Liu Wei, Ye Wanhui, Wang Zhangming, Wang Xiaorong, Tian Shiyao, Cao Honglin, Lian Jiyu
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China.
Environ Microbiol. 2006 May;8(5):858-70. doi: 10.1111/j.1462-2920.2005.00972.x.
Photorhabdus luminescens, a Gram-negative bacterium, secretes a protein toxin (PL toxin) that is toxic to insects. In this study, the effects of the PL toxin on large receptor-free unilamellar phospholipid vesicles (LUVs) of Manduca sexta and on brush border membrane vesicles (BBMVs) of M. sexta and Tenebrio molitor were examined. Cry1Ac served as a positive control in our experiments due to its known channel-forming activity on M. sexta. Voltage clamping assays with dissected midguts of M. sexta and T. molitor clearly showed that both Cry1Ac and PL toxin caused channel formation in the midguts, although channel formation was not detected for T. molitor midguts under Cry1Ac and it was less sensitive to PL toxin than to Cry1Ac for M. sexta midguts. Calcein release experiments showed that both toxins made LUVs (unilamellar lipid vesicles) permeable, and at some concentrations of the toxins such permeabilizing effects were pH-dependent. The lowest concentrations of PL toxin were more than 600-fold and 24-fold lower to induce BBMV permeability of T. molitor and M. sexta than those to induce calcein release from LUVs of M. sexta. These further support that PL toxin is responsible for channel formation in the larvae midguts. The lower concentration to induce permeability in BBMV than in LUV is, probably, attributable to that BBMV has PL toxin receptors that facilitate the toxin to induce permeabilization. Furthermore, our results indicate that the effects of PL toxin on BBMV permeability of M. sexta were not significantly influenced by Gal Nac, but those of Cry1Ac were. This implies that PL toxin and Cry1Ac might use different molecular binding sites in BBMV to cause channel formation.
发光杆菌（Photorhabdus luminescens）是一种革兰氏阴性菌，能分泌一种对昆虫有毒的蛋白质毒素（PL毒素）。在本研究中，检测了PL毒素对烟草天蛾（Manduca sexta）的无受体大单层磷脂囊泡（LUVs）以及烟草天蛾和黄粉虫（Tenebrio molitor）的刷状缘膜囊泡（BBMVs）的影响。由于Cry1Ac对烟草天蛾已知的通道形成活性，在我们的实验中它作为阳性对照。对烟草天蛾和黄粉虫的中肠进行电压钳实验清楚地表明，Cry1Ac和PL毒素都能在中肠中形成通道，尽管在Cry1Ac作用下黄粉虫中肠未检测到通道形成，并且对于烟草天蛾中肠而言，它对PL毒素的敏感性低于对Cry1Ac的敏感性。钙黄绿素释放实验表明，两种毒素都能使LUVs（单层脂质囊泡）具有通透性，并且在某些毒素浓度下，这种通透作用是pH依赖性的。诱导黄粉虫和烟草天蛾BBMV通透性的PL毒素最低浓度，分别比诱导烟草天蛾LUVs中钙黄绿素释放的浓度低600倍以上和24倍。这些进一步支持了PL毒素在幼虫中肠中负责通道形成。诱导BBMV通透性的浓度低于诱导LUV通透性的浓度，可能是因为BBMV具有PL毒素受体，有助于毒素诱导通透作用。此外，我们的结果表明，Gal Nac对PL毒素影响烟草天蛾BBMV通透性的作用没有显著影响，但对Cry1Ac有显著影响。这意味着PL毒素和Cry1Ac可能在BBMV中使用不同的分子结合位点来导致通道形成。
