INFECTION AND IMMUNITY, Sept. 1990, p. 2745-2749 0019-9567/90/092745-05$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 58, No. 9
Stimulatory Effect of Staphylococcal Leukocidin Phosphoinositide Metabolism in Rabbit Polymorphonuclear Leukocytes XIA WANG, MASATOSHI NODA,
Department of the Second Microbiology, Chiba University School of Medicine, 1-8-1 Inohana, Chiba 280, Japan Received 27 December 1989/Accepted 29 May 1990
When rabbit polymorphonuclear leukocytes (PMNs) were incubated with staphylococcal leukocidin (F and S components) in the presence of 32Pi at 37C, incorporation of 32Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) occurred after a lag phase of 10 s and reached a maximal level at 60 s of 50- and 30-fold increase, respectively, compared with that of the control in the absence of the toxin. Whereas the amount of 32P radioactivity incorporated in PIP and PIP2 decreased to control levels in a few minutes, 32p incorporation into phosphatidic acid (PA) continuously increased over 3 min. These findings suggested an early activation of phosphoinositide-specific phospholipase C in rabbit PMNs by leukocidin as shown by the rapid breakdown of PIP and PIP2 accompanied by the appearance of PA. The stimulatory effect of leukocidin on some enzymatic activities of the phosphatidylinositol pathway was further investigated by using PMN cell membrane preparations. In the presence of both the F and S components, enhanced 32p incorporation was observed not only in PIP2 and PA but also in PIP. While the F component mainly enhanced 32p incorporation into PIP2 and PA, the S component alone had no effect on 32p incorporation into PIP, PIP2, and PA. The F component alone enhanced conversion of PIP to [32P]PIP2 in the presence of unlabeled PIP and [_y-32P]ATP, through the activation of PIP kinase. PIP kinase activity was potentiated by the addition of NAD and GTP. Subsequent formation of [32P]PA was also enhanced by the F component, resulting from activation of the phosphoinositide-specific phospholipase C. These results suggested that the F component of staphylococcal leukocidin is responsible for the enhancement of phosphoinositide metabolism in rabbit PMN cell membranes. It has been reported that signal transduction between a membrane receptor and phosphoinositide-specific phospholipase C (PLC) is mediated through coupling of the receptor to a G protein as an amplifying and regulatory component of signal-transforming systems (18, 21). A role for G protein in the activation of phosphoinositide-specific PLC in membranes has been demonstrated in many systems, including human neutrophils (8, 10, 16). With this background, the present studies were designed to clarify the effects of leukocidin and its F component alone on activation of phosphatidylinositol (PI) metabolic enzymes such as phosphoinositide-specific PLC, and PI and phosphatidylinositol 4-phosphate (PIP) kinases.
Staphylococcal leukocidin consists of two components, the F component (Mr 32,000) and the S component (Mr 31,000). Whereas either component alone is inactive, they act synergistically to induce lysis of polymorphonuclear leukocytes (PMNs) and macrophages of rabbits and humans. In previous papers, Noda et al. (14, 15) reported that the S and F components of leukocidin are preferentially bound and inactivated by ganglioside GM, and phosphatidylcholine, respectively, of rabbit PMN cell membranes. Specific binding of the S component to ganglioside GM, on rabbit leukocytes stimulated rapid phospholipase A2 activity, catalyzing cleavage of membrane phospholipids to increase the binding sites of the F component (13). However, the primary synergistic action of the F and S components on the cell surface membrane remains to be solved. It is well known that cholera and pertussis toxins possess ADP-ribosyltransferase activity capable of modifying the GTP-binding proteins G. and G,, respectively, which can act either as a stimulator (G,) or as an inhibitor (Gi) of adenylate cyclase (2, 6, 22). More recently, Kato and Noda (7) reported that both the leukocidin F and S components possess ADP-ribosyltransferase activity catalyzing ADP-ribose transfer from NAD predominantly to rabbit PMN cell membrane proteins. The S component ADP-ribosylated a 37kilodalton (kDa) GTP-binding protein (G protein) involved in the transduction to activate phospholipase A2, and the F component resulted in ADP-ribosylation of a 41-kDa G protein in rabbit neutrophil membranes (7). *
