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Preliminary investigations on the molecular mode of action of the δ-endotoxin produced by Bacillus thuringiensis var. alesti
JOURNAL
OF INVERTEBRATE
Preliminary
PATHOLOGY
23, 259-261
Investigations
b-Endotoxin
Produced
(1974)
on the
Molecular
by Bacillus
The action of the 6-endotoxin produced by several varieties of Bacillus thuringiensis in Bombyx mori larvae stimulates glucose uptake in the larval midgut within 1 min after the insects ingests the toxic crystals. Maximum stimulation is reached within 5 min (P. Fast and T. Donaghue, J. Inwertebr. Pathol. 18, 135-138, 1971; P. Fast and I. Morrison, J. Invertebr. Pathol. 20, 208-211, 1972), and uptake ceases in 10 min. A general breakdown of control of permeability then occurs between 10 and 15 min as levels of K+ and other ions increase in the hemolymph (P. Fast and T. Angus, J. Invertebr. Pathol. 7, 29-32, 1965; S. Louloudes and A. Heimpel, J. Invertebr. Pathol. 14, 375380, 1969; I. Pendleton, J. Invertebr. Pathol. 16, 313314, 1970; P. Fast and I. Morrison, J. Invertebr.
Pathol.
20,
208-211, 1972).
However, K+, Na+, Ca?+, and Mg?+ are not accumulated in gut tissue during the first 10 min after toxin is given per OS, and no change is detected in the rate at which K+ turns over in the gut tissue. Approximately 20 min after the toxin is ingested by some susceptible species, there is noticeable ‘(ballooning” or extrusion of the columnar cells within 25 min that produces a severe disruption of the gut wall as the cells burst and release their contents (T. A. Angus, Proc.
4th
Int.
Colloq.
Insect.
Pathol.
183-189, 1970; G. Sutter and E. Raun, J. Invertebr.
Pathol.
Hoopingarner
9,
90-103, 1967; R.
and M. Materu,
PathoE. 6, 26-30, 1964). B. thuringiensis var. ale&i
J. Insect
(HD-1) parasporal crystals were obtained in crude powdered form from H. Dulmage, USDA, Brownsville, Texas (Abbott Laboratories
@ 1974 by of reproduction
Academic Press, Inc. in any form reserved.
thuringiensis
of Action var.
of the
alesti
lot No. 837-3424-212) and purified as described by R. Faust (J. Invertebr. Pathol.
11,466475,1968). The midguts were dissected from approximately 200 third-stage larvae of the silkworm, B. tori, that had been starved for 48 hr. The gut juice was collected over an ice-bath and then centrifuged at 15,000 g for 20 min; the supernatant fluid was decanted. Then, midgut preparations containing active mitochondria were obtained by washing the dissected midguts slowly for 30 min in 0.9% NaCl-0.001 M Na,EDTA at 4°C. The preparations were centrifuged at 1000 g for 10 min and resuspended in 0.3 M potassium phosphate-O.001 1\6 Na2EDTA buffer, pH 7.4, to give a final concentration of about 2 g/ml (wet weight). Then the material was broken into particles by grinding at slow speed with a glass tube and Teflon homogenizer-rod for 7 min. Fifty-milligram samples of parasporal crystals (LD,, = 2.8 pg/g) were mixed with 3 ml each of clarified gut juice and incubated in a Dubnoff metabolic shaking incubator at 32% for 1.5 hr. Then the suspension was centrifuged at 8000 g for 10 min, and the pellet was resuspended in 2 ml of clarified gut juice and reincubated as before. Purification and isolation of toxic components were obtained by gel filtration (Sephadex G-200: 2.54 X 100 cm column) using 0.1 M Na,CO,-NaHC03 buffer, pH 9.0, at 4%. The toxic protein fractions were further purified by gel electrophoresis using ’ Mention of a company name or proprietary product does not necessarily imply endorsement of the company or product by the U.S. Department of Agriculture.
