The lepidopteran-specific Pl ô-endotoxin of Bacillus thuringiensis var. kurstaki HD-1 was activated in vitro using insect gut proteases and found to be highly specific for the lepidopteran cell line Choristoneura fumiferana CF1 among a wide range of lepidopteran and dipteran cell lines tested. The toxicity of Pl against CF1 cells is inhibited by.’V-acetylgalactosamine (GalNAc), and the lectins soybean agglutinin (SBA) and wheat-germ agglutinin. Protein blotting was used to identify a glycoprotein of 146×103Mr in the plasma membrane of CF1 cells, capable of binding both the toxin and SBA, which is specific for GalNAc. This glycoprotein was labelled using galactose oxidase and sodium boro-[3H] hydride and solubilized in Triton X-100 before partial purification by affinity chromatography on SBA-agarose. We propose that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific Pl δ-endotoxin of B. thuringiensis var. kurstaki HD-1.

The gram-positive bacterium Bacillus thuringiensis synthesizes a proteinaceous crystalline inclusion within the sporangium during sporulation. This inclusion, known as the crystal δ-endotoxin, displays insecticidal activity towards larvae within the orders Lepidoptera, Diptera and Coleóptera and is therefore of commercial importance as a biological control agent. To date, isolates of B. thuringiensis have been classified into 22 serotypes based on their flagellar H-antigens (de Barjac et al. 1985), many of these serotypes exhibiting a different spectrum of insecticidal activity (Dulmage, 1981).

The crystal δ-endotoxin is a protoxin that requires the high pH and proteases characteristic of the insect gut for solubilization and proteolytic activation (Lecadet & Martouret, 1965; Haider et al. 1986). The target of the activated toxin is the larval midgut epithelium, whose cells show the characteristic cytopathic effects of rapid swelling and lysis (EbersoldeZ al. 1978; de Barjac, 1978; Endo & Nishiitsusuji-Uwo, 1980; Percy & Fast, 1983). This in turn results in disruption of the epithelium and a consequent lowering of the gut pH, which favours germination of B. thuringiensis spores in the nutrient-rich environment of the dead or incapacitated insect. Assay of the activated δ-endotoxin on cultured insect cells in vitro reveals similar cytopathic effects to those observed in vivo (Murphy et al. 1976; Nishiitsusuji-Uwo et al. 1979; Thomas & Ellar, 1983a).

B. thuringiensis var. kurstaki HD-1 is the current industrial standard. The crystal δ-endotoxin comprises two distinct toxic proteins: the 130× 103Mr Pl lepidopteran-specific toxin and the broader spectrum 63×103Mr P2, toxic to lepidopteran and mosquito larvae (Yamamoto & McLaughlin, 1981). In previous assays of var. kurstaki δ-endotoxin in vitro, alkaline buffers and insect or mammalian proteases were used for solubilization and activation of the crystal (Murphy et al. 1976; Johnson, 1981; Tojo & Aizawa, 1983). These regimes do not solubilize P2, which aggregates unless the pH is maintained above 10 (Yamamoto & McLaughlin, 1981). Since insect cell lines are not viable at this pH all in vitro assays so far have been restricted to the study of Pl.

The mechanism of action of B. thuringiensis δ-endotoxins is not known, but the primary target appears to be the plasma membrane of larval midgut cells, which become leaky to small ions and glucose within 2 min of toxin addition (Fast & Donaghue, 1971), and permeable to larger molecules, such as lactate dehydrogenase, after 10 min (Ebersoldet al. 1980). The electrogenic K+ pump, located in the apical membrane of insect midgut goblet cells, has been proposed as the target for B. thuringiensis var. kurstaki δ-endotoxin (Griego et al. 1979; Gupta et al. 1985), since this pump is responsible for maintaining the steep K+ gradients and the transepithelial potential difference across the midgut epithelium. However, any toxin that causes non-specific leakage across the plasma membrane is likely to show an indirect effect on this pump, and a specific effect on the K+ pump has yet to be proven.

