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The pH characterizing the cytolytic activity of Bacillus thuringiensis δ-endotoxin in the larval epithelium of Spodoptera littoralis
JOURNAL
OF INVERTEBRATE
PATHOLOGY
(1987)
s&320-321
NOTES The pH Characterizing the Cytolytic Activity of Bacillus &Endotoxin in the Larval Epithelium of Spodoptera The peritrophic membrane (PM) plays a very important role in protecting the sensitive epithelial cells in midguts of various insects (A. G. Richards and P A. Richards, Annu. Rev. Entomol. 22, 219-240, 1977). The PM enables the permeability of small molecules like glucose, amino acids, various ions, and also peptides and enzymes (D. P. Zhuzhikov, J. Insect Physiol. 10, 273-278, 1964; D. P. Zhuzhikov, J. Insect Physiol. 16, 1193-1202, 1970; M. J. Adang and K. D. Spence, Bacterial. Proc. 97, 1979). Thus, the PM protects the delicate epithelial cells from exposure to physical or chemical damage caused by large size molecules (C. R. Brandt, M. J. Adang, and K. D. Spence, J. Invertebr. Pathol. 32, 12- 14, 1978). During a study on the cytolytic effect of the 6-endotoxin produced by Bacillus thuringiensis var. entomocidus on an isolated midgut of Spodoptera littoralis (H. Yunovitz, B. Sneh, S. Shuster, U. Oron, M. Broza, and A. Yawetz, J. Invertebr. Pathol. 48, 223-231, 1978) a certain protective role of the PM in the insect midgut was demonstrated. In the absence of detergents the molecules of the toxin (64,000 M,) form large aggregates that are elluted in the void volume of Sepharose 6B gel filtration. These aggregates did not pass through the PM and did not induce bursting of the epithelial cells. It is well known (I. Ishaaya, I. Moore, and D. Joseph, J. Insect Physiol. 17, 945-953, 1971) that the pH of the midgut content in S. littoralis is above 9. Results of the present work indicate an additional protective role of the PM. The PM protects the epithelial cells from exposure to the high pH of the lumen contents. 320 0022-2011/87 Copyright All rights
$1.50
0 1987 by Academic Press, Inc. of reproduction m any form reserved.
thuringiensis littoralis
Larvae of S. littoralis (5th instar) were dissected by a longitudinal ventral skin cut. The midgut was removed, rinsed with Ringer solution, blotted on filter paper, and then was carefully cut longitudinally leaving the PM with the food content intact. The midgut was flattened and the PM plus food were carefully removed. The pH on the epithelium was measured using a flat-end electrode and was similarly measured in the lumen. The measurements were replicated five times. The results indicate that the average pH on the epithelial cell membranes was 7.56 + 0.20 while that in the lumen was 9.14 ? 0.20. The high pH in the lumen is provided by secretion of Na,CO, from the goblet cells. The mechanism by which the pH on the cell membrane is maintained at close to a neutral value of 7.5 may be the result of a constant proton movement from the midgut to the surface area of the epithelial cells (W. R. Harvey, M. Cioffi, and M. G. Wolfersberger, 1986, in “Fundamental and Applied Aspects of Invertebrate Pathology,” R. A. Samson, J. M. Valk, and D. Peters, Eds., pp. 11-14. ICIP, The Netherlands). The solubilization and activation of the parasporal crystal of B. thuringiensis require a high pH environment (R. M. Faust, J. R. Adams, and A. M. Heimpel, J. Invertebr. Pathol. 9, 488-499, 1967). The pH conditions are matched only in the lumen while on the surface of the intestinal epithelial cell membranes the pH is maintained close to neutral. In a previous study (H. Yunovitz, B. Sneh, S. Shuster, U. Oron, M. Broza, and A. Yawetz, 1986, lot. cit.), using the “isolated midgut system,” the 8-endotoxin pro-
321
NOTES
duced by B. thuringiensis var. entomocidus induced a cytolytic effect on the epithelial cells of S. littoralis larvae at pH 10. In this technique the isolated midgut is placed between two cells, one on the luminal side and one on the hemocoel side of the gut. A cytosolic enzyme, reduced glutathione-Stransferase, is released to the luminal cell medium from epithelial cells that were ruptured by the 6-endotoxin, and its activity is measured spectrophotometrically. As the present work revealed that the pH on the surface of the midgut epithelial cells is around 7.5, it was important to find out whether the Cendotoxin is active at the pH value occurring naturally at the site of action, i.e., the epithelial cell membrane. The results summarized in Table I clearly indicate that the toxin retained about 65% of toxic activity at pH 7.5. This finding expands the pH range at which the 6-endotoxin maintains its activity below the pH 10.0 value reported (W. R. Harvey and M. G. Wolfersberger, J. Exp. Biol. 83, 293-304, 1979) as the lowest pH for the toxin activity. The pH conditions under which the toxin is active affect the charge state of functional groups on both the toxin molecule and relevant interaction sites on the epithelium membrane. These aspects of the mode of action of the 6-endotoxin are currently being studied in our laboratory. KEY WORDS: Bacillus thuringiensis var.