259 Copyright ,411 rights
Mode
NOTES
260
a modification of the technique described by A. Klein, J. Chudzik, and M. SarneckaKeller (J. Chronzatogr. 53, 329-336, 1970). The gel electrophoresis was performed in 2.54 X 10 cm tubes at 20 mA per tube and at pH 9.0 in 0.1 M Na,CD-NaHCO, buffer. Salts were removed from the toxic fractions by dialysis in a Bio-Rad Beaker osmolyzer (Bio-Rad Laboratories, Richmond, California) before lyophilization. Respiratory activity of the midgut preparations was determined as described by J. Clark (“Experimental Biochemistry,” 1st Ed., pp. 154-163, Freeman, San Francisco, California, 1964). Experiments were carried out in duplicate at 32OC and pH 7.4 using a Gilson differential respirometer, with 0, consumption as the expression of activity and added succinate as the substrate. A standard midgut preparation consumed 150-200 ~10, per 30 min STP. The results in Table 1 demonstrate that the catalytic activities of the midgut prepa-
rations were inhibited by malonate, antimycin A, and KCN, and stimulated by dibromophenol and the 6-endotoxin of B. thuringiensis var. ale&i, an action typical of known uncoupling agents in some mitochondrial systems (B. Chance and G. Williams, J. Biol. Chew. 217,409, 1955; B. Chance and B. Sacktor, Arch. Biochem. Biophys. 76, 509-531, 1958). To bring together the histological and biochemical evidence of the mode of action known to date, we offer the following explanation: Removal of the reactive intermediate linked to the carrier [ ( ) -PI by an ATP-generating step is accomplished either through the formation of glucose-6-P from glucose or by hydrolysis of ATP. (H. Mahler and E. Cordes, in “Biological Chemistry,” 1st ed., pp. 612-617, 1966). One observation closely related to this phenomenon is the accleration/stimulation with abolition of respiratory chain-linked phosphorylation brought about by un-
TABLE
1
RESPIRATORY ACTIVITY OF MIDWJT HOMOGENATE FROM LARVAL Bombyx
Treatment of flask contents Midgut homogenate+ succinate(standard) Midgut homogenate+ succinate+ malonate Midgut Midgut Midgut Midgut
homogenate + succinate + KCN homogenate + succinate + antimycin A homogenate + succinate + dibromophenol homogenate + succinate + Bacillus thuringiensis 6-endotoxin of molecular weight” 230,000 67,000 30,200 <5,000 Midgut homogenate (endogenous control)d
maria
Averagepercent oxygenconsumedb 100.00 10.72 5.67 2.20 127.8
(N/A) + 4.3 rf: 1.0 If: 0.5 zk 6.4
106.21 114.64 131.43 156.05 5.00
k f + zk 2~
5.3 4.6 5.8 6.8 1.8
-Oxygen consumed was measured in a Gilson differential respirometer with standard manometric vessels. Total volume was made to 3 ml by using 0.3 M potassium phosphate buffer at pH 7.4. Final concentrations of components in the vessels were as follows: Midgut preparation, 0.66 g/ml; sodium succinate, 2.3 X 10e3 g/ml; KCN, 7.0 X 10e4 g/ml; sodium malonate, 3.7 X lo+ g/ml; antimycin A, 1.8 X lOMB g/ml; dibromophenol, 6.3 X lo+ g/ml; B. thuringiensis b-endotoxin protein moieties, 3.3 X 1O-4 g/ml. Substrate was placed in the side arm. The center well contained 0.1 ml of 6N KOH and fritted filter paper. 6 Activity is expressed as percentage 02 consumed in the first 30-min period after equilibration. Data were compiled from duplicate tests of three experiments. ~Molecular weights were estimated by molecular sieving through Sephadex G-200 (100 X 2.5 cm column at pH 9.0) with a molecular weight variance of I? 5 %. d Endogenous control vessels contained no substrate (succinate).
