Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhonpathom 73170, Thailand.
J Invertebr Pathol. 2011 Feb;106(2):230-5. doi: 10.1016/j.jip.2010.10.004. Epub 2010 Oct 20.
Bacillus sphaericus produces a mosquitocidal binary toxin composed of two subunits, BinA (42 kDa) and BinB (51 kDa). Both components are required for maximum toxicity against mosquito larvae. BinB has been proposed to provide specificity by binding to the epithelial gut cell membrane, while BinA may be responsible for toxicity. To identify regions in BinB responsible for receptor binding and for interaction to BinA, we used six BinB shorter constructs derived from both the N-terminal and the C-terminal halves of the protein. All constructs expressed as inclusion bodies in Escherichia coli, similarly to the wild-type protein. A marked decrease in larvicidal activity was observed when BinA was used in combination with these BinB constructs, used either individually or in pairs from both N and C-halves of BinB. Nevertheless, immunohistochemistry analyses demonstrate that these constructs are able to bind to the epithelium gut cell membrane, and in vitro protein-protein interaction assays revealed that these constructs can bind to BinA. These results show that fragments corresponding to both halves of BinB are able to bind the receptor and to interact with BinA, but both halves are required by the toxin to exhibit full larvicidal activity.
球形芽孢杆菌产生一种由两个亚基组成的杀蚊毒素，即 BinA（42 kDa）和 BinB（51 kDa）。这两种成分对于对蚊幼虫的最大毒性都是必需的。BinB 被认为通过与上皮肠道细胞膜结合提供特异性，而 BinA 可能负责毒性。为了鉴定 BinB 中负责受体结合和与 BinA 相互作用的区域，我们使用了源自蛋白两端的六个较短的 BinB 构建体。所有构建体在大肠杆菌中均表达为包涵体，与野生型蛋白相似。当 BinA 与这些 BinB 构建体单独或配对使用时，无论是来自 BinB 的 N 端还是 C 端，都观察到杀幼虫活性显著降低。然而，免疫组织化学分析表明这些构建体能够与肠上皮细胞膜结合，体外蛋白-蛋白相互作用测定显示这些构建体能够与 BinA 结合。这些结果表明，BinB 两端的相应片段都能够结合受体并与 BinA 相互作用，但毒素需要这两个片段才能表现出完全的杀幼虫活性。