MATERIALS AND METHODS Chemicals. PI, PIP, phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA), NAD, and GTP were obtained from Sigma Chemical Co. (St. Louis, Mo.). 32p; (28,500 Ci/mmol) and [_y-32P]ATP (4,500 Ci/mmol) were purchased from ICN Radiochemicals, Inc. (Irvine, Calif.). All other chemicals were of analytical grade. Staphylococcal leukocidin. The F and S components of staphylococcal leukocidin were purified and crystallized as described previously (12). Assay of incorporation of 32Pi into phospholipids in intact rabbit PMNs. Rabbit PMNs were isolated from rabbit peripheral blood by Ficoll-Hypaque density centrifugation (17). The purity of PMNs was more than 97% and the viability was over 98% as assessed by trypan blue exclusion and phase-contrast microscopy. The cell suspension (180 ,ul;
Corresponding author. 2745
WANG ET AL.
0 0 sQ
'1 4i J-J .,1 C." p.
10 30 60
10 30 60
10 30 60
Time (sec) FIG. 1. Time course of the incorporation of 32Pi into polyphosphoinositides and PA in rabbit PMNs. The cells (4.5 x 106/200 RI) were incubated with 30 ,Ci of 32p- in the absence (0) or presence (0) of 0.1 ,ug of the leukocidin F and S components at 37°C for the indicated times. The radioactivities of 32P-labeled PIP, PIP2, and PA were measured as described in the text. The data presented are the means of three experiments.
2.5 x 107/ml) in 10 mM Tris hydrochloride buffer (pH 7.2) containing 0.9% NaCl2 and 1 mM MgCl2 was transferred to a glass test tube and then preincubated at 37°C for 3 min. The reaction was initiated by adding 0.1 ,g of the leukocidin S and F components and 30 ,uCi of 32Pj and then incubated at 37°C. The reaction was terminated by adding 720 ,ul of cold chloroform-methanol-HCl (10:20:0.2, vol/vol). The phases were separated by the addition of 250 ,u of KCl (2 M) and chloroform followed by centrifugation at 2,000 x g for 10 min. The lower organic phase was removed, and the upper aqueous phase was washed once with 1 ml of chloroform. The combined organic extract was concentrated to 100 ,ul under a flow of N2, spotted on a Silica Gel 60 plate (layer thickness, 25 ,um; impregnated with 1% potassium oxalate containing 2 mM EDTA and activated at 110°C for 1 h; E. Merck AG, Darmstadt, Federal Republic of Germany), and developed with chloroform-acetone-methanol-acetic acidwater (40:15:13:12:8, vol/vol) (5). Separated phospholipids were localized by both iodine vapor and autoradiography. The regions of the plate corresponding to standard materials were scraped into scintillation vials containing 5 ml of Econofluor (Dupont, NEN Research Products, Boston, Mass.), and the radioactivity was counted in a liquid scintillation spectrometer. Assay of PI metabolism in PMN cell membranes. The rabbit PMN cell membranes were prepared by sonication and centrifugation (9). PMN cell membranes (100 ,ug of protein)
in 10 mM Tris hydrochloride buffer (pH 7.2) containing 1 mM MgCl2 were incubated at 37°C for 30 s with 10 ,uCi of [_y-32P]ATP and other additions as indicated. After incubation, the reaction was terminated by the addition of 720 ,lI of cold chloroform-methanol-HCl (10:20:0.2, vol/vol). Phospholipids were extracted and separated as described above. The 32P radioactivity of PIP, PIP2, and PA was determined by autoradiography. Assay of PI kinase and PIP kinase activities in PMN cell membranes. PMN cell membranes (100 ,ug of protein) in 10 mM Tris hydrochloride buffer (pH 7.2) containing 1 mM MgCl2 were preincubated with or without 100 puM GTP in ice for 20 min. To assay PI kinase and PIP kinase activities, the preincubated samples were supplemented with 50 pug of PI and PIP, respectively, and then incubated with 10 puCi of [_y-32P]ATP in the presence or absence of 0.5 p.g of leukocidin at 37°C for 30 s. The reaction was terminated by the addition of 720 pu1 of cold chloroform-methanol-HCl (10: 20:0.2, vol/vol). Phospholipids were extracted and separated as described above for the assay of incorporation of 32p; into phospholipids in intact rabbit PMNs. The radioactivities of 32P-labeled PIP and PIP2 were detected by autoradiography, and the localized regions on the plate corresponding to standard materials of PIP and PIP2 were scraped into scintillation vials containing 5 ml of Econofluor and counted in a liquid scintillation spectrometer.
