Pérez Claudia, Fernandez Luisa E, Sun Jianguang, Folch Jorge Luis, Gill Sarjeet S, Soberón Mario, Bravo Alejandra
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. postal 510-3, Cuernavaca 62250, Morelos, Mexico.
Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18303-8. doi: 10.1073/pnas.0505494102. Epub 2005 Dec 9.
Bacillus thuringiensis subsp. israelensis produces crystal proteins, Cry (4Aa, 4Ba, 10Aa, and 11Aa) and Cyt (1Aa and 2Ba) proteins, toxic to mosquito vectors of human diseases. Cyt1Aa overcomes insect resistance to Cry11Aa and Cry4 toxins and synergizes the toxicity of these toxins. However, the molecular mechanism of synergism remains unsolved. Here, we provide evidence that Cyt1Aa functions as a receptor of Cry11Aa. Sequential-binding analysis of Cyt1Aa and Cry11Aa revealed that Cyt1Aa binding to Aedes aegypti brush border membrane vesicles enhanced the binding of biotinylated-Cry11Aa. The Cyt1Aa- and Cry11Aa-binding epitopes were mapped by means of the yeast two-hybrid system, peptide arrays, and heterologous competition assays with synthetic peptides. Two exposed regions in Cyt1Aa, loop beta6-alphaE and part of beta7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop alpha8 and beta-4, which are also involved in midgut receptor interaction. Characterization of single-point mutations in Cry11Aa and Cyt1Aa revealed key Cry11Aa (S259 and E266) and Cyt1Aa (K198, E204 and K225) residues involved in the interaction of both proteins and in synergism. Additionally, a Cyt1Aa loop beta6-alphaE mutant (K198A) with enhanced synergism to Cry11Aa was isolated. Data provided here strongly indicates that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium because it produces a toxin and also its functional receptor, promoting toxin binding to the target membrane and causing toxicity.
苏云金芽孢杆菌以色列亚种产生晶体蛋白，即Cry（4Aa、4Ba、10Aa和11Aa）蛋白以及Cyt（1Aa和2Ba）蛋白，这些蛋白对人类疾病的蚊媒具有毒性。Cyt1Aa可克服昆虫对Cry11Aa和Cry4毒素的抗性，并增强这些毒素的毒性。然而，协同作用的分子机制仍未解决。在此，我们提供证据表明Cyt1Aa作为Cry11Aa的受体发挥作用。对Cyt1Aa和Cry11Aa进行的顺序结合分析表明，Cyt1Aa与埃及伊蚊刷状缘膜囊泡的结合增强了生物素化Cry11Aa的结合。通过酵母双杂交系统、肽阵列以及与合成肽的异源竞争试验对Cyt1Aa和Cry11Aa的结合表位进行了定位。Cyt1Aa中的两个暴露区域，β6-αE环和β7的一部分，可结合Cry11Aa。另一方面，Cry11Aa通过结构域II-α8环和β-4与Cyt1Aa蛋白结合，这两个结构域也参与中肠受体相互作用。对Cry11Aa和Cyt1Aa中的单点突变进行表征，揭示了参与两种蛋白相互作用和协同作用的关键Cry11Aa（S259和E266）以及Cyt1Aa（K198、E204和K225）残基。此外，还分离出了对Cry11Aa具有增强协同作用的Cyt1Aaβ6-αE环突变体（K198A）。此处提供的数据有力地表明，Cyt1Aa通过作为膜结合受体发挥作用来协同或抑制对Cry11Aa毒素的抗性。苏云金芽孢杆菌以色列亚种是一种高效的病原菌，因为它既能产生毒素，又能产生其功能性受体，促进毒素与靶膜结合并导致毒性。
