Vol. 13, No. 4 Printed in U.SA.

INFECTION AND IMMUNITY, Apr. 1976, p. 1046-1053 Copyright © 1976 American Society for Microbiology

Interaction of Purified Leukocidin from Pseudomonas aeruginosa with Bovine Polymorphonuclear Leukocytes WOLFGANG SCHARMANNI Institut far Bakteriologie und Immunologie, Universitdt Giessen, D-63 Giessen, Germany

Received for publication 2 December 1975

The interaction of purified leukocidin from Pseudomonas aeruginosa, strain 158, with polymorphonuclear leukocytes of cattle (PMLC) was studied by using '25I-labeled toxin. According to the Scatchard plot, PMLC offered two binding sites for leukocidin: one at the surface of the plasma membrane, and a second one that presumably became accessible to the toxin in the course of the cytotoxic action. Toxin once fixed to PMLC at 37 C could not be detached from the cells by either chemical or mechanical treatment. However, active leukocidin was liberated if it was bound to PMLC at 4 C and the temperature of the cell suspension was subsequently increased to 37 C. In the presence of Ca2 the velocity of toxin fixation was accelerated and the rate of fixation was increased. Preliminary investigations on the identification of the leukocidin-binding material indicated the leukocidin receptor to be an integral protein of the plasma membrane. ,

Leukocidin from Pseudomonas aeruginosa (20-23) is a cell-bound protein that exerts a cytopathogenic effect on leukocytes and various tissue culture cells, but is ineffective on erythrocytes or blood platelets (21). The cytotoxic action of leukocidin on bovine leukocytes is characterized by an increased permeability of the plasma membrane for low molecular markers (23), leading to a swelling of the cells without rupture of the cell membrane (22, 23). These results suggest the locus of cytopathogenicity to be the plasma membrane. In the present paper, studies of the interaction of purified leukocidin with plasma membrane of bovine polymorphonuclear (PMLC) leukocytes are described. MATERIALS AND METHODS Leukocidin. Leukocidin was obtained from P.

aeruginosa, strain 158, by autolysis of washed cells (20) and purified by ammonium sulfate precipitation (20% saturation) and combined gel filtration on Sephadex G-100 superfine and Bio-Gel P-100 (21). The purified toxin appeared as a single band in sodium dodecyl sulfate disc electrophoresis (21) and was devoid of hemolysin, lecithinase C, lipase, and protease. Lecithinase C was determined by egg yolk agar; lipase was determined by tributyrin agar. Protease and hemolysin were assayed as described earlier (20). Buffer. Phosphate-buffered saline, pH 7.2 (PBS), was prepared according to Dulbecco and Vogt (4) except that CaCl2 was omitted. The buffer contained, in grams per liter: NaCl, 8.0; KCl, 0.2; ' Present address: Bundesgesundheitsamt, D-1 Berlin 33 Postfach, Germany.

KH2PO4, 0.2; Na2HPO4-2H20, 1.25; MgCl2 6H20, 0.1. Preparation and purification of ['251]leukocidin. Purified leukocidin was labeled with Na 125I (Behringwerke, Marburg, Germany), using chloramineT as an oxidant according to the procedure of McConahey and Dixon (12). After iodination, the ['25 ]leukocidin was separated from the bulk of free 1251 by gel filtration on a column of Sephadex G-100 (1.5 by 30 cm), equilibrated with PBS containing 0.2 M NaCl. The radioactivity was measured in a Packard Autogamma spectrometer. Measurement of leukocidic activity. To standardize leukocidic activity, the microscopic slide adhesion method (8) was used. Serial twofold dilutions of toxin in PBS were incubated with human granulocytes at 37 C for 60 min in a moist chamber. One MLeD (minimal leukocidic dose) was the highest dilution of toxin that destroyed all leukocytes of a slide field (about 6,000 to 8,000 cells) (20). The measurement of leukocidic activity with 86Rb-labeled PMLC has been described (23). Leukocytes. The preparation of PMLC according to the method of Behrens and Esch (2) has been described (23). Homogenization of PMLC. For homogenization, PMLC were suspended in PBS and disrupted by a motor-driven, continuous-flow glass homogenizer (Buhler, Tubingen, Germany) at 4 C. Preparation of granules. Granules were isolated from homogenized PMLC by the differential sedimentation technique described by Hegner (9). Preparation of PMLC membranes. PMLC membranes were isolated by the method of Woodin and Wieneke (31). Leukocytes were homogenized and suspended in 11.6% (wt/vol) sucrose solution. Samples of 1.5 ml were layered onto a sucrose gradient that had been formed by 3 ml of 3 0% and 2 ml of 48% (wt/vol) sucrose solution. Gradients were centrifuged at 100,000 x g for 1 h. The membrane fraction

