When solubilized and proteolytically activated by insect gut proteases or proteinase K, the crystal was cytotoxic to insect and mammalian cells in vitro and was ...
JOURNAL
OF
BACTERIOLOGY, June 1989,
p.
3060-3067
Vol. 171, No. 6
0021-9193/89/063060-08$02.00/0 Copyright C) 1989, American Society for Microbiology
Purification and Properties of a 28-Kilodalton Hemolytic and Mosquitocidal Protein Toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E1O-2 FRANCIS A. DROBNIEWSKI* AND DAVID J. ELLAR Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 IQW, United Kingdom Received 1 August 1988/Accepted 1 January 1989
The mosquitocidal crystal of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2 was purified, bioassayed against third-instar Aedes aegypti larvae (50% lethal concentration, 7.5 ,ug/ml), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealing polypeptides of 125, 50, 47, and 28 kilodaltons (kDa). When solubilized and proteolytically activated by insect gut proteases or proteinase K, the crystal was cytotoxic to insect and mammalian cells in vitro and was hemolytic. By using nondenaturing polyacrylamide gel electrophoresis, a polypeptide of 23 kDa, derived from the 28-kDa protoxin, was identified which was hemolytic and cytotoxic to Aedes albopictus, A. aegypti, and Choristoneurafumiferana CF1 insect cell lines. The 23-kDa polypeptide was purified by ion-exchange chromatography and gave 50% lethal dose values of 3.8, 3.3, and 6.9 ,ug/ml against A. albopictus, A. aegypti, and C. fumiferana CF1 cells lines, respectively. Cytotoxicity in vitro was both dose and temperature dependent, with a sigmoidal dose-response curve. The cytotoxicity of the 23-kDa toxin and the solubilized and proteolytically activated 8-endotoxin was inhibited by a range of phospholipids containing unsaturated fatty acids and by triglyceride and diglyceride dispersions. An interaction with membrane phospholipids appears important for toxicity. Polyclonal antisera prepared against the 23-kDa polypeptide did not cross-react with polypeptides in the native crystals of four other mosquitocidal strains.
23-kDa polypeptide from the &-endotoxin crystal of B. thuringiensis subsp. darmstadiensis 73-E10-2 and found it to be cytolytic and hemolytic in vitro. A toxic polypeptide of 23 kDa, proteolytically derived from an inactive 28-kDa precursor, was purified by ion-exchange chromatography from the crystal. The 23-kDa toxin was found to bind phospholipids containing unsaturated fatty acids, which suggests that an initial interaction with membrane lipid is a prerequisite for the toxicity of this strain.
The gram-positive organism Bacillus thuringiensis produces a crystalline inclusion during sporulation which, in many strains, is toxic to lepidopteran larvae (22, 27). The first strain toxic to dipteran larvae, B. thuringiensis subsp. israelensis H-14, was identified a decade ago (13). Since then, several strains producing diptera-specific toxins have been identified: B. thuringiensis subsp. morrisoni PG14 (H 8a:8b) (26), B. thuringiensis subsp. darmstadiensis H-10 strains 73-E10-2 and 73-E10-16 (24, 25), Bacillus thuringiensis subsp. kurstaki HD-1, and other strains that produce a mosquitocidal P2 protein (38). The toxins occur in the inclusions as inactive protoxins that are solubilized in the alkaline environment of the insect gut and activated by gut
MATERIALS AND METHODS Bacterial strains, culture conditions, and harvesting conditions. B. thuringiensis subsp. darmstadiensis 73-E10-2 was obtained from K. Aizawa, Kyushu University, Kyushu, Japan. Growth and sporulation were essentially as described previously for B. megaterium KM (30). Briefly, six colonies from a plate culture were checked under a phase-contrast microscope for the presence of spores and crystals and inoculated into 250 ml of peptone-water-yeast extract broth for 12 h at 200 rpm and 30°C. A 1-ml sample of this culture was used to inoculate each of 8 liters of modified CCY sporulation medium (30). Cultures were then grown at 200 rpm and 30°C at a volume-to-flask ratio of 1:2 until sporulation was complete. Spores and crystals were harvested by centrifugation at 16,000 x g for 15 min at 4°C. The pellet was then washed three times in 1 M NaCl-10 mM EDTA at 4°C, and the spores and crystals were separated by differential ultracentrifugation, using sodium bromide gradients. Bands containing less than approximately 1% spore contamination as judged by phase-contrast microscopy were pooled, washed in deionized water, and stored at -20°C in 1 M NaCl. Crystals from B. thuringiensis subsp. israelensis and kurstaki were purified as described previously (31). Crystals from B. thuringiensis subspp. morrisoni PG14 and aizawai
proteases. The primary site of action of the lepidoptera-speclfic toxins is the midgut epithelium. This action leads to cell lysis, loss of permeability barriers to small ions and dyes, gut disruption, cessation of feeding, and death (reviewed in references 6, 15, and 22). Alkali-solubilized and proteolytically activated 6-endotoxins of B. thuringiensis subsp. israelensis and darmstadiensis 73-E10-2 have been shown to be cytolytic to several lepidopteran and dipteran cell lines (8, 18, 22, 31). Several mechanisms of action for different 6-endotoxins have been suggested, but recently a unifying mechanism has been proposed in which disruption of membrane integrity and eventual cytolysis occurs via a disruption of the colloid-osmotic or Donnan equilibrium (5, 7, 8, 19). Individual toxins purified or cloned from both lepidopteraand diptera-specific B. thuringiensis subspecies have been found to be larvicidal and cytolytic in vitro (3, 4, 26, 28, 35-37); in the case of a 27-kilodalton (kDa) toxin from B. thuringiensis subsp. israelensis, the protein was also observed to be hemolytic (3, 4, 35). In this study, we purified a *
Corresponding author. 3060
VOL. 171, 1989
B. THURINGIENSIS MOSQUITOCIDAL &-ENDOTOXIN
IC1 were the kind gifts of D. J. Earp and M. Z. Haider, respectively. Solubilization of crystals. Crystals were solubilized in 50 mM Na2CO3 (pH 10) with or without 10 mM dithiothreitol (DTT) for 90 min at 37°C. The supernatant remaining after centrifugation will be referred to as alkali-solubilized crystal or fraction. Gut protease activation of the alkali-solubilized crystal. The midguts of fourth-instar Pieris brassicae larvae were dissected and treated as described elsewhere (18). The guts of third-instar Aedes aegypti larvae were microdissected, homogenized in 50 mM Na2CO3 (pH 10), and cleared by centrifugation three times at 10,000 x g. Both gut extracts were stored at -20°C until required. Alkali-soluble fractions at a protein concentration of 1 mg/ml were treated with these extracts for 30 min at 37°C. For in vitro assays, proteolytic activation was stopped by addition of fetal calf serum (gut extract/fetal calf serum ratio of 2:1 [vol/vol]) before addition to cells. For sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), samples were precipitated with 12% (vol/vol) ice-cold trichloroacetic acid. After 30 min on ice, the samples were centrifuged at 10,000 x g, and the pellets were washed with acetone, recentrifuged, and dried in a stream of air. The pellets were then solubilized in gel sample buffer. Inhibition of gut protease activation. Gut proteases were incubated with 2 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, Mo.) for 10 min on ice. Treated proteases were incubated 1:1 (vol/vol) with 50 mM Tris hydrochloride (pH 7.5) for 30 min on ice and then incubated with the alkali-DTT-soluble fraction of strain 73-E10-2 as described above.