261
NOTES
couplers. It has been suggested as an explanation for the stimulation that any of the reactive intermediates of the type A - B can form a complex with an uncoupler (4) such that +p- A or +- B breaks down hydrolytically more rapidly N B. R. M. Faust (J. Invertebr. than A Pathol. 11, 464-475, 1968) and R. M. Faust, E. M. Dougherty, A. M. Heimpel, and C. F. Reichelderfer (J. Econ. Entomol. 64, 610-615, 1971) demonstrat,ed a similar phenomenon with the B. thuringiensis 6-endotoxin and various enzymes. Uncouplers such as dibromophenol, dicoumarol, and gramicidin have been shown to be capable of tight binding into certain mitochondrial proteins (H. Mahler and E. Cordes, in “Biological Chemistry,” 1st ed., pp. 612-617, 1966). R. Faust (Doctoral dissertation, University of Maryland, 1970) demonstrated that extracted B. thuringiensis &endotoxin combines with cytochrome c and causes reduction of the oxidized form. Stimulation of respiration by the B. thuringiensis 6-endotoxin might be the explanation for the stimulation of glucose uptake during the first 5 min of intoxication in B. thuringiensis 6-endotoxin susceptible insects (R. Faust, 1970). By 10 min, glucose uptake ceases, probably because respiratory chainlinked phosphorylation is ceasing (analysis
of inorganic phosphate in our tests revealed that ATP production was being inhibited in the affected systems). Thus, the energy involved in glucose uptake and other energy-requiring activities becomes limited. The initial stimulation of glucose uptake could result from the initial effect of the 6-endotoxin on the respiratory system, that is, glucose uptake increases as metabolic respiration increases but without the benefit of respiratory chain-linked ATP production. When endogenous ATP becomes limited, glucose uptake ceases and the observed changes between 15 and 25 min would follow, i.e., ion transport upset, “ballooning,” bursting from uncontrolled osmotic effects, and disintegration of the mid-gut wall. We hope that completion of curent work will give us a fuller knowledge of the bases for these effects. ROBERT M. FAUST RUSSELL S. TRAVERS GLADYS M. HALLAM Insect Pathology Laboratory Plant Protection Institute Agricultural Research Service U.S. Department of Agriculture Reltsville, Maryland to705 Received August
7, 1973
OF INVERTEBRATE
Preliminary
PATHOLOGY
23, 259-261
Investigations
b-Endotoxin
Produced
(1974)
on the
Molecular
by Bacillus
The action of the 6-endotoxin produced by several varieties of Bacillus thuringiensis in Bombyx mori larvae stimulates glucose uptake in the larval midgut within 1 min after the insects ingests the toxic crystals. Maximum stimulation is reached within 5 min (P. Fast and T. Donaghue, J. Inwertebr. Pathol. 18, 135-138, 1971; P. Fast and I. Morrison, J. Invertebr. Pathol. 20, 208-211, 1972), and uptake ceases in 10 min. A general breakdown of control of permeability then occurs between 10 and 15 min as levels of K+ and other ions increase in the hemolymph (P. Fast and T. Angus, J. Invertebr. Pathol. 7, 29-32, 1965; S. Louloudes and A. Heimpel, J. Invertebr. Pathol. 14, 375380, 1969; I. Pendleton, J. Invertebr. Pathol. 16, 313314, 1970; P. Fast and I. Morrison, J. Invertebr.
Pathol.
20,
208-211, 1972).
However, K+, Na+, Ca?+, and Mg?+ are not accumulated in gut tissue during the first 10 min after toxin is given per OS, and no change is detected in the rate at which K+ turns over in the gut tissue. Approximately 20 min after the toxin is ingested by some susceptible species, there is noticeable ‘(ballooning” or extrusion of the columnar cells within 25 min that produces a severe disruption of the gut wall as the cells burst and release their contents (T. A. Angus, Proc.
4th
Int.
Colloq.
Insect.
Pathol.
183-189, 1970; G. Sutter and E. Raun, J. Invertebr.
Pathol.