Himeno et al. (1985) reported that Na+ and K+ are essential for toxicity of var. aizawai activated δ-endotoxin in vitro, since in the presence of isotonic sucrose alone no toxicity was observed. However, these authors did not test any other ions, and their results can be explained by osmotic protection afforded by impermeant sucrose molecules (Knowles & Ellar, unpublished data). A recent publication by English & Cantley (1985) reporting an inhibitory effect of var. kurstaki δ-endotoxin on the K+-ATPase of a Manduca sexta cell line failed to demonstrate that this effect is due to the δ-endotoxin since their inhibitory factor was trypsin-sensitive. It is well established that the active portion of var. kurstaki δ-endotoxin represents a trypsinresistant core (Lilley et al. 1980; Fast, 1981; Huber & Luthy, 1981, and references therein; Chestukhina et al. 1982), thus these effects must be due to another protein factor.

The specificity of B. thuringiensis var. kurstaki Pl toxin implies a requirement for a specific receptor on the surface of susceptible cells, which is absent or masked in unaffected cells. Knowles et al. (1984) have shown that Pl can be inhibited by N- acetylgalactosamine (GalNAc) and by the lectins soybean agglutinin (SBA) and wheat-germ agglutinin, which bind this sugar on the cell surface (Lis et al. 1970; Monsigny et al. 1980), suggesting that this toxin binds a glycoconjugate receptor. The present paper describes the identification and partial purification of a GalNAc-containing glycoprotein from the membranes of a susceptible insect cell line, that appears to be a good candidate for the cellular receptor of B. thuringiensis var. kurstaki Pl δ-endotoxin.

Production and purification of crystal δ-endotoxin

B. thuringiensis var. kurstaki HD-1 was obtained from Dr H. D. Burges (Glasshouse Crops Research Institute, England). Conditions for growth and sporulation were as described for Bacillus megaterium KM (Stewart et al. 1981). The crystal δ-endotoxin was purified by ultracentrifugation on discontinuous sucrose gradients (Thomas & Ellar, 1983a).

Protein estimation

Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin (Sigma) as a standard.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

Separation of proteins on 10 % SDS-polyacrylamide gels was conducted as described by Thomas & Ellar (1983a).

Solubilization of crystal δ-endotoxin

Purified crystal δ-endotoxin was solubilized by the method of Huber et al. (1981). Crystals were incubated for 60 min at 37°C in 50mM-Na2CO3/HCl (pH 9·-5) and lOmM-dithiothreitol (DTT). Insoluble material was removed by centrifugation at 10 000gfor 5 min and the supernatant retained for subsequent assays.

Insect cell lines

Choristoneura futniferana CF1 cells (Lepidoptera, spruce budworm, trypsinized larval tissue) obtained from Dr S. S. Sohi (Canadian Forest Pest Management Institute, Ontario) were grown in Grace’s Medium.

Spodoptera frugiperda (Lepidoptera, fall army worm, pupal ovary), Heliothis zea (Lepidoptera, cotton bollworm, adult ovary), Trichoplusia ni (Lepidoptera, cabbage looper, adult ovary) and Mamestra brassicae cells (Lepidoptera, cabbage moth) obtained from Mrs T. Lescott (NERC Institute of Virology, Oxford), were grown in TC 100 medium. Aedes albopictus (Diptera, trypsinized larval tissue) obtained from Mrs T. Lescott, Aedes aegypti Aa(s), Aedes aegypti 20A (Diptera, trypsinized larval tissue) and Culex quinquefasciatus C2 cells (Diptera, larval tissue) obtained from Dr D. W. Roberts (Boyce Thompson Institute, Ithaca, USA), were grown in Mitsuhashi and Maramorosch medium. Drosophila melanogaster cells (Diptera, ovarian tissue) obtained from Mrs T. Lescott, were grown in Schneider’s Drosophila Medium.

All media contained 10% foetal calf serum (Gibco), 50 μgml-1 gentamicin (Nicholas Laboratories) and 50 μg ml-1 fungizone (Flow Laboratories). Cells were grown at 28°C.

In vitro assays

The soluble crystal fraction was activated by incubation 10:1 (v/v) with Pieris brassicae gut extract at 37°C for 15 min (Knowles et al. 1984). Activated soluble δ-endotoxin was made 10% in foetal calf serum (FCS, Gibco) prior to use in in vitro assays, in order to neutralize the effect of insect gut enzymes on the cell lines. Cells, at l×105 to 2×106ml-1 in tissue culture medium were incubated with activated toxin at 50 μg ml-1 cells. Viability was assessed by ability to exclude Trypan Blue (Thomas & Ellar, 1983a). Controls contained the appropriate volume of buffer, gut extract and-FCS.

