Rôle des facteurs physico-chimiques du micro-environnement intestinal et des boucles inter-hélicales du Domaine I dans l’activité de la toxine insecticide Cry9Ca du bacille de Thuringe
Once ingested by susceptible insects, Bacillus thuringiensis insecticidal toxins must be activated by the insect’s intestinal proteases.  Their first domain, a bundle of seven amphipathic -helices, is responsible for their insertion into the luminal membrane of midgut cells, thereby creating poorly selective pores.  The toxicity and pore-forming ability of one such toxin, Cry9Ca, its single-site mutants, R164A and R164K, and of the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using a combination of homology modeling, bioassays, osmotic swelling experiments with Manduca sexta larval midgut brush border membrane vesicles and electrophysiological measurements on isolated midguts.  Neither the single mutations nor the proteolytic cleavage altered Cry9Ca toxicity.  In low ionic strength solutions however, pore formation was highly dependent on pH: increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization.  All four toxin preparations nevertheless depolarized the apical membrane of freshly isolated midguts bathing in a solution containing 122 mM KCl at pH 10.5.  The activity of Cry9Ca, R164A and R164K was greatly enhanced when the experiments were conducted in the presence of midgut juice, the lipids extracted from an equivalent volume of midgut juice or a cocktail of water-soluble protease inhibitors.  Additionally, the role of the interhelical loops of Domain I in membrane insertion was investigated with Cry9Ca double mutants with mutations that either introduced, neutralized or reversed an electrical charge.  All but three mutants retained a toxicity and a pore-forming ability that were comparable to those of their parental toxin.  Overall, the results suggest that the midgut microenvironment contributes to minimizing the influence of surface charges carried by Domain I interhelical loop residues on B. thuringiensis toxins pore-forming ability.  They also indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.
Formation de pores Bacillus thuringiensis Manduca sexta Insecticidal toxins Pore formation Microenvironment Proteolysis Electrostatic interactions Osmotic swelling assay Electrophysiological assay Homology modelling Bacillus thuringiensis Manduca sexta