Protease inhibitors fail to prevent pore formation by the activated Bacillus thuringiensis toxin Cry1Aa in insect brush border membrane vesicles

Abstract

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.

FIG. 1.

Osmotic swelling of M. sexta midgut brush border membrane vesicles induced by various concentrations of Cry1Aa. Vesicles equilibrated in 10 mM CAPS-KOH (pH 10.5) and 1-mg/ml bovine serum albumin were rapidly mixed with an equal volume of 150 mM KCl, 10 mM CAPS-KOH (pH 10.5), 1-mg/ml bovine serum albumin, and the indicated concentrations of toxin (in picomoles of toxin per milligram of membrane protein). Percent volume recovery was calculated for each experimental point, and the values measured for control vesicles, assayed in the absence of toxin, were subtracted from those obtained in the presence of toxin. Data are means ± SEM of four experiments. For clarity, error bars are shown for every 100th experimental point.

FIG. 2.

Osmotic swelling of midgut brush border membrane vesicles induced by Cry1Aa in the presence of PMSF (A) and EDTA (B). Vesicles were mixed with a solution containing 150 mM KCl, 10 mM CAPS-KOH (pH 10.5), 1-mg/ml bovine serum albumin, and 50 pmol Cry1Aa/mg of membrane protein. The vesicle suspension and the KCl solution contained 5 mM PMSF (+PMSF), 2.5% ethanol (−PMSF), or 2 mM EDTA (+EDTA). Data are means ± SEM of three experiments. For clarity, error bars are shown for every 100th experimental point.

FIG. 3.

Effect of PMSF concentration on the rate of pore formation by Cry1Aa in brush border membrane vesicles. Vesicles were mixed with a solution containing 150 mM KCl, 10 mM CAPS-KOH (pH 10.5), 1-mg/ml bovine serum albumin, and 50 pmol Cry1Aa/mg of membrane protein. Data are means ± SEM of three experiments. Asterisks indicate a significant difference (P < 0.05) relative to controls (0 mM PMSF).

FIG. 4.

Effect of PMSF on the rate of pore formation by Cry1Aa. Vesicles were mixed with a solution containing 150 mM KCl, 10 mM CAPS-KOH (pH 10.5), 1-mg/ml bovine serum albumin, and the indicated concentration of Cry1Aa (in picomoles of toxin per milligram of membrane protein). The experiments were performed without PMSF (▪) and with 4 mM PMSF (•). Data are means ± SEM of four (without PMSF) or five (with PMSF) experiments.

FIG. 5.

Effect of PMSF additions during incubation of the vesicles with Cry1Aa. Vesicles were incubated for 60 min and mixed with a solution containing 150 mM KCl, 10 mM CAPS-KOH (pH 10.5), and 1-mg/ml bovine serum albumin. Vesicles were incubated either without (Control) or with 50 pmol Cry1Aa/mg of membrane protein. Ethanol (2%) (None) or PMSF (4 mM) was added at the beginning of the incubation period, 10 min before the addition of the toxin (Start), or after an incubation of 50 min with the toxin (End). Percent volume recovery at 3 s (B) was derived from the experimental curves shown in panel A. Data are means ± SEM of three experiments. Bars labeled with the same letter are not significantly different (P > 0.05).

FIG. 6.

Effect of EDTA and EGTA on the rate of pore formation by Cry1Aa. Vesicles were mixed with a solution containing 150 mM KCl and 10 mM CAPS-KOH (pH 10.5) (A and B) or 10 mM HEPES-KOH (pH 7.5) (C), 1-mg/ml bovine serum albumin, and 50 pmol Cry1Aa/mg of membrane protein. The vesicle suspension and the KCl solution contained the indicated concentration of EDTA (A and C) or EGTA (B). Data are means ± SEM of three experiments.

FIG. 7.

Effect of EDTA and EGTA additions during incubation of the vesicles with Cry1Aa. Vesicles were incubated for 60 min and mixed with a solution containing 150 mM KCl and 10 mM HEPES-KOH (pH 7.5) (A and C) or CAPS-KOH (pH 10.5) (B and D) and 1-mg/ml bovine serum albumin. Vesicles were incubated either without (Control) or with 50 pmol Cry1Aa/mg of membrane protein. EDTA (A and B) or EGTA (C and D) were added at a final concentration of 2 mM at the beginning of the incubation period, 10 min before the addition of the toxin (Start), or after an incubation of 50 min with the toxin (End). Data are means ± SEM of three experiments. Bars labeled with the same letter are not significantly different (P > 0.05).