Hoopingarner
9,
90-103, 1967; R.
and M. Materu,
PathoE. 6, 26-30, 1964). B. thuringiensis var. ale&i
J. Insect
(HD-1) parasporal crystals were obtained in crude powdered form from H. Dulmage, USDA, Brownsville, Texas (Abbott Laboratories
@ 1974 by of reproduction
Academic Press, Inc. in any form reserved.
thuringiensis
of Action var.
of the
alesti
lot No. 837-3424-212) and purified as described by R. Faust (J. Invertebr. Pathol.
11,466475,1968). The midguts were dissected from approximately 200 third-stage larvae of the silkworm, B. tori, that had been starved for 48 hr. The gut juice was collected over an ice-bath and then centrifuged at 15,000 g for 20 min; the supernatant fluid was decanted. Then, midgut preparations containing active mitochondria were obtained by washing the dissected midguts slowly for 30 min in 0.9% NaCl-0.001 M Na,EDTA at 4°C. The preparations were centrifuged at 1000 g for 10 min and resuspended in 0.3 M potassium phosphate-O.001 1\6 Na2EDTA buffer, pH 7.4, to give a final concentration of about 2 g/ml (wet weight). Then the material was broken into particles by grinding at slow speed with a glass tube and Teflon homogenizer-rod for 7 min. Fifty-milligram samples of parasporal crystals (LD,, = 2.8 pg/g) were mixed with 3 ml each of clarified gut juice and incubated in a Dubnoff metabolic shaking incubator at 32% for 1.5 hr. Then the suspension was centrifuged at 8000 g for 10 min, and the pellet was resuspended in 2 ml of clarified gut juice and reincubated as before. Purification and isolation of toxic components were obtained by gel filtration (Sephadex G-200: 2.54 X 100 cm column) using 0.1 M Na,CO,-NaHC03 buffer, pH 9.0, at 4%. The toxic protein fractions were further purified by gel electrophoresis using ’ Mention of a company name or proprietary product does not necessarily imply endorsement of the company or product by the U.S. Department of Agriculture.
259 Copyright ,411 rights
Mode
NOTES
260
a modification of the technique described by A. Klein, J. Chudzik, and M. SarneckaKeller (J. Chronzatogr. 53, 329-336, 1970). The gel electrophoresis was performed in 2.54 X 10 cm tubes at 20 mA per tube and at pH 9.0 in 0.1 M Na,CD-NaHCO, buffer. Salts were removed from the toxic fractions by dialysis in a Bio-Rad Beaker osmolyzer (Bio-Rad Laboratories, Richmond, California) before lyophilization. Respiratory activity of the midgut preparations was determined as described by J. Clark (“Experimental Biochemistry,” 1st Ed., pp. 154-163, Freeman, San Francisco, California, 1964). Experiments were carried out in duplicate at 32OC and pH 7.4 using a Gilson differential respirometer, with 0, consumption as the expression of activity and added succinate as the substrate. A standard midgut preparation consumed 150-200 ~10, per 30 min STP. The results in Table 1 demonstrate that the catalytic activities of the midgut prepa-
rations were inhibited by malonate, antimycin A, and KCN, and stimulated by dibromophenol and the 6-endotoxin of B. thuringiensis var. ale&i, an action typical of known uncoupling agents in some mitochondrial systems (B. Chance and G. Williams, J. Biol. Chew. 217,409, 1955; B. Chance and B. Sacktor, Arch. Biochem. Biophys. 76, 509-531, 1958). To bring together the histological and biochemical evidence of the mode of action known to date, we offer the following explanation: Removal of the reactive intermediate linked to the carrier [ ( ) -PI by an ATP-generating step is accomplished either through the formation of glucose-6-P from glucose or by hydrolysis of ATP. (H. Mahler and E. Cordes, in “Biological Chemistry,” 1st ed., pp. 612-617, 1966). One observation closely related to this phenomenon is the accleration/stimulation with abolition of respiratory chain-linked phosphorylation brought about by un-
TABLE
1
RESPIRATORY ACTIVITY OF MIDWJT HOMOGENATE FROM LARVAL Bombyx
