Département de Biologie Structurale et Chimie, Institut Pasteur, CNRS UMR 3528, 28 Rue du Dr Roux, 75724 Paris, CEDEX 15, France.
Université Paris Diderot Paris VII, 75013 Paris, France.
Pathog Dis. 2018 Nov 1;76(8):5188676. doi: 10.1093/femspd/fty085.
The adenylate cyclase toxin (CyaA) is a multi-domain protein secreted by Bordetella pertussis, the causative agent of whooping cough. CyaA is involved in the early stages of respiratory tract colonization by Bordetella pertussis. CyaA is produced and acylated in the bacteria, and secreted via a dedicated secretion system. The cell intoxication process involves a unique mechanism of transport of the CyaA toxin catalytic domain (ACD) across the plasma membrane of eukaryotic cells. Once translocated, ACD binds to and is activated by calmodulin and produces high amounts of cAMP, subverting the physiology of eukaryotic cells. Here, we review our work on the identification and characterization of a critical region of CyaA, the translocation region, required to deliver ACD into the cytosol of target cells. The translocation region contains a segment that exhibits membrane-active properties, i.e. is able to fold upon membrane interaction and permeabilize lipid bilayers. We proposed that this region is required to locally destabilize the membrane, decreasing the energy required for ACD translocation. To further study the translocation process, we developed a tethered bilayer lipid membrane (tBLM) design that recapitulate the ACD transport across a membrane separating two hermetic compartments. We showed that ACD translocation is critically dependent on calcium, membrane potential, CyaA acylation and on the presence of calmodulin in the trans compartment. Finally, we describe how calmodulin-binding triggers key conformational changes in ACD, leading to its activation and production of supraphysiological concentrations of cAMP.
腺苷酸环化酶毒素（CyaA）是百日咳博德特氏菌（百日咳的病原体）分泌的一种多功能蛋白。CyaA 参与了百日咳博德特氏菌在呼吸道的早期定植。CyaA 在细菌中被合成并酰化，然后通过专门的分泌系统分泌。细胞中毒过程涉及 CyaA 毒素催化结构域（ACD）穿过真核细胞膜的独特转运机制。一旦易位，ACD 与钙调蛋白结合并被其激活，产生大量 cAMP，从而颠覆真核细胞的生理学。在这里，我们回顾了我们在鉴定和表征 CyaA 的一个关键区域方面的工作，即转运区域，该区域是将 ACD 递送到靶细胞胞质溶胶中所必需的。转运区域包含一个具有膜活性的片段，即能够在与膜相互作用时折叠并使脂质双层穿孔。我们提出，该区域需要局部破坏膜的稳定性，从而降低 ACD 易位所需的能量。为了进一步研究易位过程，我们开发了一种连接双层脂质膜（tBLM）设计，该设计再现了 ACD 在分隔两个密闭隔室的膜上的转运。我们表明，ACD 易位严重依赖于钙、膜电位、CyaA 酰化以及钙调蛋白在跨膜隔室中的存在。最后，我们描述了钙调蛋白结合如何触发 ACD 的关键构象变化，导致其激活和产生超生理浓度的 cAMP。
