Membrane binding and oligomer membrane insertion are necessary but insufficient for Bacillus thuringiensis Cyt1Aa toxicity

Abstract

Bacillus thuringiensis Cyt proteins are pore-forming toxins that have insecticidal activity mainly against dipteran insects. However, certain Cyt proteins have toxicity to some insect orders, but not toxicity of Cyt1Aa against lepidopteran larvae has been found. Insect specificity has been proposed to rely in specific binding to certain lipids on the brush border membrane of midgut cells since no protein receptors have been described so far. To determine the molecular basis of Cyt1Aa insect specificity we compared different steps of Cyt1Aa mode of action in a susceptible insect as the dipteran Aedes aegypti and also in the non-susceptible lepidopteran Manduca sexta. Our data shows that the lack toxicity of Cyt1Aa to M. sexta larvae does not rely on protoxin processing, membrane binding interaction, and oligomerization of Cyt1Aa since these steps were similar in the two insect species analyzed.

Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade

“…preventing their fall-off into arciform oligomers. At present, we can nonetheless propose a model that fits all the information developed by us and others on Cyt1Aa pore formation [2][3][4][5][6]21,27,31,36 (Supplementary Fig. 16).…”

“…It remains uncertain whether or not the mosquito gut is a reducing environment 19 and thus under what form the protoxin is mainly released upon crystal dissolution in the natural context. We found that regardless of whether the protoxin is present in the form of a disulfide-bridged dimer or a protoxin monomer, addition of proteinase K, shown to faithfully mimic mosquito gut proteases 20 , allows activation into a 23 kDa toxin monomer, consistent with the cleavage of its first 30 and last 5 amino acids 21 ( Fig. 4b and Supplementary Fig.…”

“…Soluble protoxins were activated into toxins by the addition of proteinase K or trypsin (condition 3). For further experiments, toxin was activated by proteinase K rather than other enzymes, such as trypsin, as it was shown to better mimics the activation performed by gut enzymes in insects 20,21 . The different forms of Cyt1Aa were analyzed by two methods: SDS-PAGE and MALDI-ToF.…”

“…We therefore investigated the propensity of these three Cyt1Aa species-the disulfide-bridged dimer, the protoxin monomer and the activated toxin monomer-and of their mutated variants to assemble into membrane-bound oligomers (MBO). When monomers of either the 27 kDa protoxin or the 23 kDa proteolytically activated toxin 7,21,22 are mixed with POPC liposomes (~100 nm radius) and electrophoresed on 6% SDS-PAGE gels, a ladder pattern characteristic of Cyt1Aa MBO is observed (Fig. 4c).…”

Evaluation of Cytotoxicity, Genotoxicity and Hematotoxicity of the Recombinant Spore-Crystal Complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss Mice

“…These strains of recombinant spore-crystals were grown in Embrapa medium [ 35 ], supplemented with 6 μg mL −1 of chloramphenicol [ 36 ] for BtCry1Ba6 and BtCry10Aa, with 10 μg mL −1 erythromycin [ 37 , 38 ] for BtCry1Ia, incubated for 72 h at 28 °C, and maintained in a shaker. After growing, these were centrifuged at 12,800 × g for 30 min at 4 °C, frozen for 16 h and then lyophilized for 18 h. The colony forming units test (CFU) was carried out to quantify the viable Bt spore-crystals and followed the protocol proposed by Alves and Moraes [ 39 ].…”