Iodination of crystal δ-endotoxin and soybean agglutinin (SBA)

Iodination of whole crystals was carried out by the method of Fraker & Speck (1978) using carrier-free Na125I (Amersham International) and lodogen (Pierce). Unbound l25I was removed by extensive washing in phosphate-buffered saline (PBS; Oxoid, Dulbecco A, pH7·3) on ice. Iodinated crystals were solubilized and activated as described above. SBA (Sigma) was iodinated by the same method, but separated from free 125I by gel filtration through a Sephadex G-25 column (Pharmacia).

Preparation of a membrane fraction from insect cells

Cells were lysed in 10mM-PBS at 2°C for 30 min, and unbroken cells and nuclei removed by centrifugation at 1000gfor 5 min. The supernatant was centrifuged at lOOOOOgin a Beckman 50 Ti rotor at 2°C for 60 min to pellet the crude membrane fraction. This fraction was resuspended in PBS by sonication and boiled in gel sample buffer (Thomas & Ellar, 1983a) prior to SDS-PAGE, or solubilized in Triton X-100 before affinity chromatography as described below.

Protein blotting

Proteins separated by SDS-PAGE were transferred electrophoretically to nitrocellulose filters (Schleicher & Schüll) by the method of Towbin et al. (1979) using a Bio-Rad‘Trans Blot’ apparatus. Non-specific binding sites were blocked by incubation of the filter in 3 % (w/v) bovine serum albumin (Sigma) in PBS for 60 min, then 100(2× 10ctsmin-1) 125I-labelled activated δ-endotoxin or SBA were added for a further 3 h on a rocking platform. After six washes in PBS the nitrocellulose filter was blotted dry and autoradiographed with Fuji Rx film.

Suif ace labelling of insect cells

Cells, assessed as 98% viable by Trypan Blue exclusion, were washed twice in PBS at 106 cells ml-1, 1000 g, for 5 min and resuspended at 107 cells ml-1 in 1 ml PBS with 10 units galactose oxidase (Sigma). After incubation for 60 min at 22°C the cells were washed three times in PBS and incubated with 1 mCi sodium boro-[3H]hydride (Amersham International, 10 Ci mmol-1 sp. act.) for 20 min, 22°C. Unbound label was removed by further washes in PBS. Membrane fractions were prepared as described above.

Affinity chromatography

Affinity chromatography was carried out essentially as described by Butters & Hughes (1981). A SBA-agarose column (Pharmacia) was equilibrated with column buffer (0·5% (v/v) Triton X-100, 10 mM-Tris·HC1, pH 8, 150mM-NaCl). The crude membrane fraction from 2×107 C. fumiferana cells, surface labelled as described above, was solubilized in 0·5% (v/v) Triton X-100, 10 mM-Tris. HC1, pH8, for 30 min at 2°C. After centrifugation at 100 000 g for 60 min the supernatant was adjusted to 150 mM in NaCl and loaded onto the column. The column was washed with column buffer at 5mlh-1. Retarded material was eluted with 150mM-GalNAc (Sigma) in column buffer. Fractions (1 ml) were collected and 5 μl removed for scintillation counting. The remainder was precipitated with 12·5 % trichloroacetic acid prior to analysis by SDS-PAGE.

Surface labelling P. brassicae cells

The guts from four 5th instar P. brassicae larvae were removed and the contents washed out thoroughly with PBS. The guts and bodies were incubated separately in 0·25 % (w/v) collagenase in PBS for 30 min at 37°C to dissociate the cells. The reaction was stopped by the addition of FCS to 10% (v/v), and three washes in 0·2% FCS (v/v) in PBS. The cells were surface labelled with galactose oxidase and sodium boro-[3H] hydride as described above for C. fumiferana cells.

Fluorography

Fluorography was by the method of Chamberlain (1979).

