Atomic force microscopy imaging of Bacillus thuringiensis Cry1 toxins interacting with insect midgut apical membranes

Atomic force microscopy was used to image Bacillus thuringiensis (Bt) toxins interacting with their natural targets, Manduca sexta midgut brush border membranes (BBMs), as well as with dipalmitoylphosphatidylcholine-dioleoylphosphatidylcholine (DPPC-DOPC) solid-supported lipid bilayers. In lipid bilayers, Cry1Aa formed structures 30-60 nm wide and 3-7 nm high, mostly at the interface of domains formed by the two different lipids or at the edge of DOPC-enriched domains. BBM vesicles, in the absence of toxin, formed flat membrane fragments of up to 25 microm(2) and 4.2 nm high, with irregular embedded structures. After incubation with Cry1Aa, Cry1Ac and Cry1C, which are active against M. sexta, new structures, 35 nm wide and 5.1-6.7 nm high, were observed in some membrane fragments, sometimes only in particular regions. Their density, which reached a plateau within 4 h, was toxin- and concentration-dependent. The structures formed by Cry1Ac were often grouped into dense, two-dimensional arrangements. No such specific interactions were observed with Cry1Ba, which is inactive against M. sexta. This study provides the first visual demonstration of specific interactions of Bt toxins with insect midgut BBMs at the nanometric scale. The observed structures likely represent the protein complexes forming functional Bt pores in target membranes.