Eur. J. Biochem. 245, 797-804 (1997) 0 FEBS 1997

Analysis of the large aqueous pores produced by a Bacillus t~uringie~sis protein insecticide in Manduca sexta midgut-brush-border-membrane vesicles Joe CARROLL and David J. ELLAR

Department of Biochemistry, University of Cambridge, England

(Received 21 October 1996/26 February 1997) - EJB 96 1551/6

An osmotic swelling assay utilising carboxyfluorescein self-quenching to measure intravesicular vol- ume changes was adapted to investigate permeability changes induced by the Bacillus thuringiensis Cry 1Ac b-endotoxin in Manduca sexta midgut-brush-border-membrane vesicles (BBMV). This assay provides a more quantitative analysis of Cry-toxin-induced BBMV permeability changes, extending our previously published protocol which employed a light-scattering signal to monitor b-endotoxin activity [Carroll, J. & Ellar, D. J. (1993) EUK J. Biochem. 214, 771-7781, The fluorescence signal changes, supported by electron microscopy of the BBMV, demonstrated that Cryl Ac altered the membrane perme- ability for large non-electrolyte solutes. With this approach CrylAc was observed to induce or form pores freely permeant for raffinose (1.14 nm diameter) and using non-electrolytes of increasing size the pores were estimated to have a limiting diameter of approximately 2.4-2.6 nm under alkaline pH conditions.

Keywords: Bacillus thuringiensis; toxin ; pore; membrane; permeability.

Cytolysis of midgut cells characterises the insecticidal action of Bacillus thuringiensis Cry toxins. The molecular mechanism of action at the target cell membrane is believed to be a two- stage process of receptor binding followed by membrane integ- ration and pore formation, leading to cell death by colloid os- motic lysis (Knowles and Ellar, 1987). In vivo activity was ini- tially correlated with the presence of high-affinity receptors on target insect midgut cell membranes (Hofmann et al., 1988; Van Rie et al., 1989), but it emerged that events subsequent to this step also play a role in determining toxin potency (Wolfers- berger, 1991). Irreversible binding, indicative of toxin insertion into the membrane, quickly follows the initial toxin-receptor interaction (Van Rie et al., 1989) and is believed to be required for the formation of the active membrane lesion (Knowles and Dow, 1993). Differences in the toxin insertion step have been proposed to account for in vivo activity differences that are in- consistent with the receptor determinant model (Ihara et al., 1993; Liang et al., 1995). While the irreversible binding compo- nent correlates with toxin activity in these cases, quantitative comparisons show that in some cases a 2-4-fold decrease in the amount of irreversible binding is accompanied by a 20-400- fold decrease in activity (Chen et al., 1995; Rajamohan et al., 1995).

Following receptor binding and membrane insertion, lysis of the susceptible insect midgut cell is believed to result from a

Correspondence to D. J. Ellar, Department of Biochemistry, Univer- sity of Cambridge, Tennis Court Road, Cambridge, England CB2 1QW

Fax: +44 1223 333345. E-mail: djel @mole.bio.cam.ac.uk URL: http :Nwww.bio.cam.ac.uWdeptlbiochem/ Abbrevintions. BBMV, brush-border-membrane vesicles; Ches, 2-

(cyclohexy1amino)-ethanesulphonic acid; PEG, poly(ethy1ene glycol). Note. Cry toxins are named according to the revised Cry holotype

toxin nomenclature (WWW site: http ://epunix.biols.susx.ac.uWHome/ NeilLCrickmore/Bt/index. html).

toxin-induced change in cell membrane permeability, but the na- ture of the toxin lesion is still disputed (Knowles and Dow, 1993). Studies using artificial membranes have demonstrated that some Cry toxin preparations can form channels (Slatin et al., 1990; Schwartz et al., 1993; Grochulski et al., 1995). How- ever, the results of incorporation of target insect midgut-brush- border-membrane vesicles (BBMV) into such membranes and subsequent challenge with B. thuringiensis Cry toxin suggest that the channels formed in the presence of specific receptors may differ significantly from those formed in their absence (Martin and Wolfersberger, 1995). Such differences and the pos- sibility that altered pore characteristics may result in observable in vivo activity changes argues for a fuller understanding of the nature of the toxic lesion formed under conditions that resemble the in vivo situation.