Purification of active toxin by anion-exchange chromatography. The spore-crystal pellet harvested from 8 liters of medium (-25 g [wet weight]) was extracted in 100 ml of 50 mM Na2CO3 (pH 10) overnight at 37°C. The extract was centrifuged three times at 16,000 x g for 15 min, and the supernatant was kept on ice. The pellet was reextracted for an additional 3 h. The extracts were combined, and the volume was adjusted to give a protein concentration of =1 mg/ml (-250 ml). The extracts were frozen at -20°C or activated immediately. The supernatant was then incubated with proteinase K (BDH Ltd., Poole, United Kingdom) at 100 ,ug/ml for 15 min at 37°C. The enzyme was inhibited by addition of phenylmethylsulfonyl fluoride to a final concentration of 1 mM. The supernatant was then dialyzed against 50 mM NH4HCO3 (pH 8.1)-0.1 mM phenylmethylsulfonyl fluoride for 3 h and then against 50 mM NH4HCO3 (pH 8.1) overnight at 4°C. Some precipitation of nontoxin components occurred. The protease-treated and dialyzed extract was clarified by centrifugation three times at 16,000 x g at 4°C. The extract was applied at 32 ml/h and 4°C to a DE52 column (2 by 15 cm; Whatman Ltd., Maidstone, United Kingdom) that had been equilibrated in 50 mM NH4HCO3 (pH 8.1). The column was washed with 2 column volumes of the equilibriation buffer. The protein was eluted with a linear gradient of 125 ml each of 50 and 300 mM NH4HCO3 (pH 8.1) for toxin used for antibody production (see below) or 200 ml each of the same buffers for toxin used in in vitro and in vivo assays. The flow rate was 18 ml/h, and the fraction size was 1.6 ml. Toxic fractions were identified by cytotoxicity assays using an exclusion dye, trypan blue. Fractions were subjected to PAGE with and without SDS (SDS-PAGE and non-SDSPAGE). For toxicity assays, only the two or three central peaks were pooled; for antibody production, a wider range
3061
of fractions were pooled. In both cases, the pooled fractions were dialyzed against deionized water at 4°C overnight, lyophilized, and stored at -80°C. For antibody production, lyophilized fractions were suspended in gel sample buffer (50 mM Tris hydrochloride [pH 7.5], 1% [wt/vol] SDS, 1 mM EDTA, 15% [wt/vol] glycerol, 25 mM DTT, 0.5% [wt/vol] bromophenol blue), heated at 100°C for 5 min, and run on SDS-PAGE (see below). The toxin band was visualized with 1 M KCl, excised, and eluted into 3 ml of 50 mM NH4HCO3 (pH 8.1) for 10 h on a shaking water bath at 25°C. The eluate was exhaustively dialyzed against deionized water at 4°C and lyophilized. Antibody production. Antisera against the lyophilized B. thuringiensis subsp. darmstadiensis toxin and the purified and SDS-denatured (see below) P2 protein of B. thuringiensis subsp. kurstaki HD1 were prepared as previously described (8). Antiserum against the undenatured, active P1 protein of B. thuringiensis subsp. kurstaki HD1 was the kind gift of B. H. Knowles. Antisera to the purified and SDSdenatured 130- and 26- to 28-kDa polypeptides of B. thuringiensis subsp. israelensis were the kind gifts of T. J. Sawyer and E. S. Ward, respectively. PAGE. SDS-PAGE was performed essentially as described previously (20), using 1.5-mm-thick 13% (wt/vol) gels (except where stated). Gels were stained with 0.1% Coomassie blue R250 (Sigma) or silver stained by the method of Ansorge (1). Peptide mapping was performed as described previously (29), using a-chymotrypsin (Sigma). Nondenaturing PAGE was performed at 4°C as described elsewhere (14). Non-SDS-PAGE elution involved cutting the gel horizontally into slices of equal width (-5 mm), which were eluted by diffusion overnight at 25°C in 1 ml of 50 mM Na2CO3 (pH 10)-10 mM DTT. Protein determination. Protein concentrations were measured by the method of Lowry et al. (21), using bovine serum albumin as the standard. Bioassays. Bioassays with lepidopteran (P. brassicae) and dipteran (A. aegypti) larvae were performed as described previously (31). Larvae placed in water alone constituted the controls. Immunoblotting. Protein blotting was performed as described by Towbin et al. (33), with modifications. Samples were electrophoresed on 13% SDS-gels and blotted overnight at 150 mA and room temperature onto two sheets of nitrocellulose paper (Schleicher & Schuell GmbH, Dassel, Federal Republic of Germany). The second sheet was stained with 0.1% amido black lOB (Raymond A. Lamb, Alperton, Middlesex, United Kingdom) in methanol-glacial acetic acid-water (45:10:45) for 5 min, destained in H20, and used as a reference for the first sheet. The first sheet was blocked in 2% (wt/vol) skimmed milk powder (Marvel; Cadbury's Ltd.) in Tris-buffered saline (pH 7.4) and incubated with the prepared antibody in the blocking buffer for 2 h. The paper was then washed with Tris-buffered saline and incubated with secondary antibody (goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase [Miles Laboratories, Inc., Elkhart, Ind.]) for 90 min, followed by further washing with Tris-buffered saline. 4-Chloro-1-naphthol (Sigma) was used as the color substrate. Cell assay. In vitro cytotoxicity assays were carried out by vital staining with the exclusion dye trypan blue (Sigma) (7, 8, 18, 31). Liposomes. Multilamellar liposomes were prepared as described previously (32). All lipids, cholesterol, dicetyl phosphate, and stearylamine were from Sigma. The lipids were examined on thin-layer chromatography plates (Polygram Sil
