Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae
Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae
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DOI：
10.1074/jbc.m701314200
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发表时间：
2007-07-20
影响因子：
4.8
通讯作者：
Bravo, Alejandra
中科院分区：
文献类型：
--
作者：
Jimenez-Juarez, Nuria;Munoz-Garay, Carlos;Bravo, Alejandra
孔形成毒素是由各种生物体产生的生物武器，特别是细菌，但也由昆虫，爬行动物和无脊椎动物产生。这些蛋白质影响其靶细胞的细胞膜，破坏渗透性并最终导致细胞死亡。成孔毒素通常从可溶性单体蛋白转化为形成跨膜通道的寡聚体。由苏云金芽孢杆菌产生的Cry毒素被广泛用作杀虫剂。这些蛋白质被认为是成孔毒素，它们的主要作用是裂解目标昆虫的中肠上皮细胞。为了发挥其毒性作用，形成前孔低聚中间体，最终导致膜插入的低聚孔。为了理解Cry寡聚前孔形成在杀虫活性中的作用，我们分离了影响毒素寡聚化但不影响其与钙粘蛋白样Bt-R-1受体结合的点突变。我们发现，螺旋α-3域I包含的序列，可以形成卷曲螺旋结构的寡聚化的重要。一些单点突变体在这个螺旋结合Bt-R-1受体与野生型毒素的亲和力相似，但在寡聚化的影响，并在孔的形成和毒性对天蛾幼虫分别受损。这些数据表明，前孔寡聚体和毒素孔的形成在昆虫幼虫的Cry 1Ab毒素中毒过程中起主要作用。
Pore-forming toxins are biological weapons produced by a variety of living organisms, particularly bacteria but also by insects, reptiles, and invertebrates. These proteins affect the cell membrane of their target, disrupting permeability and leading eventually to cell death. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. The Cry toxins produced by Bacillus thuringiensis are widely used as insecticides. These proteins have been recognized as pore-forming toxins, and their primary action is to lyse midgut epithelial cells in their target insect. To exert their toxic effect, a pre-pore oligomeric intermediate is formed leading finally to membrane-inserted oligomeric pores. To understand the role of Cry oligomeric pre-pore formation in the insecticidal activity we isolated point mutations that affected toxin oligomerization but not their binding with the cadherin-like, Bt-R-1 receptor. We show the helix alpha-3 in domain I contains sequences that could form coiled-coil structures important for oligomerization. Some single point mutants in this helix bound Bt-R-1 receptors with similar affinity as the wild-type toxin, but were affected in oligomerization and were severally impaired in pore formation and toxicity against Manduca sexta larvae. These data indicate the pre-pore oligomer and the toxin pore formation play a major role in the intoxication process of Cry1Ab toxin in insect larvae.