Single Molecule Fluorescence Study of the B. Thuringiensis Toxin Cry1Aa Reveals Tetramerization

Abstract

Pore forming toxins compose a class of potent virulence factors that attack their host membrane in a 2- or 3-step mechanism. After binding to the membrane often aided by specific receptors, they form pores in the membrane either leading to cytolysis or providing a pathway to introduce enzymes effecting intracellular proteins. The major rearrangements of the toxins upon binding to the membrane are accompanied by oligomerization to the final pore forming construct. However, it remains often unknown how many monomers constitute the pore, if that number is fixed, and whether the toxins enter the membrane in monomeric or oligomeric form. Here, we used single subunit counting, to determine the number of oligomers contained in a Cry1Aa toxin as a function of the concentration. Cry1Aa is one of the δ-endotoxins of B. thuringiensis, a soil bacteria that is, because of its specificity for certain insect larvae, used as a biological alternative to chemical pesticides. Cry toxins were purified and fluorescently labeled in their monomeric form. Lipid vesicles of various compositions were incubated with labeled toxin monomers, and supported bilayers formed from the mixture. We recorded the fluorescence intensity over time of distinct fluorescence intensities in the supported bilayer and determined their photobleaching behaviour. As single fluorophores bleach in a single step, the number of steps corresponded to the minimal number of monomers in each oligomer. The oligomerization state showed a concentration dependence which was consistent with both a concentration dependent association rate between monomers and a concentration dependent distribution on the lipid vesicles during incubation. Statistical analysis of the results showed that the toxins enter the membrane in their monomeric form and diffuse laterally to form tetramers, which appears to be the pore forming unit.