3062
DROBNIEWSKI AND ELLAR
(b)
(a)
J. BACTERIOL. (c)(d)
K125K ZOOM
83K 77K 69K
50K
50K
28K
FIG. 1. SDS-PAGE polypeptide profile of 50 p.g of purified native 73-E10-2 crystal (a), probed (b) with antiserum raised against the active 23-kDa toxin in an immunoblotting system as described in Materials and Methods. A single polypeptide band of 28 kDa, the protoxin, is indicated. Native crystal (50 ,ug) was solubilized (c) in 50 mM Na2CO3 (pH 10) without DIT and probed (d) with anti-23-kDa antiserum. A single 28-kDa polypeptide was again noted.
G; Macherey-Nagel, Duren, Federal Republic of Germany), using a chloroform-methanol-H20 (65:25:4) system to ensure purity, and were used without further purification. Glyceride dispersions were prepared as described elsewhere (2). Tissue culture. Aedes albopictus, A. aegypti, Choristoneura fumiferana CF1 and 63CF1, Spodoptera frugiperda, Anopheles gambiae, Anopheles stephensi, Culex quinquefasciatus, Drosophila melanogaster, Heliothis zea, and mouse L929 cells were maintained in 25-cm2 tissue culture flasks (Nunc) at 27 or 37°C (31). Cell lipid extraction. Total cell lipid from A. albopictus cells was extracted by a modified form of the method of Folch et al. (11). The chloroform layer, which contained the extracted lipid, was dried under vacuum in a rotary evaporator at 30°C. The lipid was suspended in phosphate-buffered saline (PBS; pH 7.4), gently sonicated in a sonic cleaning bath, and used immediately. Hemolytic assay. Rabbit, human, sheep, and horse erythrocytes were serially diluted in round-bottom-well microdilution plates (Nunc) in 100 p.l of 50% (vol/vol) PBS-0.05% (wt/vol) gelatin-2.25% (wt/vol) glucose (31). Quantitative hemolysis was measured by hemoglobin release at 540 nm. Hemolytic activity in non-SDS-gels was visualized by using a blood overlay; i.e., human erythrocytes were suspended at a final concentration of 2% (vol/vol) in PBS containing 1% (wt/vol) agarose (Sigma) and 50 ,ug of ampicillin per ml, poured onto the gel to a depth of 5 mm, and allowed to set. Protease digestion. Trypsin (tolylsulfonyl phenylalanyl chloromethyl ketone treated), a-chymotrypsin, subtilisin, and pronase were obtained from Sigma; proteinase K and Staphylococcus aureus V8 protease were obtained from BDH and Miles Laboratories, respectively. The alkaii-solubilized fraction (see above), at a protein concentration of 1 nmg/ml, was treated with 100 ,ug of protease per ml for 2 to 30 min at 37°C. Purification of B. thuringiensis subsp. kurstaki P2 protein. The 8-endotoxin crystals of B. thuringiensis subsp. kurstaki were purified as previously described (31). The P1 protein was largely removed by extracting purified crystals with 50 mM Na2CO3 (pH 9.5)-10 mM DTT for 60 min (31). P2 was
purified from the remaining P1 by preparative SDS-PAGE with 7.5% (wt/vbl) slab gels and elution into 50 mM Na2CO3 (pH 10.0)-10 mM DTT-0.1% SDS. After dialysis against deionized water, the sample was lyophilized, suspended in PBS, and used to produce antibodies as described above.
RESULTS Isolation, solubilization, and activation of the 8-endotoxin. The crystal 8-endotoxin of B. thuringiensis subsp. darmstadiensis 73-E10-2 was purified by sodium bromide density gradient centrifugation and subjected to SDS-PAGE. This procedure revealed several polypeptides of 125, 83, 79, 77, 69, 50, and 28 kDa, as reported earlier (F. A. Drobniewski, T. J. Sawyer, and D. J. Ellar, Bacterial Protein ToxinsSecond European Workshop, Zentralbl. Bakteriol. [Naturwissen], vol. 15, p. 55-56, 1986) (Fig. 1). Peptide mapping indicated that the 69- to 83-kDa polypeptides were derived from the 125-kDa polypeptide through proteolysis (data not shown). Purified crystals were bioassayed against the dipteran larva A. aegypti and the lepidopteran larvae P. TABLE 1. Toxicity spectrum of solubilized and activated crystal proteins in vitro %
Cell line or erythrocyte species (106 cells/ml)
or
Aedes albopictus ........................ ................ A. albopictusb ........................................ A. albopictusc......................................... A. aegypti ........................................ A. aegyptib ........................................
Choristoneura fumiferana CF1 ................................ C. fumiferana CF1b ......................................... Heliothis zea ............... .........................
Live cells' hemolytic 5.0 99.0 6.0 4.0 99.0
40.0 100.0 44.0
Anopheles gambiae ........................................
