Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity

Hayakawa, Tohru; Miyazaki, Midoka; Harada, Syoya; Asakura, Mami; Ide, Toru

doi:10.1007/s00253-020-10893-5

Cited by 3 publications

(10 citation statements)

References 27 publications

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“…Considered together with this observation, our result suggested that the channel pore cation selectivity was another factor that affect mosquito-larvicidal activity in addition to conductance. Similar observations have been reported in mutational analyses targeting the transmembrane region of the mosquito-larvicidal Mpp46Ab toxin [ 27 , 28 ]. Mpp46Ab (formerly designated Cry46Ab), a mosquito-larvicidal toxin with an aerolysin-type architecture, is structurally quite different from Cry4Aa, but like Cry4Aa, it has been shown to act as a pore-forming toxin [ 29 ].…”

Section: Discussionsupporting

confidence: 86%

Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

Takahashi,

Asakura,

Ide

et al. 2024

Curr Microbiol

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Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae.

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Section: Discussionsupporting

confidence: 86%

Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

Takahashi,

Asakura,

Ide

et al. 2024

Curr Microbiol

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“…We previously demonstrated that Cry46Ab, an aerolysin-like mosquito-larvicidal toxin from B. thuringiensis TK-E6, forms cation-selective channel-pores in artificial lipid bilayers (Sakakibara et al 2019;Hayakawa et al 2020). Like Cry4Aa channel-pores, those formed by Cry46Ab exhibit cation preference as well, but the order differs from that of Cry4Aa channel-pores: K + > Na + , Ca 2+ > Na + , and K + > Ca 2+ (Sakakibara et al 2019;Hayakawa et al 2020). The cation preference of channel-pores may thus vary with the type of Cry toxin.…”

Section: Discussionmentioning

confidence: 99%

“…The cation preference of channel-pores may thus vary with the type of Cry toxin. Mutational analysis targeting the putative transmembrane domain of Cry46Ab has demonstrated apparent correlation between the degree of cation-selectivity of the channel-pores and the insecticidal activities (Hayakawa et al 2020). It is therefore reasonable to speculate the order of cation preference also affects insecticidal activity of Cry toxin.…”

Section: Discussionmentioning

confidence: 99%

“…The mosquito-larvicidal activity of recombinant Cry4Aa was analyzed using bioassays with third-instar mosquito larvae as described previously (Hayakawa et al 2020). Briefly, 40 μg of purified toxin was adsorbed onto 2 mg of latex beads (0.8-mm diameter, Sigma-Aldrich Corp., St. Louis, MO) for 1 h at room temperature and then administered to the mosquito larvae as a diet.…”

Section: Measurement Of Mosquito-larvicidal Activitymentioning

confidence: 99%

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Characteristics of channel pores formed by Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

et al. 2021

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Cry4Aa toxin produced by Bacillus thuringiensis subsp. israelensis exhibits specific toxicity to larvae of medically-important mosquito genera. In the present study, we analyzed the characteristics of channel-pores formed by recombinant Cry4Aa in a solvent-free planar lipid bilayer. Stable channel-currents were observed in electrophysiologic measurements, and the single-channel conductance was 187 ± 10 pS in symmetrical buffer containing 150 mM KCl. The channel-pores formed by Cry4Aa were cation selective, with an estimated PK/PCl permeability ratio of 4.9. In addition, Cry4Aa channel-pores exhibited apparent cation preference in the order Na + > K + , Na + > Ca 2+ , and K + > Ca 2+ . Although the effect was limited, the cation preference of Cry4Aa channel-pores seemed to be correlated with toxicity. Culex pipiens mosquito larvae reared in NaCl solution exhibited greater sensitivity to Cry4Aa, particularly early period 2 after exposure. The presence of cations that preferentially translocate through Cry4Aa channel-pores may facilitate excessive influx of water into the midgut cells, leading to colloid-osmotic lysis. Whereas CaCl2 had some effect on the mosquito-larvicidal activity of Cry4Aa, KCl had no effect. The effect of some cations may be mitigated by the variety of ion channels present on the midgut cell membrane.

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“…We previously speculated that the charged amino acid residues within the β-hairpin line the lumen of Mpp46Ab channel pores and that the electrical environment generated by the charged residues affects the ion permeability of the channel pores. We constructed a series of Mpp46Ab mutants in which one of the charged residues in the βhairpin was replaced with an oppositely charged residue, and the effect of the replacement on both ion permeability of the channel pores and resulting mosquito-larvicidal activity was then investigated [17]. A significant increase in cation-selectivity was observed on the channel pores formed by mutant K155E, along with increased toxicity.…”

Section: Introductionmentioning

confidence: 99%

Random Mutational Analysis Targeting Residue K155 within the Transmembrane β-Hairpin of the Mosquitocidal Mpp46Ab Toxin

Miyazaki,

Asakura,

Ide

et al. 2023

Biology

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Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from Bacillus thuringiensis TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K155 within the transmembrane β-hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using Culex pipiens mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K155 with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane β-hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab.

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Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity

Abstract: Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity.

Cited by 3 publications

References 27 publications

Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

Characteristics of channel pores formed by Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

Random Mutational Analysis Targeting Residue K155 within the Transmembrane β-Hairpin of the Mosquitocidal Mpp46Ab Toxin

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