An induced cation leakage activity has commonly been ob- served when Cry toxins interact with insect midgut preparations (Sacchi et al., 1986; Crawford and Harvey, 1988; Wolfersberger, 1989; Lorence et al., 1995). In addition Hendrickx et al. (1990) described an increased alanine permeability for Munduca sexta BBMV treated with CrylAb. Using M. sexta midgut BBMV we have previously analysed the pore-forming activity of Cryl Ac using a light-scattering assay (Carroll and Ellar, 1993). Acti- vated CrylAc was shown to increase M. sexta BBMV mem- brane permeability for cations, anions and neutral solutes al- though some selectivity between anions and cations could not be ruled out. The absolute solute hydrodynamic size of the pore was not determined, although neutral solutes of the size of sucrose (0.92 nm diameter) were shown to be permeant. While CrylAc alone forms channels in lipid bilayers (Slatin et al., 1990), Martin and Wolfersberger (1995) demonstrated that in the presence of M. sexta BBMV different channel characteristics were observed with this toxin. At pH 8.8 channels with a calcu- lated diameter of 0.9 nm were formed (Wolfersberger, 1995), whereas at pH 9.6 large conductance channels were produced

798 Carroll and Ellar (ELI): J . Biochem. 245)

with a calculated pore diameter of 2.2 nm (Martin and Wolfers- berger, 1995), clearly larger than sucrose.

The effect of solute refractive index differences, volume-in- dependent scattering changes and artefacts associated with vesi- cle motion and aggregation can limit the quantitative application of the light-scattering method (Carroll and Ellar, 1993). How- ever, it is still valuable for comparing toxins under identical ex- perimental conditions (Wolfersberger et al., 1996). In order to extend this experimental approach to allow quantitative compar- isons of membrane permeability changes to different test solutes, a method based on the self-quenching of an entrapped fluoro- phore was employed (Chen et al., 1988). Changes in vesicle volume are followed by the consequential change in fluores- cence of an entrapped fluorophore as the BBMV shrink and swell.

MATERIALS AND METHODS

Materials. M. sexta eggs were obtained from Prof. S. Rey- nolds (University of Bath, School of Biological Sciences, Eng- land). Larvae were reared on an artificial diet (Bell and Joachim, 1976). B. thuringiensis subsp. kurstaki HD73, which synthesises a single Cryl Ac toxin (Hofte and Whiteley, 1989) was obtained from USDA Northern Regional Research Laboratories (Illinois, USA). Growth conditions were as described for Bacillus rnegat- eriunz KM (Stewart et al., 1981). Carboxyfluorescein was the kind gift of Dr G. A. Smith (Department of Biochemistry, Uni- versity of Cambridge, England). Poly(ethy1ene glycol) (PEG) 1000 (Sigma), PEG 1500 (BDH) and PEG 4000 (Fisons) were used.

CrylAc purification and activation. The Cry 1 Ac crystal inclusion was purified from sporulated B. thuringiensis cultures using discontinuous sucrose gradients (Thomas and Ellar, 1983). The protein concentration was determined by the method of Lowry et al. (1951) using BSA as a standard. CrylAc was solu- bilked and activated at 1 mg/ml as described previously (Carroll and Ellar, 1993). The SDS/PAGE analysis of samples was per- formed by a modified method of Laemmli and Favre (1973) as described by Thomas and Ellar (1983). The activated toxin was quantified by gel scanning densitometry of SDWPAGE-sepa- rated and Coomassie-blue-stained protein using a Molecular Dy- namics 300s laser scanning densitometer. Cryl Ac toxin acti- vated with trypsin (enzyme/toxin 1 : 5 by mass, pH 8, 37"C, overnight) and purified on a Mono Q column (Pharmacia) (etha- nolamine pH 9.5, NaCl gradient) was used as a standard. This standard was quantified by amino acid analysis using an Alpha Plus I1 analyser (Pharmacia-LKB) prior to SDSPAGE.

BBMV osmotic swelling assays. M. sexta BBMV were iso- lated as described previously (Carroll and Ellar, 1993).

Light-scattering assay. This was performed essentially as de- scribed earlier (Carroll and Ellar, 1993), except the buffer used was 10 mM Ches (2-[cyclohexylamino]-ethanesulphonic acid)/ KOH pH 9.

Fluorescence self-quenching assay. This osmotic swelling assay was based on the method of Chen et al. (1988) with car- boxyfluorescein used as the intravesicular fluorescent dye for monitoring vesicle volume changes (Harris et al., 1990). The primary M. sexta midgut BBMV preparation was isolated in the presence of 250 mM sucrose (Carroll and Ellar, 1993). Low os- motic BBMV, also referred to as swollen BBMV, were then formed by resuspending vesicles in 10 mM Ches/KOH or Caps/ KOH, pH 6-7. Where indicated, 10 mM sucrose was also pre- sent. Carboxyfluorescein loading was performed on ice over- night at 10 mg/ml BBMV and 10 mM carboxyfluorescein. KOH was then added to raise the pH to approximately 8.7 or 9.0 for

Ches- and 9.8 for Caps-buffered BBMV. The BBMV were then diluted to 2 mg/ml in a buffedcarboxyfluorescein mixture at the required pH and left on ice for 1 h. Non-encapsulated carboxy- fluorescein was removed by passing the BBMV mixture down two successive PD-10 columns (Pharmacia) according to the manufacturer's instructions, using 10 mM ChedKOH (pH 8.7 or 9.0) or Caps/KOH (pH 9.8) containing 1 mg/ml BSA. The final BBMV suspension was diluted to 0.2 mg/ml in the same buffer/ BSA solution and kept on ice. Native BBMV refers to samples prepared throughout in the presence of an additional 250 mM sucrose so as to maintain the initial osmotic environment, pre- venting pre-swelling.