Cell assays

Of a wide range of lepidopteran and dipteran cell lines tested, only C. fumiferana cells were susceptible to activated Pl δ-endotoxin. S. frugiperda, M. brassicae, T. ni and H. zea (lepidopteran), A. aegypti, A. albopictus, A. stephensi, A. gambiae, C. quinquefasciatus and D. melanogaster (Diptera) showed no cytopathic response to the toxin.

Visualization of toxin binding

It has previously been shown that SB A competes for binding with activated Pl δ-endotoxin (Knowles et al. 1984). Visualization of 125I-labelled toxin binding to membrane components from a range of insect cell lines was carried out by incubation of nitrocellulose filters, blotted with insect membrane proteins separated by SDS-PAGE (see Materials and Methods), with 125I-labelled Pl δ-endotoxin or 125I-labelled SBA (Fig. 1). It can be seen that most of the cell lines contain one or more proteins capable of binding the toxin. Comparison of the binding of bilabelled Pl with the binding of 125I-labelled SBA to C. fumiferana membranes indicates that both ligands bind the same membrane components, confirming the hypothesis that GalNAc-bearing glycoconjugates are involved in toxin binding.

Fig. 1.

Blotting membrane proteins obtained from insect cell lines. Insect membrane proteins separated by SDS-PAGE were transferred to nitrocellulose filters, which were then incubated with 125I-labelled SBA or Pl. A. C. fumiferana proteins blotted with 125I-labelled SBA. B. Insect cell line membrane proteins blotted with I25I-labelled Pl. Track 1, C. fumiferana; 2, S. frugiperda; 3, H. zea; 4, T. ni; 5, C. quinquefasciatus; 6, A. albopictus; 7, A. aegypti Aa(s); 8, A. aegypti 20A.

Fig. 1.

Blotting membrane proteins obtained from insect cell lines. Insect membrane proteins separated by SDS-PAGE were transferred to nitrocellulose filters, which were then incubated with 125I-labelled SBA or Pl. A. C. fumiferana proteins blotted with 125I-labelled SBA. B. Insect cell line membrane proteins blotted with I25I-labelled Pl. Track 1, C. fumiferana; 2, S. frugiperda; 3, H. zea; 4, T. ni; 5, C. quinquefasciatus; 6, A. albopictus; 7, A. aegypti Aa(s); 8, A. aegypti 20A.

Labelling of cell surface Gal or GalNAc residues

In order to discover which of the GalNAc-containing glycoproteins in C. fumiferana are exposed on the surface of intact cells, and are thus available for toxin binding, cells were labelled with galactose oxidase and sodium boro-[3H]hydride. Four proteins were labelled in C. fumiferana cells: 146, 52, 47 and 42 (×103)Mr bands and a 100×103Mr‘smear’ (Fig. 2A). Thus only one of these, the 146×103Mr component, had a similar mobility to a 12sI-labelled Pl binding glycoprotein.

Fig. 2.

A. Surface labelling C. fumiferana cells. Fluorograph of C. fumiferana cells surface labelled with galactose oxidase and sodium boro-[3H]hydride, separated by SDS-PAGE. B,C. SDS-PAGE of column fractions. B. Coomassie Blue stained gel; C, fluorograph. Track 1, Mr standards; 2, column peak 2; 3, column peak 1.

Fig. 2.

A. Surface labelling C. fumiferana cells. Fluorograph of C. fumiferana cells surface labelled with galactose oxidase and sodium boro-[3H]hydride, separated by SDS-PAGE. B,C. SDS-PAGE of column fractions. B. Coomassie Blue stained gel; C, fluorograph. Track 1, Mr standards; 2, column peak 2; 3, column peak 1.

Partial purification of the Pl δ-endotoxin receptor

Affinity chromatography on a SBA-agarose column was employed to purify the Pl receptor. A membrane fraction was prepared from C. fumiferana cells surface-labelled with 3H as described in Materials and Methods. The membranes were solubilized with 0·5% Triton X-100 and run on the column as described above. Two peaks were collected (Fig. 3): the unretarded fraction containing the bulk of the protein, with the GalNAc-eluted peak containing very little protein but with the highest specific activity of 3H-labelled material, as predicted. SDS-PAGE of pooled fractions (Fig. 2B) revealed that bands of 64 and 68(×103)Mr could be faintly seen by Coomassie Blue staining of peak 2, while fluorography (Fig. 2C) revealed a strongly labelled 146 band and 100(×103)Mr smear in peak 2, with 52, 47(×103)Mr bands and a 42×103Mr band (visible on longer exposure of the fluorogram) in peak 1, and a lower amount of the 146× 103Mr component.