1046

VOL. 13, 1976

INTERACTION OF LEUKOCIDIN WITH LEUKOCYTES

1047

Inactivation of the toxin was effected only by the insoluble fraction. Therefore both plasma membranes and granules were isolated from PMLC and tested for their ability to inactivate leukocidin. It appeared that the toxin was eliminated by the membrane fraction to the same degree as by intact or homogenized leukocytes (Table 1). The granular fraction, however, did not influence leukocidic activity. Binding of leukocidin as a function of time and toxic activity. Binding of ['25I]leukocidin as a function of time is shown in Fig. 1. As can be seen, 86% of the added toxin (120 MLeD) was bound to plasma membranes of PMLC within 10 min at 37 C. Figure 2 illustrates the binding and uptake, respectively, of leukocidin to PMLC at different toxin concentrations. Using 2,250 MLeD ofleukocidin and 2 x 107 PMLC, no saturation of toxin uptake was obtained. When the data from Fig. 2 were drawn as a Scatchard plot, it became apparent that the leukocytes presumably had two sites of fixation of different affinity for leukocidin (Fig. 3). Effect of temperature on the binding of leukocidin. When equal amounts of PMLC and L'25llleukocidin were incubated at 4 and 37 C, about 65% less toxic activity was bound at 4 C than at 37 C. However, at both temperatures nearly the same activity was bound during the first minutes of incubation (Fig. 4). At 4 C, the process of binding was virtually completed after RESULTS about 20 min, whereas at 37 C maximal toxin uptake was not obtained until 2 h after the Fixation of leukocidin to leukocytes. Pre- beginning of incubation. liminary experiments had shown that the incuNo cytotoxic effect was detected at 4 C (23). bation of PMLC with leukocidin was accompa- In a mixture of PMLC and [25I]leukocidin kept nied by a loss of toxic activity in the incubation first at 4 C and then at 37 C, the following mixture. This inactivation of leukocidin might phenomenon was observed: toxin, bound to the have been caused by an irreversible fixation of leukocytes at 4 C, partly dissociated from the toxin to the cells or by a temporary interaction cells when the temperature increased to 37 C between leukocidin and cells without perma- and was subsequently fixed to the cells again. nent fixation of the toxin. To test this point, The same phenomenon occurred when the PMLC were incubated with ['251]leukocidin at cooled, toxin-carrying leukocytes were washed 37 C for various times and the radioactivity in the cell-free supernatant was determined. The TABLE 1. Inactivation of leukocidin" experiment revealed that the toxin, once exposed to leukocytes, remained fixed to the cells Leukocidic activity in the supernatant Material for at least 2 h and that the process of fixation (MLeD/ml) started immediately after adding the toxin to the granulocytes. 0 ............ Leukocytes Binding of leukocidin to the leukocyte Homogenate ........... 0 0 plasma membrane. ("Binding" means the spe- Membrane fraction ..... 450 cific fixation of toxin to the plasma membrane, Sediment .............. whereas "uptake" is an unspecific fixation to x "PMLC (2 108) in 1 ml of PBS or the components components of the damaged cell.) To determine of 2 x 108 PMLC in 1 ml of PBS were incubated with the leukocidin to of the site of fixation cell, 1,000 MLeD of leukocidin at 37 C for 60 min. After PMLC were homogenized and the homogenate centrifugation (12,000 x g, 4 min), leukocidic activwas separated into a soluble and an insoluble ity remaining in the supernatant was determined by fraction by centrifugation (100,000 x g, 60 min). the slide adhesion method.