BBMV were mixed with hyperosmotic solutes using an Ap- plied Photophysics SF.17MV stopped-flow spectrometer. Car- boxyfluorescein was excited at 490 nm and the fluorescence monitored at 90" through a 515-nm Schott glass filter. All ex- periments were performed at 20-21 "C. BBMV were either in- cubated with toxin at 20-21°C for 30 or 60 min prior to use, or equilibrated at 20-21°C for 15 min before mixing with hy- perosmotic solutions containing the toxin. M. sexta BBMV car- boxyfluorescein permeability was not significantly altered by Cryl Ac as judged by experiments performed under iso-osmotic conditions, where signal changes would be the result of carboxy- fluorescein leakage (data not shown).

Solute concentrations used were derived empirically from experiments with untreated BBMV, using an equivalent osmotic shrinkage change in the carboxyfluorescein signal as a measure of equal osmotic strength. The experimental concentrations fi- nally used were 150 mM sucrose, 145 mM raffinose, 11.75% (mass/vol.) PEG 1000, 15.75% (mass/vol.) PEG 1500 and 21 % (mass/vol.) PEG 4000. All stopped-flow solutions were de- gassed before use. Solute hydrodynamic radii were taken from Scherrer and Gerhardt (1971).

Microscopy. BBMV samples representing various stages of the light-scattering assay were analysed by electron microscopy. At times indicated in the figure legends, samples were fixed with cold glutaraldehyde at a final concentration of 2.5% (by vol.) for 4 h at 4°C. Following this step, BBMV were washed in the experimental buffer solution and post-fixed for 1 h at room tem- perature with 2% (mass/vol.) OsO, in 0.05 M sodium cacodylate pH 7.2. The samples were then dehydrated through an ethanol series and embedded in Spurr resin. Sections were cut with a glass knife mounted on a Reichert-Jung Ultracut E and stained for 30min in saturated uranyl acetate and subsequently for 2 min with Reynolds lead citrate (Reynolds, 1963). Stained sec- tions were viewed using a Philips EM300 transmission electron microscope.

RESULTS

Electron microscopy. We previously used an osmotic swelling assay coupled with light scattering to follow M. sexta BBMV volume changes (Carroll and Ellar, 1993). To improve our un- derstanding of the BBMV changes that occur, we have used electron microscopy to examine vesicles at various stages of both the toxin mixing (Fig. 1) and toxin incubation (Fig. 2) as- says.

Both villus-like rods and spherical vesicles are observed in the primary BBMV preparation (Fig. 1A). Incubation in a low osmotic environment resulted in vesicle swelling and the loss of the rod-shaped structures (Fig. 1B). Mere exposure of these vesicles to an osmotic gradient produced marked shape changes with many transformed back from the swollen spherical vesicles into compressed villus-like structures (Fig. 1 C). This shape change may account for the observed non-linear signahohme

Carroll and Ellar (Eur. J . Biochern. 245) 799

Fig. 1. Electron micrographs of thin sections of M. sextu BBMV during a CrylAc mixing osmotic swelling assay. (A) M. sexfa primary BBMV preparation; (B) low-osmotic BBMV prepared by the equilibration of a primary BBMV preparation in low-osmotic 10 mM CheslKOH, pH 9 ; (C) low-osmotic BBMV (0.4 mg/ml) 10 min after mixing 1 : 1 (by vol.) with 10 mM Ches/KOH plus 250 mM sucrose, pH 9, and a buffedenzyme control for the activated toxin (12 pl/ml); (D) low-osmotic BBMV (0.4 mg/ml) 10 min after mixing 1 : 1 (by vol.) with 10 mM Ches/KOH plus 250 mM sucrose, pH 9, and Cryl Ac toxin (12 pl/ml). Magnification 17 500X.

relationship displayed by these vesicles as they shrink (Fig. 3 inset). Unlike control vesicles (Fig. 1 C), BBMV exposed to hyperosmotic sucrose containing Cry 1 Ac returned to a swollen state, indicating an increased permeability for this solute (Fig. 1 D).

