Structural and Functional Studies of Bacterial Adhesins and Protein Nanotubes

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The ability to stick to surfaces is an essential capability of bacteria that enables growth and adaptation in various environments. Bacteria routinely produce complex nanoscale functional structures such as flagella and pili (fibre-like protein polymers) for use in motility, as well as surface and cellular adherence and to transfer DNA and other molecules across membranes. These protein-based adhesion systems are very robust and are assembled and disassembled rapidly by the cell through the use of various secretion systems, which are themselves highly specialized nanomachines. Our research program aims to understand at a structural level how these systems assemble, and how their multiple interactions affect infection and virulence, increase genetic diversity and propagate resistance strategies. One main focus of our research program is the type II secretion system (T2SS) that assembles the type IV pilus (T4P) in several organisms, including Pseudomonas aeruginosa. We are building upon our work characterizing the assembly of protein nanotubes (PNTs) from an engineered form of the P. aeruginosa type IV pilin. Specifically, our research is focusing on: (a) a structural characterization the PNT assembly process, both in solution and at surfaces; (b) exploring the integration of the PNT architecture into functional biosensors and nanodevices; and (c) characterizing the sensing and signal transduction properties of the PNTs within the context of a functional biosensor targeting specific biochemical signals/triggers. A second focus of our research program studies the T4P and T2SS assembly machinery used by the gram-negative bacteria Francisella tularensis and Coxiella burnetii. Both organisms have recently been shown to assemble T4P through associated T2SSs and that the pilus mediates adherence to cells during infection. In addition, methods for testing for Coxiella and Francisella infections are limited. Our research focuses on the structural characterization of the type IV pilins and T2SS component proteins of both F. tularensis and C. burnetii, primarily through X-ray crystallography. We will also explore the possibility of adapting these type IV pilins for biosensor applications using analogous strategies to our studies of the P. aeruginosa PNTs. Our research will provide structural and functional insights into the roles that these T4P play in surface adherence, their mechanisms of assembly, and the adaptability of these proteins for biosensor applications. We are also exploring the type IV secretion system (T4SS) of the F plasmid from E. coli. This system is a main route for the transfer of DNA among a bacterial population, a process known as conjugation, which leads to greater diversity, adaptability and resistance to challenging environments. We are interested in understanding how the proteins of the F-T4SS interact structurally to assemble the conjugative pilus. These studies will provide a clearer picture into how these systems are assembled, leading to the design of new strategies to combat the spread of DNA through T4SS-mediated conjugation. Our research program encompasses a range of biochemical, physical and analytical methods to study several bacterial adhesins and their associated assembly systems. It will lead to a greater understanding of how these structures are assembled, the specific interactions that occur at the binding interface during surface/cellular adherence, and will further advance pilin-derived PNTs for applications in biosensors.