Treatment of flask contents Midgut homogenate+ succinate(standard) Midgut homogenate+ succinate+ malonate Midgut Midgut Midgut Midgut
homogenate + succinate + KCN homogenate + succinate + antimycin A homogenate + succinate + dibromophenol homogenate + succinate + Bacillus thuringiensis 6-endotoxin of molecular weight” 230,000 67,000 30,200 <5,000 Midgut homogenate (endogenous control)d
maria
Averagepercent oxygenconsumedb 100.00 10.72 5.67 2.20 127.8
(N/A) + 4.3 rf: 1.0 If: 0.5 zk 6.4
106.21 114.64 131.43 156.05 5.00
k f + zk 2~
5.3 4.6 5.8 6.8 1.8
-Oxygen consumed was measured in a Gilson differential respirometer with standard manometric vessels. Total volume was made to 3 ml by using 0.3 M potassium phosphate buffer at pH 7.4. Final concentrations of components in the vessels were as follows: Midgut preparation, 0.66 g/ml; sodium succinate, 2.3 X 10e3 g/ml; KCN, 7.0 X 10e4 g/ml; sodium malonate, 3.7 X lo+ g/ml; antimycin A, 1.8 X lOMB g/ml; dibromophenol, 6.3 X lo+ g/ml; B. thuringiensis b-endotoxin protein moieties, 3.3 X 1O-4 g/ml. Substrate was placed in the side arm. The center well contained 0.1 ml of 6N KOH and fritted filter paper. 6 Activity is expressed as percentage 02 consumed in the first 30-min period after equilibration. Data were compiled from duplicate tests of three experiments. ~Molecular weights were estimated by molecular sieving through Sephadex G-200 (100 X 2.5 cm column at pH 9.0) with a molecular weight variance of I? 5 %. d Endogenous control vessels contained no substrate (succinate).
261
NOTES
couplers. It has been suggested as an explanation for the stimulation that any of the reactive intermediates of the type A - B can form a complex with an uncoupler (4) such that +p- A or +- B breaks down hydrolytically more rapidly N B. R. M. Faust (J. Invertebr. than A Pathol. 11, 464-475, 1968) and R. M. Faust, E. M. Dougherty, A. M. Heimpel, and C. F. Reichelderfer (J. Econ. Entomol. 64, 610-615, 1971) demonstrat,ed a similar phenomenon with the B. thuringiensis 6-endotoxin and various enzymes. Uncouplers such as dibromophenol, dicoumarol, and gramicidin have been shown to be capable of tight binding into certain mitochondrial proteins (H. Mahler and E. Cordes, in “Biological Chemistry,” 1st ed., pp. 612-617, 1966). R. Faust (Doctoral dissertation, University of Maryland, 1970) demonstrated that extracted B. thuringiensis &endotoxin combines with cytochrome c and causes reduction of the oxidized form. Stimulation of respiration by the B. thuringiensis 6-endotoxin might be the explanation for the stimulation of glucose uptake during the first 5 min of intoxication in B. thuringiensis 6-endotoxin susceptible insects (R. Faust, 1970). By 10 min, glucose uptake ceases, probably because respiratory chainlinked phosphorylation is ceasing (analysis
of inorganic phosphate in our tests revealed that ATP production was being inhibited in the affected systems). Thus, the energy involved in glucose uptake and other energy-requiring activities becomes limited. The initial stimulation of glucose uptake could result from the initial effect of the 6-endotoxin on the respiratory system, that is, glucose uptake increases as metabolic respiration increases but without the benefit of respiratory chain-linked ATP production. When endogenous ATP becomes limited, glucose uptake ceases and the observed changes between 15 and 25 min would follow, i.e., ion transport upset, “ballooning,” bursting from uncontrolled osmotic effects, and disintegration of the mid-gut wall. We hope that completion of curent work will give us a fuller knowledge of the bases for these effects. ROBERT M. FAUST RUSSELL S. TRAVERS GLADYS M. HALLAM Insect Pathology Laboratory Plant Protection Institute Agricultural Research Service U.S. Department of Agriculture Reltsville, Maryland to705 Received August
7, 1973