2.0
Culex quinquefasciatus ........................................ Drosophila melanogaster ....................................... Mouse L929 cells .............................. ........... Mouse L929 cellsb ................................... ......
4.0
Human + activated toxind ...................................... Human + activated toxine ...................................... Human (control) ........................... ............... Human + activated toxin + Aedes total cell lipidf Human + activated B. thuringiensis subsp. israelensis toxing .......................................... Horse + activated toxin ........................................ Horse (control) ...................... ................... ......
Rabbit + activated toxin ........................................ ................... Rabbit (control) ...................... Sheep + activated toxin ........................................ Sheep (control) ...................... ...................
4.0 83.0 99.0
1:256 0 1:2 1:2 1:512 1:64 0 1:32 0 0 0
a Viability was assessed by the inability to exclude trypan blue dye after 60 min of incubation at 25°C with 25 FLg of soluble b-endotoxin activated with P. brassicae gut extract per ml. b Cells were incubated as described in footnote a but with 75 ,ug of soluble, unactivated toxin only per ml. C Toxin solubilized without DTT (25 ,g/ml) and then activated as described in footnote a. d Erythrocytes were incubated at 23°C (with one exception; footnote e) for up to 12 h. The first well contained 25 ,ug of soluble, activated 8-endotoxin per ml. eErythrocytes were incubated as described in footnote d but at 4°C. f As described in footnote e except that toxin was preincubated for 120 min at 25°C with total cell lipid prepared from 106 A. Ialbopictus cells, using a modification of the method of Folch et al. (11), and suspended in 1 ml of PBS
(pH 7.4). g Erythrocytes were incubated as described in footnote f. The first well contained 15 ,ug of soluble, activated B. thuringiensis subsp. israelensis
f-endotoxin per ml.
B. THURINGIENSIS MOSQUITOCIDAL b-ENDOTOXIN
VOL. 171, 1989 b
a
68K
.
67K
63K a ~_62K
45K
_."
_ao 5 O K
36K 29 K
23K FIG. 2. SDS-PAGE polypeptide profile of 100 ,ug of crystal solubilized in 50 mM Na2CO3 (pH 10)-10 mM DTT and activated with P. brassicae (a) or A. aegypti (b) gut extract for 30 min at 37°t. Proteolytic activation was stopped by trichloroacetic acid precipitation. Similar polypeptide profiles were obtained with either activation system.
brassicae and Artica caja but exhibited toxicity only against the former (50% lethal concentration, 7.5 ,ug of crystal per ml of water). The purified &-endotoxin crystals were solubilized in 50 mM sodium carbonate buffer (pH 10)-10 mM flTT for 90 min at 37°C. This alkali-soluble material, assayed against -106 A. albopictus (diptera) or C. fumiferana CF1 (lepidoptera) cells in an in vitro system was not cytotoxic at the concentrations used (Table 1). The alkali-soluble fraction (1 mg of protein per ml) was proteolytically incubated with larval gut extracts as described in Materials and Methods, and the effects of these activation regimens on the solubilized inactive 8-endotoxin were monitored by SDS-PAGE (Fig. 2). Aedes and Pieris activation produced comparable
3063
activation profiles which gave comparable toxicities against A. albopictus and C. fumiferana CF1 cell lines (data not shown). When activated, the 73-E10-2 8-endotoxin was found to be cytotoxic to all lepidopteran and dipteran lines assayed and to a Drosophila cell line, as assessed by vital staining with trypan blue. The mouse line L929 was relatively resistant (Table 1). Visible in vitro effects consisting of cell swelling, granulation, and rounding up, occurred within minutes, followed by cell blebbing, a general breakdown of permeability, and finally lysis. The nuclear membrane remained intact almost until cell lysis (Fig. 3). These features are similar to those noted for other &-endotoxins (6, 9, 15, 31). The crystal, when solubilized in sodium carbonate buffer (pH 10) without DTT and proteolytically activated, was also found to be toxic to the cell lines tested (Table 1). Hemolytic activity. Erythrocytes showed various levels of susceptibility (human > horse > rabbit) at 23°C but were significantly less susceptible at 4°C (Table 1). Sheep erythrocytes were essentially resistant at both temperatures. Immunological relatedness of the 8-endotoxin of 73-E10-2 to the P1, P2, and 25- to 28- and 130-kDa polypeptides of B. thuringiensis subsp. israelensis. None of the crystal polypeptides of 73-E10-2 were found to cross-react with antiserum raised against the P2 polypeptide of B. thuringiensis subsp. kurstaki HD1 (38) or against the B. thuringiensis subsp. israelensis 25- to 28-kDa polypeptide (34), as determined by immunoblotting (data not shown). Cross-reactivity was observed between antiserum to the P1 polypeptide of B. thuringiensis subsp. kurstaki (18) and polypeptides of 83 and 77 kDa and also between an antiserum raised against polypeptides of 130 kDa from B. thuringiensis subsp. israelensis and the 125- and 69- to 83-kDa polypeptides in strain 73-E10-2 (data not shown). Peptide mapping of the solubilized crystal proteins with x-chymotrypsin indicated that the group of polypeptides of 69 to 83 kDa were derived from the 125-kDa polypeptide (data not shown).
FIG. 3. Inhibition of cytotoxicity of alkali-solubilized and proteolytically activated 73-EIO-2 &-endotoxin. A. albopictus cells were treated with activated toxin alone (A) or with toxin that had been preincubated with total cell lipid extracted from A. albopictus cells as described in Materials and Methods (B); 25 ,ug of toxin per ml was incubated with 106 cells for 60 min. Bars, 25 p.m.
