Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling

Abstract

The Cry11 family belongs to a large group of δ-endotoxins that share three distinct structural domains. Among the dipteran-active toxins referred to as three-domain Cry11 toxins, the Cry11Aa protein from Bacillus thuringiensis subsp. israelensis (Bti) has been the most extensively studied. Despite the potential of Bti as an effective biological control agent, the understanding of Cry11 toxins remains incomplete. In this study, five Cry11 variants obtained via DNA shuffling displayed toxic activity against Aedes aegypti and Culex quinquefasciatus. Three of these Cry11 variants (8, 23, and 79) were characterized via 3D modeling and analysis of docking with ALP1. The relevant mutations in these variants, such as deletions, insertions and point mutations, are discussed in relation to their structural domains, toxic activities and toxin-receptor interactions. Importantly, deletion of the N-terminal segment in domain I was not associated with any change in toxic activity, and domain III exhibited higher sequence variability than domains I and II. Variant 8 exhibited up to 3.78- and 6.09-fold higher toxicity to A. aegypti than Cry11Bb and Cry11Aa, respectively. Importantly, variant 79 showed an α-helix conformation at the C-terminus and formed crystals retaining toxic activity. These findings indicate that five Cry11 variants were preferentially reassembled from the cry11Aa gene during DNA shuffling. The mutations described in loop 2 and loop 3 of domain II provide valuable information regarding the activity of Cry11 toxins against A. aegypti and C. quinquefasciatus larvae and reveal new insights into the application of directed evolution strategies to study the genetic variability of specific domains in cry11 family genes.

Making 3D-Cry Toxin Mutants: Much More Than a Tool of Understanding Toxins Mechanism of Action

“…aegypti and C. quinquefasciatus, rendered a mutant (Variant 8) 3.8 times more toxic toward Ae. aegypti than the parental toxin Cry11Bb (LC 50 22.9 ng/mL), and 6.09 times more toxic than the Cry11Aa toxin (LC 50 36.9 ng/mL) [118]. DNA sequence analysis of Variant 8 showed that the mutant contained a deletion of 219 nucleotides (73 aa) at the N-terminal end of the molecule (Domain I), and 6 and 13 nucleotide substitutions in Domain II and III, respectively.…”

Generation of Cry11 Variants of Bacillus thuringiensis by Heuristic Computational Modeling

“…Values established for the Fragmentation Length were 75bp and 150bp, which correspond to experimental length values of fragments obtained through the action of DNAsaI in in vitro DNA shuffling experiments with cry11 genes. 23 …”

“…On the contrary, the values of 15 generations and 100 generations established for the parameter of number of generations correspond to values close to the range reported as favorable for the obtention of chimeric libraries, in in vitro 10 and in silico directed evolution studies. 23 This parameter allows replicating the number of cycles of directed evolution that is used as stop criteria for the execution of the HIDDEN genetic algorithm.…”

Can (We Make) Bacillus thuringiensis Crystallize More Than Its Toxins?

“…Cyt1Aa toxin from Bti has also been used to generate chimeras, expanding its host spectrum to other mosquito species when fused to the binary Tpp1Aa/2Aa (formerly BinAB) from Ls [ 74 ] and to lepidopteran when the recognition loop 3 of domain II from Cry1Ab was inserted between its loops 1 and 2 [ 75 ]. Interestingly, interdomains exchanges conducted between Cry11Aa and Cry11Ba from Bti and Btj , respectively, highlighted that while some domain combinations improved the mosquitocidal activity, others completely abolished the ability of the chimeras to form inclusions [ 76 , 77 ]. This exemplifies how different domain shuffling using two “sister” toxins, which should crystallize through a similar, albeit thus far uncharacterized, pathway and exhibit alike mode of actions, can alter the formation and stability of their crystal, along with their specificity and toxicity.…”