When BBMV were incubated with CrylAc and mixed with hyperosmotic sucrose, multivesicular structures were observed in addition to spherical BBMV (Fig. 2D). These were not seen in the absence of an imposed osmotic gradient (Fig. 2C). Again, missing from toxin-treated BBMV exposed to an osmotic gradi- ent were the compressed rod-shaped vesicles observed in control samples (Fig. 2B) indicating a toxin-induced change in BBMV permeability for sucrose. Multivesicular structures were also ob- served in control samples after exposure to an osmotic gradient (Fig. 2B), suggesting that the presence of toxin does not deter- mine their formation. The multivesicular structures seen are not obviously larger than the original single bilayer vesicles, which indicates that they are not formed by aggregation and fusion of BBMV. Instead we propose that intra-BBMV membrane fusion occurs when shrinkage forces the internal surfaces of regions of the BBMV membranes into juxtaposition. The resulting multive- sicular structures, which will exhibit different light-scattering properties from the original BBMV, may contribute to the obser- vation that the scattering signal of vesicles in the presence of a permeant solute never recovers to the initial level (Carroll and Ellar, 1993). While the BBMV structure may change, assuming no marked fragmentation of these structures, there will still be a recovery of intravesicular volume concomitant with the recov- ery in the light-scattering signal. Whether there is a reduced in- travesicular osmotic space after the formation of these structures has not been ascertained.

Light-scattering assay. Previously (Carroll and Ellar, 1993) we used non-electrolyte solutes up to the size of sucrose when ana- lysing Cryl Ac-induced permeability changes. Further experi- ments using solutes larger than sucrose revealed a marked over- shoot in the shrinkage phase, monitored as an increase in 90" light-scattering, before swelling occurs and indicated that the control BBMV shrinkage signal would not apparently reach the maximum level seen in the presence of toxin (Fig. 3). This sug- gests that CrylAc alters BBMV during the incubation step. In addition, the 90" light-scattering signal did not exhibit a simple linear change with BBMV volume, displaying a more compli- cated curvilinear relationship (Fig. 3 inset).

Fluorescence-quenching assay. Because of the complexity of the light-scattering signal and the observed BBMV morphologi- cal changes, we decided to adapt a method based on the self- quenching of an entrapped fluorophore (Chen et al., 1988) in order to extend the osmotic swelling assay to allow quantitative comparisons of different solute permeability changes. Basically, the signal from an intravesicular fluorophore decreases upon vesicle shrinkage due to self-quenching and increases with any subsequent vesicle swelling. Harris et al. (1990) showed that, while the fluorophore carboxyfluorescein exhibits a sigmoidal self-quenching fluorescence signal, the intravesicular signal is approximately linear over the carboxyfluorescein concentration range 5-20 mM. As expected, this linear signal/volume rela- tionship was observed for M. senta native BBMV loaded with approximately 10 mM carboxyfluorescein, but BBMV swollen in a low osmotic buffer exhibited a curvilinear relationship (Fig. 4), in a similar manner to the light-scattering signal (Fig. 3 inset).

800 Carroll and Ellar ( E M J . Biochern. 245)

Fig. 2. Electron micrographs of thin sections of M. sextu BBMV during a CrylAc incubation osmotic swelling assay. (A) 3 rnin after mixing low-osmotic BBMV (0.4 mg/ml), previously incubated for 60 min at 20-21 "C with a buffedenzyme control for the activated toxin (12 pl/ml), 1 : 1 (by vol.) with 10 mM CheslKOH pH 9; (B) 3 min after mixing low-osmotic BBMV (0.4 mg/ml), previously incubated for 60 rnin at 20-21 "C with a bufferlenzyme control for the activated toxin (12 pltml), 1 : 1 (by vol.) with 10 mM CheslKOH plus 250 mM sucrose, pH 9; (C) 3 min after mixing low-osmotic BBMV (0.4 mg/ml), previously incubated for 60 rnin at 20-21 "C with Cry1 Ac toxin (12 pllml), 1 : 1 (by vol.) with 10 mM Ches/KOH pH 9; (D) 3 min after mixing low-osmotic BBMV (0.4 mglml), previously incubated for 60 rnin at 20-21 "C with Cry1 Ac toxin (12 pllml), 1 : 1 (by vol.) with 10 mM CheslKOH plus 250 mM sucrose, pH 9. Magnification 17 500X.