3064
DROBNIEWSKI AND ELLAR
However, none of the antibodies was able to cause a significant decrease in toxicity when preincubated singly with the activated 73-E10-2 toxin before assay on A. albopictus or C. fumiferana CF1 cells. Furthermore, none of the P1 cross-reacting polypeptides was solubilized when DTT was absent from the alkaline buffer (Fig. lc), but the preparation was still toxic to all the cell lines. This finding suggested that the major polypeptide responsible for dipteran toxicity in vitro was one of the other polypeptides. However, a role in in vivo toxicity is not precluded. Non-SDS-PAGE. Time course activation studies in which solubilized crystal was activated with P. brassicae gut extract suggested that converting a native 28-kDa protoxin to a stable 23-kDa polypeptide could account for the toxicity of the crystal (data not shown). Further evidence of the importance of the 23-kDa polypeptide was provided by nondenaturing PAGE. Carbonate-DTT-solubilized &-endotoxin was activated with P. brassicae gut extract as described in Materials and Methods before incubation on ice. This mixture was then electrophoresed immediately on a non-SDS-gel, polypeptides were eluted from the gel as described in Materials and Methods, and 25 RI of each eluate was assayed against 100 RI of three cell lines (Fig. 4). The same eluates were toxic to all cell lines tested. An overlay of human erythrocytes onto the non-SDS-gel produced a band of hemolysis corresponding to the position from which the toxic eluates were derived (data not shown). Eluted cytolytic fractions run on SDS-gels contained a pair of bands of 23 and 25 kDa (Fig. 4). The crystal was also solubilized in carbonate buffer without DTT and treated in the same fashion. Toxic eluates were produced from gel slices whose mobility on SDS-PAGE corresponded to the mobilities of the toxic fractions derived from carbonate-DTT solubilization, i.e., 23 to 25 kDa (data not shown). Purification of the active toxin. Activation of the 5-endotoxin by using purified serine proteases and metalloproteases was also attempted as described in Materials and Methods. Treatment of the alkali-soluble fraction with proteinase K, subtilisin, or pronase for 2 to 30 min yielded a preparation as cytotoxic to A. albopictus as that produced with P. brassicae gut treatment. SDS-PAGE of the preparations revealed a polypeptide of 23 kDa, among others, and a hemolytic band on a denaturing gel overlaid with human erythrocytes with the same mobility as that obtained when gut-activated toxin was used (data not shown). The migration of this toxic polypeptide close to the anode on the gel suggested that under the pH conditions used, the polypeptide was negatively charged and might be separable from the other, more positively charged polypeptides in the mixture by anionexchange chromatography. This was performed as described in Materials and Methods. The toxin was eluted, and 25 pI of eluted fractions was assayed against A. albopictus cells. The A280 for each fraction was also noted (Fig. 5). The toxic 23-kDa polypeptide was eluted at "175 mM NH4HCO3 with proteinase K, appearing largely in the wash fractions. Fractions spanning protein peaks were subjected to SDS-PAGE, and those corresponding to the single toxicity peak (fractions 79 to 88) were pooled and rerun on SDS-PAGE (Fig. 5) to reveal a single band at 23 kDa. In vitro toxicity of the 23-kDa polypeptide. The 50% lethal concentrations for A. albopictus, A. aegypti, Anopheles stephensi CF1, and H. zea were 3.8, 3.3, 3.5, 6.9, and 5.2 ,ug/ml, respectively. These values represent the concentrations of proteinase K-activated 23-kDa polypeptide killing 50% of the cells in 60 min, as judged by trypan blue staining.
J. BACTERIOL.
23
456
7
8 9 10 S 11
68K
45K 36K 29K
_,_
-ooo
23K f.M 24K .-
-
20K
-oo
14K
100
90 0)
70L
(0 60
soL
30 F 20 10 0
2
4
6
8
10
12
14
Horizontal slice FIG. 4. SDS-PAGE profile of solubilized and protease-treated 73-E10-2 crystal separated on a non-SDS-gel. The non-SDS-gel was divided into horizontal slices, and eluates from gel slices 2 to 11 were subjected to SDS-PAGE, with corresponding cytotoxicity against 106 A. albopictus [Aa(s)], Culex quinquefasciatus, and C. fumiferana CF1 cells. Lane S, Molecular size markers of 68 (bovine serum albumin), 45 (ovalbumin), 36 (glyceraldehyde-3-phosphate dehydrogenase), 29 (carbonic anhydrase), 24 (trypsinogen), 20 (trypsin inhibitor), and 14.2 (a-lactalbumin), kDa.
The dose-response curves were steep and sigmoidal, which may reflect multihit kinetics. Doses causing 50% hemolysis against human and sheep erthrocytes were 3.6 and 8.5 jig/ml, respectively. Bioassay of the 23-kDa polypeptide. Pooled, dialyzed, and freeze-dried toxic fractions were diluted into a total of 3 ml of distilled water into which were placed 25 A. aegypti larvae. Fifty percent of the larvae were killed by 8 ,ug of soluble 23-kDa polypeptide per ml in 24 h. Antibody production, inhibition of 23-kDa polypeptide toxicity, and immunoblotting. Antiserum to the purified 23-kDa polypeptide was used in an immunoblotting system to identify the inactive precursor from which the polypeptide was proteolytically derived, by either gut or proteinase K activation, and to examine inhibition of the toxin by immunoprecipitation. Purified toxin blotted onto nitrocellulose and probed with 23-kDa antiserum showed a single band corresponding to the mobility of the 23-kDa polypeptide (data not shown). Probing of the native crystal and the Na2CO3
3065
B. THURINGIENSIS MOSQUITOCIDAL &-ENDOTOXIN
VOL. 171, 1989
TABLE 2. Inhibition of activated 73-E10-2 toxicity against 106 A. albopictus cells by preincubation with different phospholipids at 25°C
100)
% of
Toxin/lipid
Lipid'
0 U)J
C-)
U) cn
a 0
-_ CD)
-0
Unsaturatedc 11
0D
Toxin alone Phosphatidylcholine aloneb Phosphatidylcholine Phosphatidylethanolamine Phosphatidyl inositol Phosphatidyl glycerol Phosphatidyl serine Sphingomyelin a-L-Soybean phosphatidylethanolamine Unsaturated
I
(U)
ratio
a-L-Dipalmitoyl phosphatidylethanolamine (saturated) a-L-Dioleoyl phosphatidylcholine (unsaturated) a-L-Dipalmitoyl phosphatidylcholine (saturated)
.
