Protease inhibitors fail to prevent pore formation by the activated Bacillus thuringiensis toxin Cry1Aa in insect brush border membrane vesicles.
To investigate whether membrane proteases are involved in the activity of Bacillus thuringiensis insecticidal toxins, the rate of pore formation by trypsin-activated Cry1Aa was monitored in the presence of a variety of protease inhibitors with Manduca sexta midgut brush border membrane vesicles and by a light-scattering assay. Most of the inhibitors tested had no effect on the pore-forming ability of the toxin. However, phenylmethylsulfonyl fluoride, a serine protease inhibitor, promoted pore formation, although this stimulation only occurred at higher inhibitor concentrations than those commonly used to inhibit proteases. Among the metalloprotease inhibitors, o-phenanthroline had no significant effect; EDTA and EGTA reduced the rate of pore formation at pH 10.5, but only EDTA was inhibitory at pH 7.5. Neither chelator affected the properties of the pores already formed after incubation of the vesicles with the toxin. Taken together, these results indicate that, once activated, Cry1Aa is completely functional and does not require further proteolysis. The effect of EDTA and EGTA is probably better explained by their ability to chelate divalent cations that could be necessary for the stability of the toxin's receptors or involved elsewhere in the mechanism of pore formation.
Kirouac
Martin
M
Groupe d'Etude des Protéines Membranaires, Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, Quebec H3C 3J7, Canada. raynald.laprade@umontreal.ca.
Vachon
Vincent
V
Quievy
Delphine
D
Schwartz
Jean-Louis
JL
Laprade
Raynald
R
D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000633:toxicity*; D001427:Bacterial Toxins / Q000633:toxicity*; D004064:Digestive System / Q000187:drug effects / Q000378:metabolism; D004731:Endotoxins / Q000633:toxicity*; D006460:Hemolysin Proteins; D018402:Manduca / Q000187:drug effects* / Q000378:metabolism; D008871:Microvilli / Q000187:drug effects* / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011480:Protease Inhibitors; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010664:Phenylmethylsulfonyl Fluoride