1 s 100 s

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Fig.3. Effect of CrylAc on M. sextu BBMV light scattering when exposed to hyperosmotic raffinose. BBMV (0.4 mglml), equilibrated with 10 mM ChedKOHl0.1 % (masslvol.) BSA pH 9, were incubated with Cry? Ac (8 pliml) or a buffdenzyme control for the activated toxin, for 60 rnin at 20-21 "C. The change in 90" light scattering at 450 nm was observed after mixing an equal volume of BBMV with 10mM Ches/KOH/O.l % (masslvol.) BSA plus 100 mM raffinose pH 9 at 20- 2 1 "C using a stopped-flow spectrometer. Each trace represents the average of two determinations. Inset: dependence of swollen M . sexfa BBMV light scattering on vesicle volume. The light-scattering signal change was measured after BBMV were mixed with 10 mM ChesiKOH pH 9 plus different KCI concentrations. This was plotted against relative volume, calculated from the extravesicular osmolarity. Each point is the average of two or three determinations. The zero point represents no signal change observed with an iso-osmotic buffer (Carroll and Ellar, 1993).

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Fig. 4. Dependence of intravesicular fluorescence on M. sextu BBMV volume. The signal change of intravesicular carboxyfluorescein was measured after BBMV were mixed with t 0 m M Ches/KOH plus dif- ferent sucrose concentrations, pH 9. This was plotted against relative volume, calculated from the extravesicular osmolarity. Data for native BBMV prepared in the presence of 10mM Ches/KOH plus 250mM sucrose, pH 9 (V), and swollen BBMV equilibrated in 10 mM Chesl KOH, pH 9 (O), are shown.

Osmotic changes occurring during carboxyfluorescein load- ing of swollen BBMV may result in a lower than expected in- travesicular fluorophore concentration, shifting the self-quench- ing signal to a non-linear region (Harris et al., 1990). This would be particularly acute when any non-encapsulated carboxy-

Carroll and Ellar ( E m J . Biochem. 245) 801

fluorescein is finally removed, the buffer/carboxyfluorescein bathing solution with an osmolarity similar to a 10 mM buffer/ 50 mM sucrose solution (data not shown) being exchanged for a lower osmotic 10 mM buffer in which the vesicles could swell. This idea was supported by preliminary experiments using car- boxyfluorescein-loaded liposomes (data not shown). By raising the osmotic pressure of the column wash buffer used when re- moving non-encapsulated carboxyfluorescein, the liposomes ex- hibited a less exaggerated curvilinear signal/volume relationship. However, the membrane-permeabilising effect of the Cry toxin on BBMV would complicate this experimental approach, as was also apparent in the native BBMV experiments described below.

The signal/volume relationships and the added effect of toxin-induced permeability changes complicate both the light- scattering and fluosescence-quenching assays. However, the ma- jor advantages of the latter is that the fluorescence signal is po- tentially less sensitive to a change in vesicle state and solute refractive index differences, both of which can influence a light- scattering signal. The new method therefore can provide a quan- titative comparison of different solute permeabilities (Chen et al., 1988).

Analysis of the CrylAc-induced membrane permeability change. To investigate the membrane permeability changes re- sulting from CrylAc activity the toxin incubation assay was em- ployed to allow time for effective toxin action. The toxin mixing assay is more complex due to the need for receptor binding, toxin insertion into the membrane and permeabilising activity to occur before swelling results, any stage of which may be limit- ing relative to solute conductance through the pore and may be affected by different test solutes. CrylAc did not appear to re- lease encapsulated carboxyfluorescein from swollen BBMV as judged by the absence of any significant signal change in toxin- treated BBMV fluorescence under iso-osmotic conditions (data not shown). Any release after exposing BBMV to an osmotic gradient would contribute to the increased fluorescence signal representing subsequent vesicle swelling. The formation of multivesicular structures observed after exposing swollen vesi- cles to an osmotic gradient (Fig. 2) could result in release of some intravesicular contents. However, studies on the process of vesicle fusion suggest that it occurs with minimal leakage of contents, this only being observed if the membrane structures eventually break down (Wilschut and Hoekstra, 1986). In addi- tion, as suggested above, the multivesicular structures observed in our study may arise from intravesicular membrane rearrange- ment.

CrylAc increases M. sexta BBMV permeability for sucrose (Carroll and Ellar, 1993). Fig. 5 shows that carboxyfluorescein- loaded native BBMV incubated with CrylAc exhibited the ex- pected shrinkhwell trace upon mixing with hyperosmotic sucrose, the controls only demonstrating the decreased fluores- cence signal as the BBMV shrink. However, the initial signal for CryIAc-treated BBMV was displaced to a higher fluores- cence signal, suggesting that during the toxin incubation step the BBMV had swollen relative to the control vesicles. For native BBMV, where the bathing solution contains 250 mM sucrose, this probably results from sucrose entry through a toxin pore followed by water. Because of this complication, the swollen low-osmotic BBMV were preferred over native BBMV for fur- ther experiments studying toxin-induced BBMV permeability changes.