collected from the sucrose/sucrose interface and identified by a positive 5'-nucleotidase reaction (14). Determination of binding of ['251]leukocidin to PMLC (standard conditions). Unless otherwise stated, the reaction was started by mixing prewarmed [125I]leukocidin (100 MLeD/ml) with prewarmed PMLC (2 x 107 cells/ml). The suspension was gently shaken at 37 C. Aliquots (0.2 ml) were withdrawn at intervals and spun at 12,000 x g for 20 s in an Eppendorf centrifuge (Eppendorf Geratebau, Hamburg, Germany). The supernatant was decanted and the cell pellet was washed two times in PBS. The radioactivity of the pellets was determined in a Packard Autogamma spectrometer. Antiserum. The preparation of antiserum to leukocidin has been described (21). Leukocidin toxoid. For the preparation of toxoid, purified leukocidin (5,000 MLeD/ml) was incubated with 0.4% formalin at 37 C for 2 days. Reagents. Collagenase (Clostridium perfringens) was obtained from Worthington Biochemicals, Freehold, N.J. Neuraminidase (Vibrio cholerae) was a preparation of Behringwerke, Marburg, Germany. Phospholipase D (EC 3.1.4.4) was a product of Calbiochem, Lucerne, Switzerland. Phospholipase A2 (EC 3.1.1.4) and phospholipase C (EC 3.1.4.3) were from Boehringer, Mannheim, Germany. Trypsin, pancreatic lipase, hyaluronidase (sheep testes), sodium dodecyl sulfate, and Triton X-100 were obtained from Serva, Heidelberg, Germany. All other reagents were purchased from Merck, Darmstadt, Germany.

was

1048

INFECT. IMMUN.

SCHARMANN 60 C

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FIG. 1. Binding of ['25I]Ieukocidin to plasma membranes from PMLC as a function of time. Plasma membranes (0.44 mg of protein, suspended in 0.6 ml of PBS) were mixed with 120 MLeD of['25I]leukocidin (in 0.6 ml ofPBS) and incubated at 37 C.

300 200

yCG°O ; 3- loo 0

1500

1000

500

2000

Leukocidin (MLeD/ml) FIG. 2. Binding and uptake, respectively, of leukocidin to PMLC as a function of toxin concentration. PMLC (2 x 1071ml of PBS) were incubated with ['25I]leukocidin (in PBS) at 37 C for 20 min.

,:_- 0.6 00

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100

150

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Cell-bound (cpmx10-3 per 2 107 PMLC)

FIG. 3. Scatchard plot of the binding of ['25I]leukocidin to PMLC (data taken from Fig. 2).

INTERACTION OF LEUKOCIDIN WITH LEUKOCYTES 1049 100 37*C liberated by raising the incubation temperature, other methods were tried to release the cell-bound leukocidin. When testing different substances, only 6 M urea was found to be r 10 successful in the detachment of bound 4°C partially o(Table 2). However, the released toxin was no more toxic for leukocytes. Inactivation of the leukocidin by urea could be excluded, since leukocidin proved to be stable in 6 M urea a oo 1 0 for several hours. ["25I]leukocidin fixed to 0 Time (minutes) PMLC plasma membrane could not be disFIG. 4. Time course of binding and uptake, re- placed by the 10-fold concentration of "cold" spectively, of[125Illeukocidin at 4 and 37 C. Standard toxin either at 4 C or at 37 C. Homogenization conditions. of leukocidin-carrying PMLC by ultrasonic treatment at 4 C was also unsuccessful in rewith PBS before raising the temperature (Fig. leasing the toxin. 5). Only about half of the detached toxic activEffect of leukocidin antiserum and leukociity was rebound to the leukocytes. Detached din toxoid on the binding of toxin. A specific leukocidin exerted the same cytotoxic effect on antiserum to leukocidin inhibited the cytotoxic PMLC (22) as leukocidin before incubation. Lib- action of leukocidin (23) but not the fixation of eration of toxin occurred in the same manner if toxin to the cells. When PMLC were first incuplasma membranes of PMLC were used instead bated with toxoid of leukocidin and subseof leukocytes. When PMLC were first treated quently with native toxin, the binding capacity with leukocidin and then with a specific leuko- of the cells for native toxin was reduced by 76% cidin antiserum (both at 4 C), toxin was not (Table 3). This shows that the binding site of detached from the cells upon increasing the the formalin-treated leukocidin molecule was temperature. partly preserved, although the toxicity of the Parallel experiments at different tempera- molecule had been completely lost. It would tures revealed that the liberation of toxin could seem, therefore, that the capacities for binding first be detected between 20 and 25 C (Fig. 6a). on the one hand and of cytotoxic action on the The cytotoxic action of leukocidin on 86Rb-la- other were localized at different sites of the beled PMLC started between 20 and 25 C as leukocidin molecule. Likewise, as has been well (Fig. 6b). No cytotoxic effect occurred at 10 shown by the experiment with antiserum, the or 15 C. antigenic determinant of the toxin molecule Since toxin bound to PMLC at 4 C could be might not be identical with its binding site. Effect of Ca2+ on the binding of leukocidin. VOL. 13, 1976

