Molecular mechanisms of bacterial toxin translocation across membranes

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Protein translocases are ubiquitous structures in nature that enable transport of proteins across biological membranes. These membrane-embedded structures are critical for a number of important biological processes in eukaryotic cells including protein secretion, membrane protein integration into membranes, and organelle biosynthesis. Certain microbial pathogens secrete protein toxins, which naturally enter target cells where they cause damage to the host. Toxins such as diphtheria, botulinum, tetanus and the large Clostridial toxins possess a protein translocase domain, which inserts itself into the membranes of host cells to create membrane-spanning pores. These pores are thought to facilitate translocation of an associated toxic enzyme into the cytosol of the host cell, resulting in host cell death. In my laboratory, we are interested in understanding the process of protein translocation across membranes using bacterial toxins as model systems. Bacterial toxins are ideal systems to study this complex process as they: (1) possess all of the necessary elements for translocation within a single protein, (2) are highly efficient translocases; and (3) can be produced in large quantities to facilitate biophysical characterization. *** The objectives of the proposed research program are to elucidate the structural architecture and the mechanism of translocation for Clostridium difficile toxin B (TcdB), a prototypic translocating toxin. Under on-going NSERC Discovery Grant funding (2012-2017) we identified both the pore-forming region, and, unexpectedly, and a receptor-binding region within the translocation domain of TcdB. These findings allowed us to propose a model for the toxin translocase (Zhang et al. PNAS; Chumbler et al. Nature Micro, Hamza et al. Pathog Dis), and the binding site for a newly discovered receptor (Zhang et al. in preparation). Together, these findings have advanced our understanding of the structure and function of a toxin translocase, and at the same time have served to generate new hypotheses, which form the foundation of the current proposal. In the current proposed program of research, we seek to build on these discoveries to understand how the translocase “pore”, is assembled and how it then ushers proteins across membranes. We will employ a series of biochemical and biophysical techniques to address these questions. The proposed work is expected to extend our understanding of TcdB translocation into cells and further help define the principles that underlie protein transport - a ubiquitous process in all living systems that remains poorly characterized.