Molecular Mechanisms of Bacterial Toxin Translocation Across Membranes

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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.