Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

37-POS Board 37 Structural Studies of a Toxin Loaded Bacterial Type VI Secretion (T6S) Effector Complex Using Electron Microscopy Dennis Quentin 1 , John Whitney 2 , Joseph Mougous 2 , Stefan Raunser 1 . 1 Max-Planck-Institute of Molecular Physiology, Dortmund, Germany, 2 University of Washington School of Medicine, Seattle, WA, USA. Participating in interbacterial competition and mediating virulence are two major tasks of almost every bacterium in order to survive in their respective environmental niche. To do so Gram- negative bacteria developed sophisticated protein secretion machineries for translocating a variety of effector proteins across their two membranes into the cytoplasm/periplasm of the host cell. One of the latest discovered secretion systems is the T6S apparatus, contributing to the virulence of several human pathogens like V. cholera and P. aeruginosa. It uses a unique translocation mechanism, which is functionally and structurally related to the effector delivery of bacteriophages. Upon contraction of an outer sheath, an inner tube, consisting of stacked Hcp hexameric rings, is propelled outwards and finally pierces the target cell. Located at the tip of the inner tube, is a tapering VgrG trimer attached, building the basis for the secreted effector complex. In this study, we show for the first time the architecture of a Tse6 (Type VI secretion exported 6, a predicted transmembrane protein)-loaded VrgG complex using negative stain EM. Surprisingly, Tse6-mediated toxicity requires the binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). Furthermore we show, that a putative chaperoning protein, EagT6, is part of the complex. The assignment of the subunit localization within the complex is supported by nanogold and antibody labeling experiments. Additionally, we observed the adoption of different conformations in the presence/absence of detergent hinting to a conformational change upon membrane contact. Our electron microscopic studies combined with biochemical results provide structural and mechanistic insights into these medically relevant protein complexes and will equip us with a better understanding of disease transmission, finally laying the foundation for the development of new therapeutic strategies and treatment options for patients.

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