Molecular Mechanisms of Bacterial Toxin Translocation Across Membranes

许多细菌病原体分泌有毒的蛋白质，称为毒素，进入宿主细胞，通过扰乱对其生存至关重要的过程而造成损害。作用于细胞内的毒素的一个显着特征是，它们能够穿透包裹所有细胞的原本不可渗透的膜。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的管或通道，毒素结构域将通过该管道或通道进入细胞内部。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上组装，并能够识别和运输化学上不均匀且结构复杂的未折叠蛋白质，目前还知之甚少。我们在这里提出了一系列实验来揭示TcdB易位传递结构域的分子机制和结构决定因素，TcdB是艰难梭菌分泌的一种原型细菌毒素。易位的分子决定因素最初将使用功能丧失方法来揭示，其中将评估突变组成递送结构域的每个单独氨基酸对毒素介导的细胞死亡的影响。单独突变的蛋白质还将被标记上分子信标，这些信标将报告易位每个阶段的局部结构环境和特定氨基酸的位置。总之，这些研究将揭示TcdB的基本机制，其作用与最近在加拿大爆发的毁灭性艰难梭菌有关。此外，这项拟议的研究有望加强我们对蛋白质通过蛋白质通道跨脂双层运输的更一般过程的理解--这是一个普遍存在但却鲜为人知的过程，被所有生命系统使用。

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.