Oestergaard Jesko, Ehlers Ralf-Udo, Martínez-Ramírez Amparo C, Real Maria Dolores
Institute for Phytopathology, Department of Biotechnology and Biological Control, Christian Albrechts University, Hermann-Rodewald Str. 9, 24118 Kiel, Germany.
Appl Environ Microbiol. 2007 Jun;73(11):3623-9. doi: 10.1128/AEM.01056-06. Epub 2007 Apr 6.
Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.
苏云金芽孢杆菌以色列亚种（B. thuringiensis subsp. israelensis）产生四种杀虫晶体蛋白（ICPs）（Cry4A、Cry4B、Cry11A和Cyt1A）。利用沼泽大蚊（双翅目：长角亚目）的一龄幼虫测定了仅表达其中一种毒素的重组苏云金芽孢杆菌以色列亚种菌株的毒性。Cyt1A是毒性最强的蛋白，而Cry4A、Cry4B和Cry11A实际上无毒。当Cry4A和/或Cry4B与Cyt1A组合时记录到协同效应，但与Cry11A组合时未记录到协同效应。结合和孔形成是苏云金芽孢杆菌以色列亚种ICPs作用模式中的关键步骤。分析了对蚊子毒性最强的蛋白Cry11Aa和Cyt1Aa与沼泽大蚊刷状缘膜囊泡（BBMVs）的结合及孔形成活性。Cry11Aa溶解后产生两个片段，表观分子量分别为32 kDa和36 kDa。未检测到36 kDa片段与沼泽大蚊BBMVs的结合，而32 kDa片段与沼泽大蚊BBMVs结合。在竞争实验中仅观察到该片段结合的部分减少，表明结合的特异性较低。与对蚊子的结果相反，Cyt1Aa蛋白特异性结合沼泽大蚊的BBMVs，表明存在一种基于受体介导作用的杀虫机制，正如Cry蛋白所述。Cry11Aa和Cyt1Aa毒素都能够在沼泽大蚊BBMVs中形成孔。先前报道分别用胰蛋白酶和蛋白酶K处理可激活Cry11Aa和Cyt1Aa毒素，但结果却相反。用蛋白酶K处理Cyt1A时观察到更高的孔形成效率，而用胰蛋白酶处理的Cry11Aa活性降低。结合和孔形成的结果与ICPs在沼泽大蚊中的毒性及与Cyt1Aa的协同效应结果一致。