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RESULTS Time course of incorporation of 32Pi into polyphosphoinositides and PA in rabbit PMNs. When rabbit PMNs (4.5 x 106 cells per 200 pl) were incubated with 0.1 ,Lg of the leukocidin F and S components in the presence of 32Pi at 37°C, 32p was rapidly incorporated into polyphosphoinositides (PIP and PIP2). After a short lag phase of 10 s, the incorporation reached a maximal level at 60 s and the radioactivities in PIP and PIP2 increased by 50- and 30-fold, respectively, compared with that of the control in the absence of the toxin. Subsequently, 32P-labeled PIP and PIP2 gradually decreased to control levels accompanying a continuous formation of labeled PA over 3 min (Fig. 1). Stimulatory effects of leukocidin on 32p incorporation into PIP, PIP2, and PA were observed to depend on toxin dose and were abolished by prior boiling (data not shown). Also, 0.1 ,ug of leukocidin induced 10 and 100% cytolysis of PMNs at 3 and 5 min, respectively (data not shown). However, the level of 32P-labeled PI in PMNs was not affected by treatment with the leukocidin F and S components. Neither the F nor the S component alone induced an increase of 32P-labeled PIP, PIP2, or PA. Furthermore, incorporation of 32Pi into other phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine was not influenced by
leukocidin (data not shown). Activation of PLC in rabbit PMN cell membranes by leukocidin F component. The stimulatory effect of the leukocidin components on PI metabolism in rabbit PMNs was further investigated by using a cell membrane preparation. When PMN cell membranes were incubated with 0.5 ,ug of both F and S components in the presence of 10 ,M NAD and [y-32P]ATP at 370C for 30 s, 32Pi was significantly incorporated into PIP, PIP2, and PA (Fig. 2, lane 3). This increased 32p incorporation was not observed in the absence of leukocidin (Fig. 2, lane 1), even when supplemented with 10 ,uM NAD (data not shown). The F component alone, but not the S component alone, enhanced incorporation of 32p; into PA and PIP2 in the presence of 10 ,uM NAD (Fig. 2, lanes 4 and 5). Although high concentrations (5 ,ug) of both the F and S components or the F component alone in the absence of NAD induced an increase of 32P-labeled PA in a dosedependent manner (data not shown), 0.5 p,g of leukocidin was used for the experiment to observe the effect of additions on the toxin. The 0.5 ,ug of leukocidin had little effect on 32p incorporation into PIP, PIP2, and PA in the absence of NAD (Fig. 2, lane 2) and had a strong effect on 32p incorporation into them in the presence of NAD (Fig. 2, lane 3). Addition of GTP (100 ,uM) to the reaction mixture containing leukocidin and NAD increased incorporation of 32Pi into PIP, PIP2, and PA (Fig. 3, lanes 2 and 4). GTP (100 ,uM) and NAD (10 ,uM) did not increase 32Pi labeling of PIP, PIP2, or PA in PMN cell membranes in the absence of leukocidin (Fig. 3, lane 3). Activation of PI and PIP kinases by leukocidin. To determine which component, F or S, stimulates PI and PIP kinases in rabbit PMN cell membranes, either PI or PIP as a substrate of PI or PIP kinase, respectively, was added to the reaction mixtures. 32P-labeled PIP was enhanced twofold by both the F and S components in the presence of PI and NAD but was not affected by the F or S component alone (Table 1). This incorporation was increased 3.9-fold by the further addition of 100 ,M GTP. On the other hand, when unlabeled PIP was added to the reaction mixture, the F component alone or both the F and S components induced a threefold increase in 32P-labeled
