Bacillus thuringiensis Cry1A toxins are versatile proteins with multiple modes of action: two distinct pre-pores are involved in toxicity

Abstract

Cry proteins from Bacillus thuringiensis are insecticidal PFTs (pore-forming toxins). In the present study, we show that two distinct functional pre-pores of Cry1Ab are formed after binding of the protoxin or the protease-activated toxin to the cadherin receptor, but before membrane insertion. Both pre-pores actively induce pore formation, although with different characteristics, and contribute to the insecticidal activity. We also analysed the oligomerization of the mutant Cry1AbMod protein. This mutant kills different insect populations that are resistant to Cry toxins, but lost potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, but not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. These data are relevant for the future control of insects resistant to Cry proteins. Our data support the pore-formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target membrane. We propose that not only different insect targets could have different receptors, but also different midgut proteases that would influence the rate of protoxin/toxin activation. It is possible that the two pre-pore structures could have been selected for in evolution, since they have differential roles in toxicity against selected targets, increasing their range of action. These data assign a functional role for the protoxin fragment of Cry PFTs that was not understood previously. Most PFTs produced by other bacteria are secreted as protoxins that require activation before oligomerization, to finally form a pore. Thus different pre-pores could be also part of the general mechanism of action of other PFTs.

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin

“…So far, the step at which the Bt-Cry oligomerization pathway might occur, either in solution (18,(23)(24)42) or in a membrane-bound state (13,20), remains to be clearly elucidated. In this study, we demonstrated that no defined oligomeric complex was observed for the purified trypsin-activated Cry4Ba toxin in carbonate-based solution (toxin solubilization buffer (pH 9.0)), as analyzed via a seminative PAGE system and size exclusion chromatography.…”

“…We have previously employed detergent dialysis-driven two-dimensional crystallization to directly demonstrate, for the first time, a symmetrical trimeric structure of the L-␣ -dimyristoylphosphatidylcholine (DMPC) lipid-associated Cry4Ba toxin complex (21). Although the trimeric form has been supported by other studies (22,23), the tetrameric complex of Cry toxin-induced pores has also been reported recently through other approaches (20,24). In this study, further attempts were made to provide more critical insights into our trimeric pore model by employing two direct rendering techniques, i.e.…”

Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA

“…It should be noted that our bioassays were conducted on a hybrid toxin in which the C-terminal region was provided by another toxin. While the C-terminal regions of the larger Cry toxins are known to be important for toxin expression/packaging (26) and may influence toxicity (27), there is no convincing evidence that they influence specificity, and thus we believe that it is reasonable to attribute the observed activity to the newly cloned portion of the toxin.…”

Mechanism of action of Bacillus thuringiensis insecticidal toxins and their use in the control of insect pests

“…Different PFTs produced by bacteria are able to bind to their specific receptors as protoxins, and this binding interaction triggers a conformational change that induced a cleavage of small fragments and leads to oligomerization [56]. Cry1A protoxin is able to bind to cadherin receptors with similar affinity as the monomeric activated toxin [57,58]. It was shown that binding of protoxin to cadherin in the presence of midgut proteases induces a conformational change in the toxin, facilitating an extra cleavage where helix α-1 is eliminated [59].…”