TABLE 1 EFFECTOFMEDIUM pH ONTHERELEASEOF GSH-S-TRANSFEFLASE FROM Spodoptera littoralis MIDGUTEPITHELIALCELLSRUFNREDBYTHE ENDOTOXIN OF Bacillus thuringiensis var. entomocidus AFTER 15 MIN INCUBATION Endotoxin (50 t&ml)
Medium pH
Absent Present Absent Present
10.0 10.0 7.5 7.5
GSH-S-transferase activity” 0.170 0.720 0.130 0.470
2 + 2 k
0.200 0.450 0.014 0.057
0 Nanomoles 1-chloro-2,4-dinitrobenzene conjugated per 50 pJ of luminal cell medium per minute. The procedure and the results at pH 10 were from 10 determinations as previously described (H. Yunovitz, B. Sneh. S. Shuster, U. Oron, M. Broza, and A. Yawetz, 1986, lot. cit.), and those for pH 7.5 were of 6 determinations. The results are means + SD.
entomocidus; Spodoptera urn pH.
“isolated littoralis;
midgut system”; surface epitheli-
This research was partially supported by Biotechnological Applications Ltd., Jerusalem, Israel. HERMONA YUNOVITZ BARUCH SNEH AMINADAV YAWETZ Institute for Nature Conservation Research George S. Wise Faculty of Life Sciences Tel-Aviv University Tel Aviv 49978, Israel Received October 1, 1986; accepted May 4, 1987
OF INVERTEBRATE
PATHOLOGY
(1987)
s&320-321
NOTES The pH Characterizing the Cytolytic Activity of Bacillus &Endotoxin in the Larval Epithelium of Spodoptera The peritrophic membrane (PM) plays a very important role in protecting the sensitive epithelial cells in midguts of various insects (A. G. Richards and P A. Richards, Annu. Rev. Entomol. 22, 219-240, 1977). The PM enables the permeability of small molecules like glucose, amino acids, various ions, and also peptides and enzymes (D. P. Zhuzhikov, J. Insect Physiol. 10, 273-278, 1964; D. P. Zhuzhikov, J. Insect Physiol. 16, 1193-1202, 1970; M. J. Adang and K. D. Spence, Bacterial. Proc. 97, 1979). Thus, the PM protects the delicate epithelial cells from exposure to physical or chemical damage caused by large size molecules (C. R. Brandt, M. J. Adang, and K. D. Spence, J. Invertebr. Pathol. 32, 12- 14, 1978). During a study on the cytolytic effect of the 6-endotoxin produced by Bacillus thuringiensis var. entomocidus on an isolated midgut of Spodoptera littoralis (H. Yunovitz, B. Sneh, S. Shuster, U. Oron, M. Broza, and A. Yawetz, J. Invertebr. Pathol. 48, 223-231, 1978) a certain protective role of the PM in the insect midgut was demonstrated. In the absence of detergents the molecules of the toxin (64,000 M,) form large aggregates that are elluted in the void volume of Sepharose 6B gel filtration. These aggregates did not pass through the PM and did not induce bursting of the epithelial cells. It is well known (I. Ishaaya, I. Moore, and D. Joseph, J. Insect Physiol. 17, 945-953, 1971) that the pH of the midgut content in S. littoralis is above 9. Results of the present work indicate an additional protective role of the PM. The PM protects the epithelial cells from exposure to the high pH of the lumen contents. 320 0022-2011/87 Copyright All rights
$1.50
0 1987 by Academic Press, Inc. of reproduction m any form reserved.