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Fixation of leukocidin to hypotonically 30 60 93 swollen leukocytes. Suspending cells in a hybuffer causes stretching of the plasma potonic '-0 . a---~-~----membrane (15), thus uncovering parts of the

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membrane that are physiologically not accessiPMLC were preincubated in 0.0085% NaCl, pH 6.5, for 10 min at 22 C before treatment with leukocidin, within 2 min three times as much toxin was fixed to the cells than was fixed to leukocytes in PBS (Fig. 8). After 60 min of incubation, the uptake of leukocidin by hypotonically swollen cells was

| ble from outside. When

PMLC and ['25I]leukocidin (standard conditions) were maintained at 4 C for 30 min. Subsequently only 13% higher than in control cells. cells were washed two times in PBS at 4 C, resusPreliminary experiments for the determipended in ice-cold PBS, and placed in a water bath at nation of the leukocidin receptor. To obtain 37 C. Symbols: 0, Cell-bound radioactivity.Ol, Celtbound radioactivity. Leukocidin antiserum was further information on the distribution of leuadded to the['25]leukocidin-carrying cells at 4 C kocidin-binding material in the leukocytes, before heating. Radioactivity in the cell-free super- PMLC were treated by various methods. Prein-

natant.

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cubation of PMLC with the phospholipases A2,

INFECT. IMMUN.

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4 5 10 Time (minutes) FIG. 6. (a) Dissociation of [125I]leukocidin from PMLC at different temperatures. PMLC and leukocidin (standard conditions) were maintained at 4 C for 60 min. After the leukocyte suspension was washed two times with PBS at 4 C, cells were filled into tubes and immersed in water baths of corresponding temperatures. (b) Cytotoxic action of leukocidin on 56Rb-labeled PMLC at different temperatures. Standard conditions. Ordinate represents degree of release of 55Rb+, calculated as percentage of the maximal release obtained by 0

1

2

3

4

5

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0

1

2

3

Time (minutes)

treating the cells with Triton X-100 (0.2%, vollvol). TABLE 2. Experiments on the liberation of cellbound toxin by different substances" Counts/min Substance Sediment Supernatant 370 3,132 KCI, 1 M ........... 962 2,612 Urea, 6 M .......... 316 3,360 EDTA, 0.05 M ......

TABLE 3. Effect of leukocidin toxoid on the binding of leukocidin" Counts/min Substance Sediment Supernatant

dodecyl Sodium 500 3,210 sulfate, 0.001% ..... 650 Control ............. 3,048 x in 1 ml with were mixed of PBS PMLC (1 10w) 't 600 MLeD of 1'25I]leukocidin and maintained for 30 min at 4 C. Subsequently the cells were washed two times with PBS and filled (0.2 ml) into tubes. After the addition of one of the substances cited above, the tubes were kept again at 4 C for 30 min and centrifuged (12,000 x g, 4 min), and the radioactivity in the supernatant (0.1 ml) was determined.

Cells pretreated with 97,291 25,709 toxoid ............. Control ............. 60,012 102,516 " PMLC (4 x 10') in 0.1 ml of PBS were incubated with 0.1 ml of toxoid (from 500 MLeD of native leukocidin) at 37 C for 90 min. Subsequently the cells were washed two times with PBS, resuspended in 0.1 ml of PBS, and incubated with 0.1 ml of ['251]leukocidin at 37 C for 90 min. Cells were then centrifuged (12,000 x g, 30 s), and 0.1 ml of the supernatant was removed for measuring the radioactivity. The sediment was washed two times with PBS, and the radioactivity was determined. Control cells contained PBS with 0.4% formalin instead of toxoid.

