In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

Abstract

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered “net like” structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

Bacterial Toxins Active against Mosquitoes: Mode of Action and Resistance

“…This interaction depends on Cry11Aa toxin oligomerization since the non-toxic mutant Cry11Aa-E97A, affected in its oligomerization, showed an inverted array when tested with Cyt1Aa. This dynamic organization pattern in the cell microvilli is consistent with the model of Cyt1Aa acting as a receptor of Cry11Aa [ 35 ]. It was also observed that Cyt1Aa can interact with other Cry toxins such as Cry2Aa, which is naturally active against lepidoptera, resulting in a slightly higher toxicity against Cx.…”

“…The Cyt1Aa toxin has a single α-β domain that contains two α-helix surround β-sheet [75]. This toxin interacts directly with phospholipids from the midgut cells; t fore, its action is independent of the presence of specific protein receptors [36,76,77] localization pattern of the Cyt1Aa toxin on the cell microvilli along the whole larvae gut shown by recent studies corroborates its unspecific binding to the cell memb [35,78]. Two models of action were proposed for Cyt1Aa.…”

“…The first major steps described in the Bti’s crystal mode of action are their ingestion by mosquito larvae, the crystals’ solubilization in the alkaline pH of the midgut lumen, and the protoxin activation by midgut proteases ending in pore formation into the midgut cells [ 32 , 33 , 34 ]. The ingestion of the crystals is important for the mode of action since it was observed that larvae treated with soluble toxins did not display mortality [ 35 ]. Both Cry and Cyt are pore-forming toxins that destroy the epithelium midgut cells causing larval death.…”

“…Some important regions involved on the binding between Cry toxins and their midgut receptors were identified [ 68 , 69 , 70 , 71 ]. It was previously reported that the APN, ALP, and CAD receptors are located on the epithelial cells from the caeca and posterior midgut, but a recent work showed that the Cry11Aa toxin also associates with the epithelium from anterior and medium midgut regions, indicating that other molecules could be involved in this interaction [ 35 ]. After intoxication with Bti toxins, some histopathological effects such as severe vacuolization of the cytoplasm, microvilli damage, columnar cell fragmentation, massive degradation of the caeca gut structure, and cell lysis were observed [ 35 , 72 , 73 , 74 ].…”

“…aegypti larvae since Cyt1Aa mutants affected in oligomerization were still able to synergize with Cry11Aa [ 86 ]. The in vivo localization of the Cry11Aa and Cyt1Aa toxins during their synergistic interaction was analyzed at a nanoscale resolution [ 35 ]. These proteins showed an ordered array in the microvilli, where Cry11Aa was found below Cyt1Aa, facing the cell cytoplasm.…”