Effect of insect larval midgut proteases on the activity of Bacillus thuringiensis Cry toxins.
To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.
Fortier
Mélanie
M
Groupe d'Etude des Protéines Membranaires, Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, Quebec H3C 3J7, Canada.
Vachon
Vincent
V
Frutos
Roger
R
Schwartz
Jean-Louis
JL
Laprade
Raynald
R
D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D004064:Digestive System / Q000378:metabolism; D010450:Endopeptidases / Q000633:toxicity*; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D007313:Insecta; D007814:Larva
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010450:Endopeptidases
Appl Environ Microbiol. 2000 Apr;66(4):1553-8
10742241
Appl Environ Microbiol. 2000 Dec;66(12):5174-81
11097886
J Biol Chem. 2001 Sep 21;276(38):35546-51
11466307
Appl Environ Microbiol. 2001 Oct;67(10):4488-94
11571147
J Econ Entomol. 2001 Oct;94(5):1135-41
11681676
FEBS Lett. 2002 Feb 27;513(2-3):242-6
11904158
Appl Environ Microbiol. 2002 Apr;68(4):1509-15
11916662
Biochemistry. 2002 May 14;41(19):6178-84
11994014
J Membr Biol. 2003 Nov 1;196(1):51-9
14724756
Biochim Biophys Acta. 2004 Nov 17;1667(1):38-46
15533304
Appl Environ Microbiol. 1988 Aug;54(8):2010-7
16347711
Appl Environ Microbiol. 2006 Jan;72(1):506-15
16391085
J Membr Biol. 2005 Nov;208(1):77-87
16596448
J Invertebr Pathol. 2006 Jul;92(3):172-7
16777138
J Invertebr Pathol. 2006 Jul;92(3):160-5
16831627
Biochem J. 1990 Jul 15;269(2):507-12
2167072
Appl Environ Microbiol. 1990 May;56(5):1378-85
2339890
Eur J Biochem. 1989 Dec 8;186(1-2):239-47
2557209
Oecologia. 1986 Dec;71(1):133-137
28312095
Mol Microbiol. 1994 Dec;14(5):851-60
7715447
Appl Environ Microbiol. 1997 May;63(5):1679-84
9143102
Microbiol Mol Biol Rev. 1998 Sep;62(3):775-806
9729609