Low-osmotic BBMV incubated with CrylAc exhibited a shrinkage signal overshoot when mixed with hyperosmotic raffi- nose (Fig. 6) in a similar manner to that observed for the light- scattering experiment. This again indicates a toxin-induced change in the BBMV during the incubation step. In both cases,

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Fig.5. Effect of CrylAc on M. sextu native BBMV intravesicular carboxyfluorescein signal when exposed to hyperosmotic sucrose. Carboxyfluorescein-loaded BBMV (0.2 mglml) equilibrated with 10 mM Ches/KOH/O.l% (mass/vol.) BSA plus 250 mM sucrose, pH 9, were incubated with CrylAc (5.3 pl/ml) or a buffedenzyme control for the activated toxin, for 30 min at 20-21 "C. The fluorescence signal changes were observed after mixing an equal volume of BBMV with 10 mM Ches/KOH/O.l % (mass/vol.) BSA plus 750 mM sucrose pH 9 at 20-21 "C using a stopped-flow spectrometer. Each trace represents the average of three determinations. Inset: the same conditions as described above except BBMV were not equilibrated with toxin but were mixed with the sucrose solution containing CrylAc (4 pl/ml) or a buffeden- zyme control for the activated toxin. Each trace represents the average of two determinations.

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Fig.6. Effect of CrylAc on swollen M . sextu BBMV intravesicular carboxyfluorescein signal when exposed to hyperosmotic raffinose. Carboxyfluorescein-loaded BBMV (0.2 mg/ml) equilibrated with 10 mM Ches/KOH/O.l% (mass/vol.) BSA plus 10 mM sucrose, pH 9, were incubated with CrylAc (4 pllml) or a buffedenzyme control for the activated toxin, for 30min at 20-21°C. The fluorescence signal changes were observed after mixing an equal volume of BBMV with 10 mM Ches/KOH/O.l % (masshol.) BSA plus 100 mM raffinose pH 9 at 20-21 "C using a stopped-flow spectrometer. Each trace represents the average of three determinations. Inset: the same conditions as de- scribed above except BBMV were not equilibrated with toxin but were mixed with the raffinose solution containing CrylAc (4 pl/ml) or a buffedenzyme control for the activated toxin. Each trace represents the average of two determinations.

a toxin-induced decrease in BBMV volume during incubation, relative to control vesicles, could result in a subsequent shrink- age overshoot due to the curvilinear signal change (Fig. 3 inset and Fig. 4). Alternatively toxin action may alter the ability of BBMV to resist extreme shrinkage. The idea that CrylAc in-

802 Carroll and Ella ( E m J. Biochem. 245)

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Fig.7. Effect of CrylAc on swollen M . sexta BBMV intravesicular carboxyflnorescein signal when exposed to hyperosmotic solutes. Carboxyfluorescein-loaded BBMV (0.2 mglml) equilibrated with 10 mM Caps/KOWO.l% (masslvol.) BSA pH 9.8 were incubated with CrylAc (124 pmollmg BBMV) for 60 min at 20-21°C. The fluores- cence signal changes were observed after mixing an equal volume of BBMV with 10 mM CapdKOH plus 150 mM sucrose (A), 145 mM raf- finose (V), 11.75% (masdvol.) PEG 1000 (O), 15.75% (masshol.) PEG 1500 (m) or 21 % (masslvol.) PEG 4000 (+), pH 9.8, at 20-22 "C using a stopped-flow spectrometer. Each trace represents the average of two determinations.

duces BBMV changes during the incubation step that manifest themselves when the vesicles are exposed to an osmotic gradient is supported by the observation that the initial shrinkage signal was not affected when untreated BBMV were mixed with hyper- osmotic solute solutions containing toxin (Figs 5 and 6 insets). The subsequent increase in the fluorescence signal for BBMV mixed with toxin is consistent with CrylAc inducing or forming a pore permeable for sucrose and raffinose which results in vesi- cle swelling.

The Cryl Ac-induced permeability change in M. sexta BBMV was investigated using non-electrolyte solutes of increasing hydrodynamic diameter. Sucrose, raffinose and PEG of increasing molecular mass were tested on control BBMV and the concentrations giving an equivalent osmotic signal change were used for testing toxin-induced permeability changes. Monosaccharides (Carroll and Ellar, 1993) and PEG 600 (data not shown) are markedly permeant for untreated BBMV and so were not used. Prior incubation with CrylAc was used to allow pores to form and the concentration used was in each case con- sidered to be saturating (> 100 pmol/mg BBMV; Carroll and Ellar, 1993). Fig. 7 shows shrinWswel1 traces for Cryl Ac-treated M. sexta BBMV at pH 9.8 when exposed to different hyperos- niotic solutes. BBMV permeability to sucrose and raffinose is significant, with PEG 1000 more permeant than PEG 1500 and PEG 4000. A measure of toxin action, using difference traces that subtract the control performed in the absence of toxin from toxin-treated BBMV swelling changes, could not be performed because of the described differences between control and toxin- treated BBMV (Fig. 6). Instead, if no significant difference is observed between re-swelling traces for two solutes in the pres- ence of toxin, it follows that these are effectively excluded from the toxin-induced pore and this may help delineate the pore size. The same principle underlies the cell lysis protection assay (Weiner et al., 1985) and the liposome swelling assay (Yoshihara et al., 1988) used to size other pore forming agents. At a time point when the sucrose recovery signal reached a maximum level the relative signal recoveries for the test solutes were cal- culated. Using a value of l and 0 for sucrose and PEG 4000

