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

Abstract

Abstract. The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K + permeability. A fluorometric assay performed with 3,3¢-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K + -selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

“…The oligomer and pore formation activities of other Cry toxins have also been reported (Cry1Aa, Cry1Ca, Cry1Da, Cry 1Ea, and Cry1Fa) [30], showing that samples containing the oligomeric toxin showed higher pore-formation activity than samples activated with trypsin that only contain monomeric toxin [30]. This data shows that the oligomeric structure of Cry toxins, but not the monomer, is the intermediate that is responsible for insertion into the membrane and pore formation.…”

“…Cry1Ca [18,30], Cry1Da, Cry1Ea, Cry1Fa [30]; coleopteran specific, Cry3Aa, Cry3Ba, Cry3Ca [37], and dipteran specific, Cry4Ba [26] and Cry11Aa (Pérez and Bravo, unpublished results). Protein samples containing Cry1Ab pre-pore oligomer were more toxic to M. sexta larvae than protein samples containing only monomeric Cry1Ab toxin [13].…”

“…Protein samples containing Cry1Ab pre-pore oligomer were more toxic to M. sexta larvae than protein samples containing only monomeric Cry1Ab toxin [13]. In addition, Cry toxin samples containing oligomeric structures correlated with high pore activity in contrast to monomeric samples that showed marginal pore-formation activity [13,30,36]. The Cry1Ab pre-pore oligomer is membrane insertion competent in contrast to monomeric toxin that showed low membrane insertion rates [36].…”

“…After proteolytic activation of Cry1A protoxin by insect midgut proteases, the activated toxin binds to the Bt-R 1 receptor, and this interaction facilitates additional cleavage of the N-terminal end of the toxin, eliminating helix α-1, resulting in the formation of a pre-pore oligomeric structure [13]. The oligomeric structure has been observed in several Cry toxins [1,13,18,26,30,33,36,37,40]. The pre-pore binds to a second receptor, the GPI-anchored aminopeptidase, due to its higher affinity to this receptor [3], facilitating the insertion of the oligomer into membrane lipid rafts and forming pores [3,46].…”

“…One approach analyses changes in ion permeability using a fluorescent dye that is sensitive to changes in membrane potential, 3,3'-dipropylthiodicarbocyanine iodide (diS-C 3 (5)) [2,27,43]. Using this strategy it was reported that oligomeric Cry1Ab induced a dose-dependent K + permeability and that the oligomeric Cry1Ab structure has a much higher pore-forming activity than the corresponding monomeric structure [30].…”