38th Annual Meeting of the Society for Invertebrate Pathology

The effect of various Bacillus thuringiensis toxins on the permeability of the luminal membrane of Manduca sexta midgut columnar epithelial cells to a range of charged and uncharged solutes was monitored with an osmotic swelling assay and membrane potential measurements. Membrane permeability in the presence of a given toxin is strongly influenced by several biophysical and biochemical factors, including pH, ionic strength and divalent cations, suggesting an important role for electrostatic interactions, as well as by proteolytic enzymes. The influence of these factors can differ greatly, however, depending on the toxin being studied, even for closely related toxins such as Cry1Ac and Cry1C. Lowering temperature gradually decreased the rate of pore formation, but had little effect on the permeability of vesicles pre-incubated with toxin at room temperature. The formation of new pores, following incubation of the vesicles with toxin, could thus be almost abolished by rapidly cooling the vesicles to 2°C before the onset of the osmotic swelling experiments. Using this approach, changes in the rate of pore formation could be more easily distinguished from alterations in the properties of the pores formed, thus allowing a more detailed analysis of the kinetics and mechanism of pore formation.