Fig. 3.

Separation of C. fumiferana proteins by SBA-agarose affinity chromatography. Peak 1 was eluted with column buffer. At the fraction indicated by the open arrow, elution with 150mM-GalNAc in column buffer was started. Absorbance at 278nm (▴) and radioactivity (•) were monitored for each fraction.

Fig. 3.

Separation of C. fumiferana proteins by SBA-agarose affinity chromatography. Peak 1 was eluted with column buffer. At the fraction indicated by the open arrow, elution with 150mM-GalNAc in column buffer was started. Absorbance at 278nm (▴) and radioactivity (•) were monitored for each fraction.

Potential receptors in larval gut cells

Surface labelling freshly prepared P. brassicae gut and body cells (Fig. 4) revealed at least 15 bands in the gut ceils labelled with galactose oxidase and sodium boro-[3H]hydride; while the body cells only contained a few labelled proteins. A labelled band of 47×103Mr was noted in all three cell types, and a 146×103Mr band seen in C. fumiferana cells and P. brassicae gut cells was absent from body cells.

Fig. 4.

Fluorograph of insect cells labelled with galactose oxidase and sodium boro-[3H] hydride, separated by SDS-PAGE. Track 1, M, markers (×10−3); 2, C. fumiferana cell line; 3, freshly prepared P. brassicae gut cells; 4, freshly prepared P. brassicae body cells.

Fig. 4.

Fluorograph of insect cells labelled with galactose oxidase and sodium boro-[3H] hydride, separated by SDS-PAGE. Track 1, M, markers (×10−3); 2, C. fumiferana cell line; 3, freshly prepared P. brassicae gut cells; 4, freshly prepared P. brassicae body cells.

The two δ-endotoxins of B. thuringiensis var. kurstaki (Pl and P2) were separated by their differential solubility in alkaline reducing buffer, and all subsequent experiments were carried out using the lepidopteran-specific Pl. Pl, activated in vitro with P. brassicae gut extract, retains its specificity for Lepidoptera; it is not toxic to mice on injection, nor does it lyse mammalian erythrocytes (Knowles et al. 1984). The results described here indicate that the activated Pl toxin kills only C. fumiferana cells in vitro, and is not toxic towards the other lepidopteran and dipteran cell lines tested. This is in striking contrast to the δ-endotoxin of B. thuringiensis var. israelensis, which when solubilized at high pH lyses all eukaryotic cells tested and is lethal to mice on injection (Thomas & Ellar, 1983a). It was shown (Thomas & Ellar, 1983b) that B. thuringiensis var. israelensis δ-endotoxin binds phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine. The presence of these phospholipids in the plasma membrane of all eukaryotic cells explains the broad spectrum of activity of this toxin. The narrow specificity of B. thuringiensis var. kurstaki Pl toxin suggests that this toxin binds a receptor found only in a limited range of cell types.

In our hands, the T. ni TN-368 cell line showed no cytopathic response to activated var. kurstaki δ-endotoxin at 50 μg ml−1. This is in contrast to the results of Murphy et al. (1976), who reported 25 % damage to toxin-treated cells (with 680 μg toxin ml-1) compared with 16% damage to control cells.

The in vitro assays described in this paper involved control cells treated with concentrations of Na2CO3/HCl, pH9·5, DTT, larval gut extract and FCS that caused less than 5 % cell mortality during the course of the assay. Toxin-treated T. ni cells incubated with 50 μgml −1δ-endotoxin in the same concentrations of buffer, gut extract and FCS as the controls, showed no cytopathic effect. It is possible that cells stressed by changes in pH, osmotic concentration, and by addition of digestive enzymes, show an artificially high response to the toxin and for this reason every care should be taken that control cells in in vitro assays are subjected to the minimum of damage.