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Fig. 8. Relative BBMV fluorescence signal recovery as a function of the hydrodynamic diameters of non-electrolyte solutes. Conditions as described for Fig. 7. Signal recoveries were calculated at a point where sucrose recovery reached a maximum level. Each point represents the mean -C SD, n = 6. CrylAc concentrations were between 119- 156 pmoVmg BBMV. (A) pH 8.7 and (B) pH 9.8.

signal recoveries, respectively, the toxin-induced pore size was estimated from the point where the signal recovery remained unchanged, which corresponds to the x-axis intercept at zero signal recovery (Fig. 8). From these data it was estimated that CrylAc forms or induces pores with internal diameters of ap- proximately 2.4 nm and 2.6 nm at pH 8.7 and 9.8, respectively. PEG 1000 and 1500 were slightly permeant for control BBMV (< lo%), the level of which was subtracted from the toxin re- covery data.

DISCUSSION Using an intravesicular fluorescence signal in combination

with an osmotic swelling assay, we have demonstrated directly that CrylAc induces large pores in M. sextcl BBMV permeant for non-electrolyte solutes including raffinose and PEG 1000. Importantly, this assay characterises Cry toxin activity using target insect membranes which contain factors that influence the type of Cry toxin pore observed (Martin and Wolfersberger, 1995). The new assay has important advantages over our previ- ous method which used a light-scattering signal for monitoring BBMV volume changes in target insect midgut BBMV (Carroll and Ellar, 1993). The light-scattering assay can be used to show differences in the pore-forming activities of Cry toxins and changes as a result of mutations generated in a specific toxin protein (Wolfersberger et al., 1996) but only allows a qualitative comparison of different solute permeabilities (Carroll and Ellar, 1993). The fluorescence assay described here provides a quanti- tative approach for analysing toxin-mediated permeability changes for different solutes. Potential differences in the size of Cry-toxin-induced membrane permeability changes and any relationship with in vivo toxicity can now be investigated with greater confidence.

While the intravesicular fluorescence signal eliminates po- tential light-scattering artifacts, the signal changes seen were again complex. A curvilinear signal/volume relationship for car- boxyfluorescein-loaded BBMV (Fig. 4) and the effects of prior incubation with toxin both interfere with a simple interpretation of the signal changes. The toxin incubation step altered the prop- erties of BBMV relative to control vesicles. From the data pre- sented in Figs 5 and 6, we have proposed that toxin-treated vesi- cles may either shrink or swell depending upon the vesicle incu- bation bathing solution. When subsequently exposed to an os- motic gradient, the volume changes that occur are influenced by the ability of the toxin to alter the membrane permeability for the added solute.

Carroll and Ellar (Eur: J. Biochem. 245) 803

Osmotic gradients and a toxin incubation step are often used when amino acid uptake is employed to investigate toxin action (Sacchi et al., 1986). Our data suggest that changes in the in- travesicular volume of toxin-treated BBMV should be consid- ered in addition to a direct effect of a toxin on the BBMV per- meability for the test hyperosmotic solute or amino acid, or inhi- bition of the amino acid transporter. Giordana et al. (1993) ruled out the possibility that Bombyx mori BBMV incubated with B. thuringiensis subsp. aizuwui &endotoxin had a reduced osmoti- cally active space. However, the succinate-uptake experiment used to establish this was performed under different incubation conditions from those for the amino acid uptake assay. Also a possible difference in the permeability of a toxin-induced pore for the succinate anion and the amino acid histidine used in their study should be considered. The idea of different substrate per- meabilities can equally apply to the reported differential effect of Cryl Ac on aspartic acid and leucine uptake in M. sexta BBMV (Reuveni and Dunn, 1991).

