Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, 3-1-1 Tsushima-Naka, Kita-Ku, Okayama, 700-8530, Japan.
Curr Microbiol. 2024 Jan 28;81(3):80. doi: 10.1007/s00284-023-03602-8.
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.
Cry4Aa 由苏云金芽孢杆菌亚种 israelensis 产生，对医学上重要的蚊子属幼虫具有特异性毒性。Cry4Aa 作为一种孔形成毒素起作用，结构域 I 中的螺旋发夹（α4-环-α5）被认为是形成毒素孔的跨膜结构域。孔形成被认为是 Cry4Aa 作用的一种中心模式，但孔形成与毒性之间的关系尚不清楚。在本研究中，我们构建了 Cry4Aa 突变体，其中 I 型跨膜α4 螺旋中的每个极性氨基酸残基均被谷氨酸取代。使用库蚊幼虫进行生物测定，以及随后使用对称 KCl 溶液进行离子通透性测量，结果显示大多数 Cry4Aa 突变体的毒性与毒素孔导性之间存在明显的相关性。相比之下，突变体 H178E 是一个明显的例外，其几乎失去了毒性，但仍表现出约 60%野生型的中等电导。此外，突变体 N190E 形成的孔的电导（约为野生型的 50%）接近 H178E，但毒性明显高于 H178E。使用不对称 KCl 溶液进行的离子选择性测量显示，与 N190E 相比，H178E 形成的毒素孔的阳离子选择性显著降低。我们的数据表明，Cry4Aa 的毒性主要与孔相关。形成高离子渗透性和高阳离子选择性的毒素孔可能会促进阳离子和水流入靶细胞，从而促进蚊子幼虫的最终死亡。