0
ZR0 -o
i
Dioleind Trioleind a
;I,,
Except
as
specified
in footnotes b and
c,
all
A.
albopictus
cells alive after 60 min
1:25 1:25 1:25 1:25 1:25 1:25 1:30
34 72 78 98 93 90 84 67
1:25 1:25 1:25
78 82 39
1:25
87
1:25
33
1:50 1:50
47 57
liposomes
were
prepared
phospholipid/cholesterol/dicetyl phosphate ratio of 2:1.5:0.5. b Phosphatidylcholine liposomes prepared without cholesterol phosphate.
with
a
60)
" F r a c t io n N u m b e r
UiLotLyl rIIlIsc I1h ILo conlt;iering rrinnII alU aaitionaUll ^citizLV alicetyl pnos2pnadte posititve cnartge;
extract of the native crystal gave a single band corresponding to the 28-kDa polypeptide in the native crystal (Fig. 1). No cross-reactivity was observed with any polypeptides from native crystals purified from B. thuringiensis subsp. kurstaki (HD1), aizawai IC1, morrisoni PG14, and, most importantly, israelensis (data not shown). Furthermore, the 23-kDa antiserum was able to protect A. albopictus cells from the cytotoxic effects of purified 23-kDa toxin (82% of cells were alive after 60 min of incubation with 10 ,ug of 23-kDa toxin per ml and antiserum, compared with only 5% of cells incubated with toxin and preimmune sera) and protect A. albopictus cells from the alkali-soluble and gut-activated 8-endotoxin (99% of A. albopictus cells were alive after 60 min of incubation with 20 ,ug of gut-activated 5-endotoxin per ml, compared with 64% of cells incubated with toxin and preimmune sera). Lipid receptor. Several observations pointed to a lipid receptor for the activated 73-E10-2 5-endotoxin. Preincubation of activated 5-endotoxin with a lipid dispersion prepared from A. albopictus cells prevented hemolysis of human erythrocytes (Table 1) and cytolysis of A. albopictus cells (Fig. 3). In a series of experiments, multilamellar liposomes formed from chromatographically pure phosphatidylcholine, phosphatidylethanolamine, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, and phosphatidyl glycerol were shown to inhibit gut-activated 73-E10-2 5-endotoxin (Table 2). Liposomes prepared from phosphatidylcholine alone or phosphatidylcholine with cholesterol were equally inhibitory (Table 2), which indicated that cholesterol was not the receptor for the toxin.
dicetyl
thL ine iiposome. on1 Glycerides were dissolved in acetone at 95°C and dispersed in PBS (pH 7.4) at 30°C as described in Materials and Methods. iLiLIII4I
FIG. 5. Cytotoxicity against A. albopictus cells of fractions from ion-exchange chromatography of solubilized and proteinase Kactivated B. thuringiensis subsp. darmstadiensis 73-E10-2 proteins. A 100-,ul sample of A. albopictus cells (106/ml) was incubated with 25 p.l of each fraction for 60 min at 25°C. Inset, 10 ,ug of ionexchange-purified active 23-kDa toxin.
or
Phosphatidylethanolamine liposomes prepared with stearylamine instead
c
(Jl
li
d
The net charge on the liposomes did not appear to be a significant factor either, since phosphatylethanolamine liposomes on which additional negative or positive charge had been conferred by the inclusion of dicetyl phosphate or stearylamine, respectively, were equally effective in neutralizing the activated toxin (Table 2). Bacillus subtilis protoplasts, which contain no significant amounts of unsaturated fatty acid (32), were unaffected by 25 ,ug of gut-activated 5-endotoxin per ml (data not shown), which suggested that the degree of unsaturation may be a significant factor in toxicity. Of the liposomes prepared with the synthetic phospholipids (cx-L-dipalmitoyl phosphatidylethanolamine and phosphatidylcholine and a-L-dioleoyl phosphatidylcholine), only those possessing unsaturated fatty acids were inhibitory to the toxin (Table 2). Inhibition experiments using dispersions of di- and triglycerides were also performed. Some protection was provided by both glyceride preparations, with triolein inhibiting more than diolein (Table 2). The importance of membrane fluidity was demonstrated in inhibition experiments using dimyristoyl phosphatidylcholine at temperatures at which the phospholipid would be above or below its phase transition temperature of 23°C. No protection against the 73-E10-2 toxin was obtained at temperatures below or above the transition temperature (Fig. 6). This finding did not, however, exclude the possibility that the requirement for membrane fluidity was secondary to an absolute requirement for unsaturated fatty acids. Since phosphatidylcholine dimyristoyl is saturated, this secondary requirement could be masked by the absence of unsaturated fatty acids. The cryoprotectant dimethyl sulfoxide is believed to stabilize membranes by decreasing their fluidity (23). Therefore, if membrane fluidity was a significant factor, then after
3066
DROBNIEWSKI AND ELLAR
J. BACTERIOL. TABLE 3. Lipid inhibition of toxicity of the 23-kDa polypeptidea
100
Lipid' Ljpjdb
90
Toxin alone 4°C 20°C 250C 370'C
a)
CIO 91) C.)
(9)
No lipid PC dielaidoyl PC dioleoyl
X -
PC dipalmitoyl PC dilinoleoyl
A-v PE dipalmitoyl
-o
0.-
PE (from soybean)
0
.0
(9)
Toxin/lipid
ratio (wt/wt)
1:100 1:50 1:100 1:100 1:50 1:100 1:150 1:100 1:150 1:50 1:100 1:150
% of cells alive after:
30
mi
17 66 75 86 19 62 74 84 23 28 76 87 100
60
mi
9 ND 53 75 19 55 66 79 5 15 51 79 99
"The 23-kDa polypeptide (5 pLg) was incubated with lipid at 25'C for 60 min. All assays were performed in duplicate, using 106 A. albopictus cells per ml. ND, Not determined. b PC, Phosphatidylcholine; PE, phosphatidylethanolamine.