C, and D and with pancreatic lipase, collagenase, and hyaluronidase did not alter the capacity of PMLC to bind leukocidin. Upon preincubation of PMLC with neuraminidase or trypsin and Pronase, respectively, the rate of toxin fixation increased by 11 (neuraminidase) or 22% (trypsin or Pronase). The combination of neuraminidase with trypsin caused no further increase of toxin fixation. PMLC were also prein-

cubated with periodate (0.01 M), ethylenediaminetetraacetic acid (EDTA) (0.1 to 0.001 M), phytohemagglutinin M (Difco) (1:8), or KCl (0.5 to 3.0 M) (18) at 37 C for 30 min and subsequently washed with PBS. In all cases the capacity of toxin fixation remained unchanged. Lipid was extracted from PMLC by chloroform-methanol (2:1) according to Folch et al. (6). The insoluble cell residue retained 60%

VOL. 13, 1976

INTERACTION OF LEUKOCIDIN WITH LEUKOCYTES

.=

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100

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20

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Time (minutes)

FIG. 7. Effect of Ca2+ on the binding and uptake, respectively, of ['251]leukocidin to PMLC. Standard conditions. Symbols: 0, Cells in PBS; *, cells in PBS + 1 mM Ca2+.

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TimFe (minutes) FIG. 8. Time course of fixcation of['251]leukocidin to hypotonically swollen leukocytes. Standard conditions. Symbols: *, Cells in 0.0J085% NaCI, pH 6.5; 0, cells in PBS.

of its original leukocidin-binding capacity, whereas the lipid phase did not influence leukocidic activity. When ['25Illeukocidin-carrying PMLC were extracted in the same way, 92% of the radioactivity remained fixed to the cellular sediment and only 1.3 % ofthe radioactivity was found in the extract. To remove the inositides from the plasma membrane, additional lipid extractions were performed at acid pH (29), but the leukocidin-binding capacity remained connected with the cell. In another experiment, PMLC were treated with Triton X-100 (10) and sodium deoxycholate (each 1% in PBS). Now 50% of the binding capacity was detected in the dialyzed cellular extract. Upon extraction of ['25I]leukocidincarrying PMLC by the same method, 2% of the original radioactivity was found to be connected with the cellular debris and 97% was determined in the extract. Finally, PMLC were lyophilized, dried in a vacuum, and extracted with 98% formic acid

(3). Then the cellular extract was mixed with acetone. The resulting precipitate contained 30 to 40% of the original binding capacity of the

leukocytes. All methods described above resulted in a lipid-free substance whose capacity to fix and inactivate leukocidin was more or less preserved. However, this preparation was not soluble in neutral or slightly alkaline buffer. The addition of extracted lipids did not increase its solubility. Only in the presence of 8 M urea was a considerable dissolution of the toxin-binding substance achieved, although it precipitated again upon dialysis against PBS. Nevertheless, the use of urea may make it possible to identify and characterize the leukocidin receptor in the plasma membrane. DISCUSSION Earlier experiments indicated (23) that the site of action of leukocidin is presumably the cell membrane. The results reported here sup-

1052

SCHARMANN

INFECT. IMMUN.

port this hypothesis, since the toxin was bound gral protein do not rule out this hypothesis. to and inactivated by isolated plasma mem- Though transitions from the gel to the liquid branes or membrane fractions of PMLC. Ac- phase involve the lipid part of the membrane cording to the Scatchard plot, bovine leukocytes directly, the close structural and functional offer two binding sites for leukocidin. This connection between the lipid bilayer and the could explain the time course of the fixation of integral proteins (26) causes also a structural leukocidin to PMLC, which proceeded as a change of the embedded proteins. Various exrapid phase of binding within the first 10 min of amples illustrate the lateral movement of