From ionic-conductance data Martin and Wolfersberger (1995) calculated that at pH 9.6 CrylAc induces channels with a pore diameter of 2.2 nm in planar lipid bilayers fused with M. sextu BBMV. Similarly we found that at pH 9.8 CrylAc induces or forms 2.6-nm-diameter pores in M. sexta BBMV (Fig. 8B). A significant difference is seen, however, when results at pH 8.8 (Martin and Wolfersberger, 1995) and 8.7 (Fig. 8A) are com- pared. Our data indicate that the toxin-induced pore at both pH values has a similar size, whereas channel conductance measure- ments suggest that at pH 8.8 CrylAc produces a pore with a diameter of only 0.9 nm (Wolfersberger, 1995). The osmotic swelling assay presented here uses non-ionic solutes to charac- terise the membrane permeability changes and is therefore less sensitive to changes in the charge distribution on a toxin-induced pore. In contrast, pH-induced changes in channel organisation or charge distribution may potentially alter the observed ionic conductance of a toxin-mediated pore. Large differences in mea- sured channel conductances for latrotoxin were not accompanied by a significant change in pore size when analysed using a method employing non-electrolytes (Krasilnikov and Sabirov, 1992). Martin and Wolfersberger (1995) used vesicle fusion to lipid bilayers as their system for analysing toxin effects on bi- layer permeability. If the type of vesicle and the vesicle fusion rate varied under the different conditions used and assuming a direct participation of midgut BBMV specific factors in toxin activity, this could influence the type of toxin-induced pore ob- served.

Alternatively, while every effort was made to ensure the so- lution pH was comparable with that of Martin and Wolfersberger (1995), these bulk pH values may not be the same at the BBMV membrane surface under the low ionic conditions used in our system (Ehrenberg, 1986). If the pH was similar at the mem- brane surface under the conditions described here, this could account for the similar pore size being observed. Other factors complicating the estimation of the toxin pore size are: (a) our use of signal changes observed in control experiments to correct for background solute permeab es in the presence of toxin is problematical due to the different signal/volume relationships of control and toxin-treated BBMV (see Results); (b) the non-lin- ear signalivolume relationship for BBMV swelling (see Results) and (c) any polydispersion of the PEG compounds could result in an apparent permeability due to the movement of smaller mol- ecules in the preparation only (Scherrer and Gerhardt, 1971). Therefore, while raffinose (1.14 nm diameter) is obviously per- meant for the CrylAc pore (Fig. 7), at the exclusion limits of the pore where the levels of solute permeability are low, the above factors are relevant. Shrinkage signals were greater for BBMV at pH 8.7 and, unlike pH 9.8 (Fig. 7), they exhibited a

swelling signal over 101 s for the larger sotutes in the presence of CrylAc (data not shown). However, this would not alter our estimation of the toxin pore size as there was still no apparent difference between PEG 1500 and 4000 at 101 s.

Although the BBMV permeability changes we previously demonstrated for dissociated salts require the movement of both ions, with the slower-moving ion determining the rate of re- swelling, we have never ruled out some ionic selectivity (Carroll and Ellar, 1993). Lorence et al. (1995) observed a number of different sized-conductance channels (0.05 - 1.9 nS) induced by CrylC when applied to lipid bilayers containing Spodoptera frugiperdu midgut BBMV. While not commented upon, their IN data indicated that the large conductance channels exhibit lower ionic selectivity. With a reversal potential of approxi- mately -20 mV, these toxin-induced channels have a permeabil- ity ratio P K ~ : P c , - around 6: 1, under the conditions used. It is possible that when using hyperosmotic salt solutions in the os- motic swelling assay we only measure changes occurring due to a sub-population of large conductance channels with a low ionic selectivity, formed by CrylAc in the presence of receptor mole- cules. The Cryl Ac-induced channels observed by Martin and Wolfersberger (1995) in lipid bilayers containing fused M. sextu BBMV had macroscopic conductances ranging from 13 nS to greater than 50 nS.

At the present time the possibility that different toxin-vesi- cle interactions can result in different channel activities cannot be ruled out. This may be particularly acute if the toxin forms or activates a channel involving the receptor which itself may be a transmembrane protein, the identity of which varies for different toxin-insect interactions. What is clear from the pre- sent study is that, at alkaline pH, the CrylAc toxin will alter M. sextu BBMV permeability for large non-electrolyte solutes, allowing the free movement of raffinose (1.14 nm diameter) by inducing or forming a pore with an estimated internal diameter greater than 2 nm.

We thank Mr D. Hill for technical assistance in the electron micro- scopic examination of BBMV, Mr T. Sawyer for general technical help and Mr R. Summers and Ms K. Rowsell for photograhic work. Amino acid analysis was performed by Mrs J. Jacoby, supported by the Wel- come Trust. Dr J. Gray is gratefully acknowledged for the use of his gel scanning densitometer. This work was supported by the Biotechnology and Biological Research Council.

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