Abstract
Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane β-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane β-hairpin region.
Key points
• Cry46Ab mutants were constructed by targeting the putative transmembrane β-hairpin region.
• Charged residues within the β-hairpin control the flux of ions through channel pores.
• Channel-pore cation selectivity is correlated with insecticidal activity.
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Acknowledgments
Eggs of C. pipiens were kindly supplied by the Research and Development Laboratory at Dainihon Jochugiku Co., Ltd., Osaka, Japan.
Funding
The present work was supported in part by a research grant from the OSHIMO foundation (2019) and a JSPS KAKENHI grant (number JP18K05675).
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TH and TI conceived and designed research. TH and SH constructed mutants and analyzed their biological activity. TH, MM, MA, and TI contributed electrophysiologic experiments. TH, MM, and TI analyzed data. TH wrote the manuscript. All authors read and approved the manuscript.
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Hayakawa, T., Miyazaki, M., Harada, S. et al. Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity. Appl Microbiol Biotechnol 104, 8789–8799 (2020). https://doi.org/10.1007/s00253-020-10893-5
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DOI: https://doi.org/10.1007/s00253-020-10893-5