incubation and a subsequent phase of slow toxin uptake. The rapid phase could be interpreted as a primary reaction in which the toxin was bound to the plasma membrane. It is not evident from our studies whether a second binding site of identical or different structure is accessible to leukocidin from the beginning of incubation or becomes only available to the toxin by destruction of the cells. The hypothesis of two binding sites for leukocidin is supported by two further results. (i) Nearly the same toxic activity was fixed to leukocytes at 4 and 37 C during the first 10 min of incubation. Since the action of leukocidin bound to PMLC at 4 C can be inhibited by antiserum, the toxin is presumably bound at the surface ofthe leukocyte membrane. (ii) The fixation of leukocidin to hypotonically swollen cells was much more increased in the early phase of fixation when compared with cells suspended in isotonic buffer. Preliminary investigations on the identification of the leukocidin-binding material showed that the toxin was not bound to the cellular lipid. Since the treatment of PMLC with periodate did not alter the capacity of cells for the fixation of toxin, the leukocidin receptor was presumably not a glycoprotein. The receptor could also not be split off by proteases or extracted by high-molar salt solution or EDTA. It would seem, therefore, that the leukocidinbinding material did not belong to the peripheral proteins according to the membrane model of Singer and Nicolson (24), but was embedded in the phospholipid bilayer. This idea is supported by the insolubility of the leukocidinbinding material in neutral aqueous buffers and by the increased capacity of PMLC to bind leukocidin after the treatment of cells with neuraminidase or protease. Toxin once fixed to PMLC could not be detached by chemical or mechanical treatment of leukocytes. However, active toxin was liberated if it was bound to PMLC at 4 C and the temperature of the cell suspension was subsequently increased to 37 C. An explanation for this phenomenon is offered by the thermal-phase change in biological membranes (16, 17, 25). Our preliminary results indicating the toxin receptor to be an inte-

membrane proteins within the plane of the membrane in response to changes of temperature (5, 11, 27). Based on these assumptions, it is supposed that at 4 C in the solid-like gel state of the membrane, the toxin molecule can react with the leukocidin receptor but not with the membrane locus of the cytotoxic action. The phase transition may result in a rearrangement of the receptor, causing temporary detachment of the bound leukocidin. The liquid phase of the membrane may also permit the cytotoxic action of the leukocidin molecule. The so-called transition temperature of the lipids surrounding the leukocidin receptor may be 20 to 25 C according to my results. Since leukocidin showed a tendency for aggregation, the detachment of bound toxin may also have been dependent upon a depolymerization of aggregated leukocidin. Arbuthnott et al. (1) reported for staphylococcal a-hemolysin and Woodin (30) for the F component of staphylococcal leukocidin that certain phospholipids of the plasma membrane induced polymerization of the above-mentioned toxins. However, these polymers were disaggregated by high-molar salt solution, whereas bound Pseudomonas leukocidin could not be detached by this method. In the presence of Ca2+ (Mg2+ was ineffective), the velocity of toxin fixation was accelerated and the rate of fixation was increased. These findings could be a reflection of a structural alteration of the membrane due to Ca2 . Rubalcava et al. (19) and Vanderkooi and Martonosi (28) reported that the binding of fluorescent probes to the erythrocyte membrane was substantially increased in the presence of Ca2 . An alternative hypothesis is based on the observation that leukocidin, dissolved in PBS, precipitated upon the addition of Ca2+ (0.5 M). Since the precipitate could be redissolved only by dialysis against EDTA (12 h) or 0.15 M NaCl, pH 7.2 (36 h), the precipitate was presumably not effected by salting out, but due to a reaction of the leukocidin with Ca2+. This reaction could be important in the binding process of the toxin to leukocytes. In a medium without Ca2+, the cells will lose a part of their membrane-bound Ca2+ (12). Thus, if Ca2+ is an essential factor for the binding of the toxin to leukocytes, cells suspended in Ca2+-free PBS

VOL. 13, 1976

INTERACTION OF LEUKOCIDIN WITH LEUKOCYTES

would bind less leukocidin than cells in PBS with Ca2+. ACKNOWLEDGMENT This investigation was supported in part by Deutsche Forschungsgemeinschaft. LITERATURE CITED 1. Arbuthnott, J. P., J. H. Freer, and B. Billcliffe. 1973.

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