D004 pIpPI PE)
3 4 5 FIG. 2. Autoradiogram of the incorporation of 32Pi into polyphosphoinositides and PA in rabbit PMN cell membranes. PMN cell membranes (100 p,g of protein) with or without NAD (10 ,M) were incubated with 10 ,Ci of [-y-32P]ATP in the presence or absence of the leukocidin components (0.5 ,ug) at 37°C for 30 s. The autoradiogram of the thin-layer chromatography plate used for separating phospholipids is shown. The encircled regions (0) corresponded to the migration of the standard materials. The autoradiogram shown represents one of six experiments performed.
PIP2 in the presence of NAD compared with the control (Table 1). This was not seen with the S component alone. The increase of [32P]PIP2 induced by the F component alone or both the F and S components was further raised fivefold by the addition of 100 p,M GTP. DISCUSSION A characteristic feature of the polyphosphoinositide (PIP and PIP2) turnover involved in transmembrane signalling is a rapid, stimulus-induced increase in the synthesis of PIP and PIP2 (11). Therefore, we measured the incorporation of 32p; into PIP and PIP2 in response to the leukocidin F and S components. Stimulation of rabbit PMNs by the leukocidin F and S components resulted in increased incorporation of 32p; into PIP, PIP2, and PA. Both 32P-labeled PIP and PIP2 reached a maximal increase within 60 s, and then the radioactivities of both PIP and PIP2 gradually decreased to control levels, whereas 32P-labeled PA continuously increased over 3 min. These findings suggested that leukocidin stimulated the PLC-induced hydrolysis of the phosphoinositides, with the subsequent synthesis of PIP and PIP2. The aim of the present study was to determine the effect of the leukocidin F and S components on the activation of PI metabolic enzymes associated with rabbit PMN cell membranes, especially PI and PIP kinases. Recently, several studies showed that GTP analogs enhance the PIP kinase activities of placental or brain cell membranes incorporating
WANG ET AL.
l 1l ) NAINt)
TABLE 1. Stimulatory effects of leukocidin on the activities of PI and PIP kinases in rabbit PMN cell membranes
iI9~ iis I.1 l +
(0.5 ,ug) -
S+F S F S+F S F
32P radioactivity in: incorporated (cpm)b
FIG. 3. Autoradiogram of leukocidin-induced 32p incorporation into polyphosphoinositides and PA in PMN cell membranes with the addition of GTP. PMN cell membranes (100 ,ug of protein) were preincubated with or without 100 p.M GTP in ice for 20 min. The membranes with or without NAD (10 ,uM) were incubated with 10 ,uCi of [_y-32P]ATP in the presence or absence of the leukocidin components (0.5 jig) at 37°C for 30 s. The autoradiogram shown represents one of six experiments performed.