The fact that B thuringiensis var. kurstaki δ-endotoxin kills H. zea, S. frugiperda and M. brassicae larvae in vivo (Krieg & Langenbruch, 1981) but does not affect cell lines derived from these species may indicate that receptors for the toxin are not present in all cells from a susceptible insect, but only in specific cell types such as gut cells. The C. fumiferana CF1 cell line was derived from whole trypsinized larvae (Sohi, 1973), therefore it is not known which cell types are represented in this culture. Most of the other cell lines used in this study were derived from adult or pupal ovarian tissue, and thus might be expected to lack the receptors for a gut epithelial toxin. This underlines the difficulties inherent in the interpretation of results obtained from cell lines in vitro. However, since the midgut of lepidopteran larvae is a complex system in which precise assay conditions are difficult to control, and since the C. fumiferana CF1 cell line is susceptible to the Pl toxin, we feel justified in using it to study toxin-receptor interactions under controlled conditions. Characterization and identification of the toxin receptor in this system will allow us to return to the in vivo system in order to confirm the nature of the receptor mediating the insecticidal activity of the toxin in vivo.

The observation that 125I-labelled toxin binds to proteins present in all cell lines tested suggests that binding alone might not be sufficient for expression of toxicity, or that the conditions of the binding assay reveal binding sites not normally exposed on the cell surface. All of the cell lines used in this assay were agglutinated by SBA (data not shown), indicating that all have at least one GalNAc-bearing glycoconjugate exposed at the cell surface. Since SBA does not cause the cytopathic effects associated with the Pl toxin it seems likely that after binding its receptor the toxin is involved in an additional step that results in disruption of the plasma membranes.

3H-labelling of GalNAc or Gal residues exposed on the surface of intact C. fumiferana cells labels glycoproteins of 146, 52, 47 and 42 (×103) Mr. The 66 and 68(×103)Mr proteins that bind 125I-labelled Pl and 125I-labelled SBA are not labelled in this procedure, suggesting that they are not exposed on the cell surface of intact cells, or that labelling was incomplete. Avigad (1985) showed that some theoretically available terminal galactosyl residues of glycoproteins are incompletely oxidized by galactose oxidase, although removal of sialic acid from these glycoproteins caused a marked increase in the rate of oxidation. Since insect glycoconjugates lack sialic acid (Warren, 1963) it seems probable that all available galactosyl residues are labelled in this study.

The only glycoprotein labelled by both 12sI-labelled Pl and NaB3H4 is the 146× 103Mr component, leaving this as the most likely candidate for the Pl receptor. Affinity chromatography on SBA-agarose retards this component, while the 52, 47 and 42 (×103)Mr glycoproteins are unretarded, suggesting that these may contain terminal Gal rather than GalNAc.

The possibility that the Pl receptor is a glycolipid has not been investigated in this study. Huber-Lukac (1984) demonstrated binding of var. thuringiensis toxin to insect lipids, but we have been unable to demonstrate specific binding of 125I-labelled Pl to similar lipid preparations (data not shown).

Because very little is known about the structure or function of the components of insect plasma membranes, it is not possible to correlate the 146× 103Mr glycoprotein with any known enzyme, ion channel or other constituent. The partial purification of this 146×103Mr glycoprotein is the first step in elucidation of its function in the insect plasma membrane, and hence an understanding of the precise mechanism of action of B. thuringiensis var. kurstaki Pl toxin.

The comparison between glycoproteins of different cellular origin illustrates the difficulties in interpretation of such results: glycoproteins run anomalously on SDS-PAGE because of the charge on the sugar moieties, therefore it is not possible to state that the 146×103Mr glycoproteins in C. fumiferana cells and P. brassicae gut cells are the same, or that the 47×103Mr bands present in all three cell types are related. However, since very little is known about insect plasma membrane components, this direct demonstration of the large number of gut cell membrane glycoproteins containing terminal Gal or GalNAc moieties is of interest, and suggests that the in vitro system is more simple. Having characterized a Pl receptor from insect cell lines in vitro, the next stage will be to identify the physiological receptor in vivo.

We thank Mrs Audrey Symonds for invaluable help and Mrs Jillian Clements for secretarial assistance. This research was supported by grants from SERC and AFRC.

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