Molecular Mechanisms of Bacterial Toxin Translocation Across Membranes

许多细菌病原体会分泌一种叫做毒素的有毒蛋白质，这种蛋白质会进入宿主细胞，破坏宿主细胞生存所必需的过程，从而造成损害。毒素在细胞内作用的一个显著特征是它们能够穿透包围所有细胞的不透水的膜。为了突破这一保护屏障，毒素被赋予或伴随着一个专门的“易位”结构域，该结构域可以形成跨膜管或通道，有毒结构域通过这些结构域进入细胞内部。然而，为了通过狭窄的跨膜通道，有毒结构域必须从其能量稳定的折叠构象中展开，并以展开的线性链的形式穿过蛋白质通道。通道形成结构域如何在宿主膜中组装，并能够识别和运输未折叠的蛋白质，这些蛋白质在化学上是不均匀的，结构上是笨重的，目前还不清楚。我们在此提出了一系列的实验来揭示TcdB易位传递域的分子机制和结构决定因素，TcdB是一种由艰难梭菌分泌的原型细菌毒素。易位的分子决定因素最初将使用功能丧失方法来揭示，其中将评估构成递送结构域的每个单个氨基酸突变对毒素介导的细胞死亡的影响。单独突变的蛋白质将被标记为分子信标，这将报告局部结构环境和特定氨基酸在每个易位阶段的位置。总之，这些研究将揭示TcdB的基本机制，其行为与加拿大最近毁灭性的艰难梭菌爆发有关。此外，拟议的研究有望增强我们对通过蛋白质通道跨脂质双分子层的蛋白质运输的更一般过程的理解-这是一个普遍存在的，但对所有生命系统使用的过程知之甚少。

Many bacterial pathogens secrete poisonous proteins called toxins that get into host cells and cause damage by disrupting processes that are essential for its survival. A remarkable feature shared by toxins acting inside cells is their ability to penetrate the otherwise impermeable membrane that surrounds all cells. To breach this protective barrier, toxins are endowed with, or are accompanied by, a specialized 'translocation' domain that can form membrane-spanning tubes or channels, through which the toxic domains destined for the cell interior travel. To negotiate the narrow transmembrane channels, however, the toxic domains must unfold from their energetically stable folded conformations and thread through the protein channels as unfolded, linear chains. How the channel-forming domains assemble in the host membrane, and are able to recognize and transport the unfolded proteins, which are chemically heterogeneous and structurally unwieldy, is poorly understood. We propose here a series of experiments to uncover the molecular mechanisms and structural determinants of the translocation delivery domain of TcdB, a prototypic bacterial toxin secreted by Clostridium difficile. The molecular determinants of translocation will be uncovered initially using a loss-of-function approach, where the effects of mutating each of the individual amino acids that comprise the delivery domain, on toxin-mediated cell death will be assessed. Individually mutated proteins will additionally be labeled with molecular beacons that will report the local structural environment and location of a particular amino acid at each stage of translocation. Together, these studies will uncover the fundamental mechanism TcdB, the actions of which have been linked to the devastating recent C. difficile outbreaks in Canada. Additionally, the proposed research is expected to enhance our understanding of the more general process of protein transport across lipid bilayers through protein channels - a ubiquitous, yet poorly understood process used by all living systems.