Oligomerization is a key step in Cyt1Aa membrane insertion and toxicity but not necessary to synergize Cry11Aa toxicity in Aedes aegypti larvae

Abstract

Summary Bacillus thuringiensis produces insecticidal Cry and Cyt proteins that are toxic to different insect orders. In addition, Cyt toxins also display haemolytic activity. Both toxins are pore-forming proteins that form oligomeric structures that insert into the target membrane to lyse cells. Cyt toxins play an important role in mosquitocidal activity since they synergize Cry toxins and are able to overcome resistance to Cry toxins. Cry and Cyt toxins interact by specific epitopes, and this interaction is important to induce the synergistic activity observed. It was proposed that Cyt toxins do not interact with protein receptors but directly interacting with the specific midgut cell lipids. Here, we analysed if oligomerization and membrane insertion of Cyt1Aa are necessary steps to synergize Cry11Aa toxicity. We characterized Cyt1Aa helix α-C mutants that were affected in oligomerization, in membrane insertion and also in haemolytic and insecticidal activities. However, these mutants were still able to synergize Cry11Aa toxicity indicating these steps are independent events of Cyt1Aa synergistic activity. Furthermore, the data indicate that formation of stable Cyt1Aa-oligomeric structure is a key step for membrane insertion, haemolysis and insecticidal activity.

Alanine-162 ofBacillus thuringiensisCyt2Aa2 toxin is essential for membrane binding and oligomerisation

“…Additionally, electrostatic surface analysis of Cyt was carried out, and the residues V150, N159, L160 and D209 were suggested to contribute to the lipid-binding pocket (Rigden, 2009). The residues V122 and V126, which are located on helix αC of Cyt1Aa, were important for toxin oligomerisation, membrane insertion and toxicity (López-Diaz, Cantón, Gill, Soberón, & Bravo, 2013). This study aimed to investigate the putative Figure 3.…”

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

“…Recently, it was shown that the nontoxic mutant of Cyt1Aa affected in oligomerization was still able to synergize the insecticidal activity of Cry11Aa toxin against A. aegypti [28]. However, the synergism of these mutants was reduced compared to the native Cyt1Aa toxin [28]. These data indicate that the most efficient condition to synergize Cry activity by Cyt1Aa is in the membrane-bound state of the Cyt toxin.…”

“…The region of the toxin that is involved oligomerization is the amino terminal domain, which is composed mainly of α-helices. Helix α-C is directly involved in oligomerization since point mutations in this region affected oligomer formation and membrane insertion [28]. These mutants lost hemolytic and insecticidal activities, indicating that oligomerization is a crucial step in the mechanism of action of Cyt toxins [26][27][28].…”

“…Helix α-C is directly involved in oligomerization since point mutations in this region affected oligomer formation and membrane insertion [28]. These mutants lost hemolytic and insecticidal activities, indicating that oligomerization is a crucial step in the mechanism of action of Cyt toxins [26][27][28]. The C-terminal domain, which is mainly composed by long β-sheets, is involved in membrane insertion [29].…”

“…It was then proposed that Cyt1A could function as a Cry toxin receptor [35] since Cyt1Aa binds Cry11A and Cry4Ba and induces their oligomerization [37]. Recently, it was shown that the nontoxic mutant of Cyt1Aa affected in oligomerization was still able to synergize the insecticidal activity of Cry11Aa toxin against A. aegypti [28]. However, the synergism of these mutants was reduced compared to the native Cyt1Aa toxin [28].…”