(9) 'a) a)
*R 10
0
40
80
120
160
220
Time (min) FIG. 6. Effect of preincubation of toxin with phosphatidylcholine dimyristoyl liposomes. Soluble and activated 73-E10-2 crystal 8-endotoxin (50 ,.tg/ml) was incubated at temperatures above and below the phase transition temperature of the lipid (23°C) before centrifugation and incubation with 106 A. albopictus cells.
treating A. albopictus cells with increasing concentrations of dimethyl sulfoxide we might expect to see correspondingly decreasing levels of cytotoxicity. This was the case regardless of whether the cells were preincubated with dimethyl sulfoxide before or simultaneously with toxin addition (73% of A. albopictus cells were alive after treatment of cells with 10% [vol/vol] dimethyl sulfoxide and 50 jig of gut-activated &-endotoxin per ml, compared with 36% of cells treated with toxin alone). Membrane fluidity is therefore a factor in the toxicity of this strain, as suggested for the B. thuringiensis subsp. israelensis toxin (32). In vitro cytotoxicity of the 23-kDa polypeptide to A. albopictus cells was similarly inhibited by lipids containing unsaturated fatty acids. The use of both phosphatidylcholine dielaidoyl and dioleoyl indicated that the single double bond could be in either cis or trans orientation. Phospholipids containing saturated fatty acids provided no protection (Table 3). DISCUSSION In this study, we isolated the mosquitocidal crystals of B. thuringiensis subsp. darmstadiensis 73-E10-2 and identified and purified a cytotoxic, hemolytic, and mosquitocidal polypeptide within it. The crystal polypeptide profile is in agreement with that presented in a preliminary report (see Results) and with the values recently reported (10) for this crystal, with one exception: the crystal described here lacked the 144-kDa polypeptide noted previously. The majority of these toxins are plasmid encoded, and the putative plasmid encoding the 144-kDa toxin could have been lost during culturing.
Separation of the gut-activated alkali or alkaliDTT-soluble fraction on nondenaturing gels also indicated that the "'23-kDa polypeptide was responsible both for cytotoxicity against lepidopteran and dipteran lines and for hemolytic activity. Ion-exchange-purified 23-kDa toxin showed the characteristic cytolytic, hemolytic, and larvicidal properties of the strain. The 23-kDa B. thuringiensis subsp. darmstadiensis polypeptide was derived from an inactive 28-kDa protoxin but was immunologically distinct from the 26- to 28-kDa polypeptide of B. thuringiensis subsp. israelensis in blotting experiments. Kim et al. (17) reported that a 67-kDa toxin in strain 73-E10-2 was mosquitocidal. They raised a polyclonal antiserum to this polypeptide and showed that it did not cross-react with the =65-kDa mosquitocidal polypeptide from B. thuringiensis subsp. israelensis (17). Therefore, the crystal from strain 73-E10-2 may contain two mosquitocidal polypeptides (67 and 23 kDa), with only the smaller polypeptide possessing cytolytic and hemolytic activity, as appears to occur in the B. thuringiensis subsp. israelensis crystal (4, 7, 16, 17, 28, 32, 35, 37). Cytotoxicity could be inhibited by preincubating the gutactivated alkaline-DTT-solubilized fraction with multilamellar liposomes prepared from phospholipids consisting of unsaturated fatty acids. The purified 23-kDa polypeptide was similarly inhibited only by liposomes prepared with unsaturated fatty acids. Membrane fluidity was not the sole determining factor. The head group was not significant; polar and acidic groups were both effective in neutralizing toxicity. This finding is in agreement with results of recent work demonstrating the importance of fatty acids in the syn-2 position (which are usually unsaturated) of membrane phospholipids in the hemolytic activity of B. thuringiensis subsp. israelensis 8-endotoxin (12). Since antiserum prepared against the B. thuringiensis subsp. israelensis 26- to 28-kDa polypeptide did not crossreact with any polypeptides of strain 73-E10-2, the 23-kDa polypeptide, which is proteolytically derived from a 28-kDa precursor, is a second mosquitocidal protein that appears to kill cells in vitro by a cytolytic mechanism similar to that observed for B. thuringiensis subsp. israelensis. ACKNOWLEDGMENTS This work was supported by the Science and Engineering Research Council and the Agriculture and Food Research Council.