32p from [_y-32P]ATP into PIP in these membranes and that a G protein may be involved in activating PIP kinase (20, 23). PI kinase was activated by the combination of the F and S components of leukocidin in the presence of NAD (Table 1). The F or S component alone had no effect on the PI kinase activity even in the presence of NAD. The activation of PI kinase induced by the combination of the two components of leukocidin was enhanced by the treatment of the PMN cell membranes with GTP. The F or S component alone had no stimulatory effect on the PI kinase of GTP-treated PMN cell membranes in the presence of NAD. These data suggested that both components of leukocidin are required for the activation of PI kinase in PMN cell membranes and that the putative G proteins (54 and 59 kDa) which are ADP-ribosylated by the combination of the F and S components of leukocidin in the presence of GTP (7) regulate the activity of PI kinase. PIP kinase activity was also measured (Table 1). NAD-induced PIP kinase was activated by the F component alone as well as by both the S and F components. These results showed that the leukocidin F component stimulated GTP-dependent activation of PIP kinase and suggested that the putative G protein (41 kDa) which is ADP-ribosylated by the F component of leukocidin in the presence of GTP (7) regulates the activity of PIP kinase in rabbit PMN cell membranes. Phosphoinositide-specific PLC was activated by the combination of the F and S components of leukocidin or the F component alone in the presence of NAD (Fig. 2, lanes 3 and 5). It has been proposed that activation of certain cell surface receptors leads to stimulation of phosphoinositide-specific PLC activity which may involve the participa-
NAD (10 ,uM)
GTP (100 ,uM)
+ + +
± ± ± ±
10 (100) 5 (140) 10 (102) 10 (102)
44 ± 125 ± 50 ± 118 ±
10 10 10 10
± 10(101) ± 5 (191) ± 10(100)
40 ± 142 ± 52 ± 137 ±
129 244 128 131
159 ± 213 ± 158 ± 162 ±
160 ± 495 ± 158 ± 162 ±
5 (386) 10(123) 10 (127)
+ + + +
S+F S F
+ + + +
+ + + +
128 178 131 130
S+ F S F
± 10 (102)
10(124) 10(123) 10 (127)
73 ± 175 ± 68 ± 180 ± 67 ± 243 ± 81 ± 229 ±
(100) (284) (114) (268)
10(118) 10 (311)
10(166) 5 (398)
10(155) 5 (409)
10(152) 5 (552)
5 (520) a PMN cell membranes (100 ,ug of protein) were preincubated with or without 100 FM GTP in ice for 20 min. The preincubated samples were supplemented with 50 pLg of PI or PIP and then incubated at 37°C for 30 s with 10 p.Ci of [y-32P]ATP and other additions as indicated. The radioactivity of 32P-labeled PIP or PIP2 was measured as described in the text. b Numbers in parentheses are percentages compared with the value obtained when there was no addition to the assay mixture, which was defined as 100%. Values are means ± standard deviations for six experiments. C PI (50 ,ug) was added and the radioactivity incorporated in PIP was measured after incubation. d PIP (50 jig) was added and the radioactivity incorporated in PIP2 was measured after incubation.
tion of G proteins (4, 19). The putative G protein that regulates the activation of PLC has been called Gp(s) (1, 3). Therefore, activation of a phosphoinositide-specific PLC in PMN cell membranes by the F component alone or both the F and S components can be considered as an additional argument favoring the hypothesis that the putative G protein, Gp(s), corresponding to the G proteins (41, 54, and 59 kDa) ADP-ribosylated by leukocidin (7) may be involved in the functional receptors for phosphoinositide-specific PLCs in rabbit PMN cell membranes. Although the mechanism of leukocytolysis by staphylococcal leukocidin is complex, accumulation of PA and its precursor diacylglycerol may play an important role in destroying the lipid bilayer of leukocytes. These important questions remain to be addressed by further experimentation. ACKNOWLEDGMENTS This work was supported by grants 61870023, 63480150, and 01570228 from the Ministry of Education, Science and Culture of Japan. We thank Michiko Hatano for expert secretarial assistance. LITERATURE CITED 1. Banga, H. S., R. K. Walkere, L. K. Winberry, and S. E. Rittenhouse. 1988. Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation: effects on thrombin-mediated responses. Biochem. J. 252:297-300. 2. Gilman, A. G. 1984. G proteins and dual control of adenylate
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