Analysis of the Properties of Bacillus thuringiensis Insecticidal Toxins Using a Potential-sensitive Fluorescent Probe

Abstract

A potential-sensitive fluorescent probe, 3,3'-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K(+) ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K(+) ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

“…The response time of the measurement is short [12,13], and changes in fluorescence level are directly associated to changes in membrane potential induced by the changes in ionic flux. However, the use of this system to determine pore formation by Cry toxins has been questioned [15]. Here, we validated the use of diSC 3 (5), showing that under specific ionic conditions, where the surface membrane potential is shielded and the intrinsic permeability is inhibited, the ionic strength and osmolarity have no effects on the analysis of membrane potential of M. sexta membrane vesicles and changes in fluorescence can be correlated with ionic permeability induced by Cry toxins.…”

“…3A, B). A previous report from Kirouac et al [15] suggested that osmolarity and ionic strength have a dramatic effect on diSC 3 (5) dye sensibility and on the estimations of changes in membrane potential. It is important to mention that these authors did not take into account the effects that pH and ionic strength have on the surface potential.…”

“…A previous report suggested that osmolarity and ionic strength have a dramatic effect on cyanine diSC 3 (5) dye sensibility and on the estimations of the changes in membrane potential [15]. In order to analyze the effect of ionic strength and osmolarity in our determinations of BBMV membrane potential, we analyzed the K + permeability induced by valinomycin under steady-state conditions where background response is reduced by the presence of divalent cations in the external solution.…”

Effect of Insect Larval Midgut Proteases on the Activity of Bacillus thuringiensis Cry Toxins

“…The lower activity of Cry1Aa and Cry1Ab is rather surprising, in view not only of the in vivo toxicity but also of the previous in vitro studies. Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ea had similar pore-forming activities in brush border membrane vesicles, as observed using an osmotic-swelling assay (36) or a potential-sensitive fluorescent dye (17). The lower pore-forming ability estimated for Cry1Ca using the osmoticswelling assay (36) compared with that measured using the potential-sensitive dye (17) was explained by the effect of ionic strength on the activity of this toxin (11).…”

“…1) or from their ability to increase the permeability of vesicles prepared from this membrane (17,36). In the electrophysiological assay, the pore-forming ability of the toxins is measured in living cells in which the presence of a strong membrane potential, which is absent during the incubation of the vesicles with the toxin prior to the osmotic swelling (36) and the fluorescence assay (17), could influence pore formation. However, this possibility does not appear to be sufficient to explain why the relative toxicity and in vitro activity of the toxins do not follow a similar pattern.…”