VOL. 171, 1989
B. THURINGIENSIS MOSQUITOCIDAL 8-ENDOTOXIN
We thank A. Symonds, T. J. Sawyer for preparation of most of the purified f-endotoxin crystals, W. E. Thomas for demonstrating the micro-gut dissection techniques, and B. H. Knowles for demonstrating tissue culture techniques. LITERATURE CITED 1. Ansorge, W. 1985. Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J. Biochem. Biophys. Methods 11:13-20. 2. Arbuthnott, J. P., J. H. Freer, and B. Billcliffe. 1973. Lipidinduced polymerisation of staphylococcal oa-toxin. J. Gen. Microbiol. 75:309-319. 3. Armstrong, J. L., G. F. Rothman, and G. S. Beaudreau. 1985. Delta endotoxin of Bacillus thuringiensis subsp. israelensis. J. Bacteriol. 161:39-46. 4. Davidson, E. W., and T. Yamamoto. 1984. Isolation and assay of the toxic component from the crystals of Bacillus thuringiensis var. israelensis. Curr. Microbiol. 11:171-174. 5. Drobniewski, F. A., and D. J. Ellar. 1988. Investigation of the membrane lesion induced in vitro by two mosquitocidal endotoxins of Bacillus thuringiensis. Curr. Microbiol. 16:195199. 6. Ebersold, H.-R., P. Luthy, P. Geiser, and L. Ettlinger. 1978. The actions of the delta-endotoxin of Bacillus thuringiensis: an electron microscope study. Experientia 34:1672. 7. Ellar, D. J., B. H. Knowles, F. A. Drobniewski, and M. Z. Haider. 1986. The insecticidal specificity and toxicity of Bacillus thuringiensis delta-endotoxins may be determined respectively by an initial binding to membrane-specific receptors followed by a common mechanism of cytolysis, p. 7-10. In R. A. Samson, J. M. Valk, and D. Peters (ed.), Fundamental and applied aspects of invertebrate pathology. Grafisch bedrijf Ponsen and Looijen, Wageningen, The Netherlands. 8. Ellar, D. J., W. E. Thomas, B. H. Knowles, E. S. Ward, J. Todd, F. Drobniewski, J. Lewis, T. Sawyer, D. Last, and C. Nicholls. 1985. Biochemistry, genetics, and mode of action of Bacillus thuringiensis delta-endotoxins, p. 230-240. In J. Hoch and P. Setlow (ed.), Molecular biology of microbial differentiation. American Society for Microbiology, Washington, D.C. 9. Faust, R. M., R. S. Travers, and G. M. Hallam. 1974. Preliminary investigations on the molecular mode of action of the delta-endotoxin produced by Bacillus thuringiensis var. alesti. J. Invertebr. Pathol. 23:259-261. 10. Federici, B. A., J. E. Ibarra, and L. E. Padua. 1988. Parasporal body of mosquitocidal subspecies of Bacillus thuringiensis, p. 115-131. In K. Maramorosch (ed.), Biotechnology advances in invertebrate pathology and cell culture. Academic Press, Inc., San Diego. 11. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. 12. Gill, S., G. J. P. Singh, and J. Hornung. 1987. Cell membrane interaction of Bacillus thuringiensis subsp. israelensis cytolytic toxins. Infect. Immun. 55:1300-1308. 13. Goldberg, L. J., and J. Margalit. 1977. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti and Culex pipiens. Mosq. News 37:355-358. 14. Hedrick, J. L., and A. J. Smith. 1968. Size and charge isomer separation and estimation of molecular weight of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126:155-164. 15. Heimpel, A. M., and T. A. Angus. 1960. Bacterial insecticides. Bacteriol. Rev. 24:266-288. 16. Hurley, J. M., S. G. Lee, R. E. Andrews, Jr., M. J. Klowden, and L. A. Bulla, Jr. 1985. Separation of the cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis. Biochem. Biophys. Res. Commun. 126:961-965. 17. Kim, K.-H., M. Ohba, and K. Aizawa. 1984. Purification of the toxic protein from Bacillus thuringiensis serotype 10 isolate demonstrating a preferential larvicidal activity to the mosquito. J. Invertebr. Pathol. 44:214-219. f-
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18. Knowles, B. H. and D. J. Ellar. 1986. Characterisation and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific f-endotoxin. J. Cell Sci. 83:89-101. 19. Knowles, B. H., and D. J. Ellar. 1987. Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis f-endotoxins with different insect specificity. Biochim. Biophys. Acta 924:509-518. 20. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 21. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 22. Luthy, P. 1980. Insecticidal toxins of Bacillus thuringiensis. FEMS Lett. 8:1-7. 23. Lyman, G. H., H. D. Preisler, and D. Papahadjopoulos. 1976. Membrane action of DMSO and other chemical inducers of Friend leukaemic cell differentiation. Nature (London) 262: 360-363. 24. Ohba, M., K. Aizawa, and T. Furusawa. 1979. Distribution of Bacillus thuringiensis serotypes in Ehime prefecture. Appl. Entomol. Zool. 14:340-345. 25. Padua, L. E., M. Ohba, and K. Aizawa. 1980. The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae. J. Invertebr. Pathol. 36:180-186. 26. Padua, L. E., M. Ohba, and K. Aizawa. 1984. Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae. J. Invertebr. Pathol. 44: 12-17. 27. Somerville, H. J. 1978. Insect toxin in spores and protein crystals of Bacillus thuringiensis. Trends Biochem. Sci. 3: 108-110. 28. Sriram, R., H. Kamdar, and K. Jayaram. 1985. Identification of the peptides of the crystals of Bacillus thuringiensis var. israelensis involved in larvicidal activity. Biochim. Biophys. Res. Commun. 132:19-27. 29. Stewart, G. S. A. B., and D. J. Ellar. 1983. Precursor processing during the maturation of a spore coat protein in Bacillus megaterium KM. Biochem. J. 210:411-417. 30. Stewart, G. S. A. B., K. Johnstone, E. Hagelberg, and D. J. Ellar. 1981. Commitment of bacterial spores to germinate. A measure of the trigger reaction. Biochem. J. 196:101-106. 31. Thomas, W. E., and D. J. Ellar. 1983. Bacillus thuringiensis var. israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo. J. Cell Sci. 60:181-197. 32. Thomas, W. E., and D. J. Ellar. 1983. Mechanism of action of Bacillus thuringiensis var. israelensis insecticidal delta-endotoxin. FEBS Lett. 154:362-368. 33. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 34. Ward, E. S., and D. J. Ellar. 1986. Bacillus thuringiensis var. israelensis delta-endotoxin. Nucleotide sequence and characterisation of the transcripts in Bacillus thuringiensis and Escherichia coli. J. Mol. Biol. 191:1-11. 35. Ward, E. S., A. R. Ridley, D. J. Ellar, and J. A. Todd. 1986. Bacillus thuringiensis var. israelensis delta-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis. J. Mol. Biol. 191:13-22. 36. Wu, D., and F. N. Chang. 1985. Synergism in mosquitocidal activity of 26 and 65 kDa proteins from Bacillus thuringiensis subsp. israelensis crystals. FEBS Lett. 190:232-236. 37. Yamamoto, T., T. lizuka, and J. N. Aronson. 1983. Mosquitocidal protein of Bacillus thuringiensis subsp. israelensis: identification and partial isolation of the protein. Curr. Microbiol. 9:279-284. 38. Yamamoto, T., and R. E. McLaughlin. 1981. Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var. kurstaki toxic to the mosquito larva, Aedes taeniorhynchus. Biochem. Biophys. Res. Commun. 103:414-421.
