Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin‐like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins

Abstract

Directed evolution of a Cry1Aa toxin using phage display and biopanning was performed to generate an increased binding affinity to the Bombyx mori cadherin-like receptor (BtR175). Three mutant toxins (371WGLA374, 371WPHH374, 371WRPQ37425) with 16-, 16-, and 50-fold higher binding affinities, respectively, for BtR175 were selected from a phage library containing toxins with mutations in domain II loop 2. However, the observed toxicities of the three mutants against B. mori larvae and cultured cells expressing the BtR175 toxin-binding region did not increase, suggesting that increased binding affinity to cadherins does not contribute to the insecticidal activity. Affinity maturation of a Cry toxin to a receptor via directed evolution was relatively simple to achieve, and seems to have potential for generating a toxin with increased insecticidal activity.

PC, a Novel Oral Insecticidal Toxin from Bacillus bombysepticus Involved in Host Lethality via APN and BtR-175

“…PC has an LC 50 value of 77 μg/per larvae. In contrast, Cry1A toxin has an LC 50 value of 1 ng per square cm or a few hundred nanograms for B. mori instar larvae (less than 200 ng/cm 2 ) indicating that PC is at least 400-fold less toxic 8 46 . The shortcoming of low toxicity of PC may be a limitation for its utility in pest control.…”

“…Toxins are important virulence factors for bacterial pathogenicity. Interactions with single receptors are a general strategy, although several toxins such as diphtheria, Cry, or aerolysin toxins target more than one surface molecule in their binding activities and action modes 8 . Here, we demonstrated that binding to BmAPN4 provokes the pathogenicity of PC ( Figs 2 and 3 ), although the kinetic parameters of the interaction between PC with APN have not been fully analysed.…”

“…Bacterial toxins secreted by pathogens diffuse towards the target cell by binding to specific receptors that are ultimately inserted in cell membranes and that form a pore 8 . Aerolysin, a pore-forming toxin, initially interacts with a high-abundance and low-affinity molecule and subsequently interacts with a low-abundance and high-affinity binding site 41 .…”

“…Clostridium perfringens enterotoxin 6 and Vcc from Vibrio cholerae 7 are members of the β-PFT family. Additionally, many bacterial toxins that do not fully qualify as PFT are known as AB toxins; in these toxins, the B subunit is responsible for binding to the target cell and for translocating the A subunit into the cytoplasm 8 .…”

“…The modes of action of bacterial toxins include damaging cell membranes 2 , inhibiting protein synthesis 9 , activating second messenger pathways 10 , and activating the host immune response 11 . Generally, PFTs bind to a specific receptor, diffuse towards target cell membranes, form multimers, and undergo a conformational change leading to the formation of a pore in the target cell membrane that damages the extracellular matrix 8 . This damage not only results in the loss of membrane function and direct cell lysis but also facilitates bacterial dissemination throughout tissues.…”