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.

中文翻译：

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通道孔阳离子选择性是苏云金芽孢杆菌Cry46Ab灭蚊活性的主要决定因素。

苏云金芽孢杆菌TK-E6的Cry46Ab是一种新的具有气溶素型结构的灭蚊毒素，有望用作新型生物杀虫剂。Cry46Ab充当功能性的成孔毒素，并且已对所得通道孔的特性（包括离子选择性）进行了分析。然而，通道-孔离子选择性与杀虫活性之间的关系仍有待阐明。为了阐明带电荷的氨基酸残基对通道孔离子渗透性和所产生的杀虫活性的影响，在本研究中，我们构建了Cry46Ab突变体，其中假定的跨膜β-发夹区内的带电荷的氨基酸残基被替换为带相反电荷的残基。使用淡色库蚊蚊幼虫的生物测定表明，该突变改变了灭蚊活性。K155E Cry46Ab突变体的毒性明显高于野生型Cry46Ab，但E159K和E163K突变体的毒性降低。离子选择性测量表明，由野生型和突变型Cry46Ab形成的通道孔均具有阳离子选择性，并且它们的阳离子偏好性也相似。但是，在由K155E突变体形成的通道孔中，阳离子选择性的程度显然更高，并且在E159K和E163K突变体中观察到选择性降低。我们的数据表明，通道孔阳离子选择性是Cry46Ab灭蚊活性的主要决定因素，并且可以通过针对跨膜β-发夹区的诱变来控制阳离子选择性。要点：•Cry46Ab突变体是通过靶向假定的跨膜β-发夹结构构建的。•β-发夹内的带电残基控制离子通过通道孔的流量。•通道孔阳离子选择性与杀虫活性相关。