thuringiensis littoralis
Larvae of S. littoralis (5th instar) were dissected by a longitudinal ventral skin cut. The midgut was removed, rinsed with Ringer solution, blotted on filter paper, and then was carefully cut longitudinally leaving the PM with the food content intact. The midgut was flattened and the PM plus food were carefully removed. The pH on the epithelium was measured using a flat-end electrode and was similarly measured in the lumen. The measurements were replicated five times. The results indicate that the average pH on the epithelial cell membranes was 7.56 + 0.20 while that in the lumen was 9.14 ? 0.20. The high pH in the lumen is provided by secretion of Na,CO, from the goblet cells. The mechanism by which the pH on the cell membrane is maintained at close to a neutral value of 7.5 may be the result of a constant proton movement from the midgut to the surface area of the epithelial cells (W. R. Harvey, M. Cioffi, and M. G. Wolfersberger, 1986, in “Fundamental and Applied Aspects of Invertebrate Pathology,” R. A. Samson, J. M. Valk, and D. Peters, Eds., pp. 11-14. ICIP, The Netherlands). The solubilization and activation of the parasporal crystal of B. thuringiensis require a high pH environment (R. M. Faust, J. R. Adams, and A. M. Heimpel, J. Invertebr. Pathol. 9, 488-499, 1967). The pH conditions are matched only in the lumen while on the surface of the intestinal epithelial cell membranes the pH is maintained close to neutral. In a previous study (H. Yunovitz, B. Sneh, S. Shuster, U. Oron, M. Broza, and A. Yawetz, 1986, lot. cit.), using the “isolated midgut system,” the 8-endotoxin pro-
321
NOTES
duced by B. thuringiensis var. entomocidus induced a cytolytic effect on the epithelial cells of S. littoralis larvae at pH 10. In this technique the isolated midgut is placed between two cells, one on the luminal side and one on the hemocoel side of the gut. A cytosolic enzyme, reduced glutathione-Stransferase, is released to the luminal cell medium from epithelial cells that were ruptured by the 6-endotoxin, and its activity is measured spectrophotometrically. As the present work revealed that the pH on the surface of the midgut epithelial cells is around 7.5, it was important to find out whether the Cendotoxin is active at the pH value occurring naturally at the site of action, i.e., the epithelial cell membrane. The results summarized in Table I clearly indicate that the toxin retained about 65% of toxic activity at pH 7.5. This finding expands the pH range at which the 6-endotoxin maintains its activity below the pH 10.0 value reported (W. R. Harvey and M. G. Wolfersberger, J. Exp. Biol. 83, 293-304, 1979) as the lowest pH for the toxin activity. The pH conditions under which the toxin is active affect the charge state of functional groups on both the toxin molecule and relevant interaction sites on the epithelium membrane. These aspects of the mode of action of the 6-endotoxin are currently being studied in our laboratory. KEY WORDS: Bacillus thuringiensis var.
TABLE 1 EFFECTOFMEDIUM pH ONTHERELEASEOF GSH-S-TRANSFEFLASE FROM Spodoptera littoralis MIDGUTEPITHELIALCELLSRUFNREDBYTHE ENDOTOXIN OF Bacillus thuringiensis var. entomocidus AFTER 15 MIN INCUBATION Endotoxin (50 t&ml)
Medium pH
Absent Present Absent Present
10.0 10.0 7.5 7.5
GSH-S-transferase activity” 0.170 0.720 0.130 0.470
2 + 2 k
0.200 0.450 0.014 0.057
0 Nanomoles 1-chloro-2,4-dinitrobenzene conjugated per 50 pJ of luminal cell medium per minute. The procedure and the results at pH 10 were from 10 determinations as previously described (H. Yunovitz, B. Sneh. S. Shuster, U. Oron, M. Broza, and A. Yawetz, 1986, lot. cit.), and those for pH 7.5 were of 6 determinations. The results are means + SD.
entomocidus; Spodoptera urn pH.
“isolated littoralis;
midgut system”; surface epitheli-
This research was partially supported by Biotechnological Applications Ltd., Jerusalem, Israel. HERMONA YUNOVITZ BARUCH SNEH AMINADAV YAWETZ Institute for Nature Conservation Research George S. Wise Faculty of Life Sciences Tel-Aviv University Tel Aviv 49978, Israel Received October 1, 1986; accepted May 4, 1987