Equipe Matériaux Avancés pour la Catalyse et la Santé, Unité Mixte de Recherche 5253, Centre National de la Recherche Scientifique-Ecole Nationale Supérieure de Chimie de Montpellier-Université Montpellier 2-Université Montpellier 1, Institut Charles Gerhardt Montpellier, 34296 Montpellier Cedex 5, France.
Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):20473-8. doi: 10.1073/pnas.1312975110. Epub 2013 Dec 2.
Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study's in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.
许多细菌毒素可以穿过生物膜到达哺乳动物细胞的细胞质，在那里发挥其细胞毒性作用。我们的模型毒素是来自百日咳博德特氏菌的腺苷酸环化酶（CyaA），它能够通过将其催化结构域直接穿过靶细胞的质膜来入侵真核细胞。为了描述其原始的转运过程，我们设计了一种基于仿生膜模型的体外测定法，其中连接的脂质双层（tBLM）组装在衍生有钙调蛋白（CaM）的胺金表面上。组装的双层形成一个连续的、蛋白质不可渗透的边界，完全将下面的钙调蛋白（trans 侧）与上面的介质（cis 侧）隔开。通过表面等离子体共振（SPR）光谱监测 CyaA 与 tBLM 的结合。通过酶活性揭示的 CyaA 与固定化 CaM 的结合，可作为跨双层转运毒素的高度敏感报告器。发现 CyaA 催化结构域的转运严格依赖于钙的存在，并且还依赖于负电势的施加，这在真核细胞中早已得到证实。因此，CyaA 能够在不需要除 CaM 之外的任何真核成分的情况下将其催化结构域递送至生物膜中。这表明钙依赖性 CyaA 转运可能部分由跨膜电场驱动。本研究在体外证明了 CyaA 穿过 tBLM 的转运，为在精确定义的实验条件下研究蛋白质跨生物膜转运的分子机制提供了机会。
