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.
Highlights
Bacillus thuringiensis (Bt), a Gram-positive bacterium characterized by the production of Cry δ-endotoxins capable of killing insects, has been used since the late 1930s as a biological control agent (Melo et al, 2016)
In variant 8, two hydrogen bonds between R491 of the variant protein and N259 of ALP1 as well as three hydrophobic interactions of R491, V492, and Q493 of the variant protein with V258, G257, and G 261 of ALP1 were formed (Figure 5C). Directed evolution approaches such as phage display, DNA shuffling and staggered extension process shuffling combined with Red/Et homologous recombination have been proposed to increase the activity of Bt Cry toxins (Lucena et al, 2014)
Other approaches based on phage-assisted continuous evolution (PACE) (Badran et al, 2016), in vitro template-change PCR (Shu et al, 2016), site-directed mutagenesis, and errorprone PCR have been used successfully to identify novel receptors expressed on the surface of insect midgut cells and to understand the effects of different cry gene mutations on the mechanism of action of Cry toxins (Lucena et al, 2014)
Summary
Bacillus thuringiensis (Bt), a Gram-positive bacterium characterized by the production of Cry δ-endotoxins capable of killing insects, has been used since the late 1930s as a biological control agent (Melo et al, 2016). The signaling pathways is a recent proposed mechanism in which the activation of signaling cascades, leads to increased cyclic AMP and protein kinase activities, resulting in cell death (Zhang et al, 2006). In the signaling pathway mechanism, how Cry toxin-receptor interactions mediate toxic activity is unclear (Melo et al, 2016) In this context, it has been suggested that Cry toxins can cause death based on their ability to induce both pore formation and ion channel activation (Zhang Q. et al, 2017)
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