and B. B. Diena. 1974. Pathogenic and immunological properties of culture filtrates of Pseudomonas aeruginosa. Can. J. Microbiol. 20:14171422. 30. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406-4412.
