Grant: CIHR Fellowship

Many pathogenic bacteria (Pseudomonas aeruginosa, Vibrio cholerae, Neisseria gonorrhoeae, Legionella pneumophila, etc) use long cell-surface filaments called the type IV pili (T4P) to sense contact with surfaces and to initiate infections. Using the strongest biological motor, T4P can undergo rapid cycles of extension and retraction. This repeated assembly (extension) and disassembly (retraction) of the grappling-hook-like T4P filaments helps bacteria crawl along surfaces towards various niches for colonisation and to capture free DNA that can include antibiotic resistance genes. The T4P motor is a multi-protein complex within a larger nanomachine that spans the bacterial cell envelope. Although individual components of the T4P nanomachine have been intensively studied, how they work together to assemble and disassemble the T4P filaments remains one of the most important unresolved questions in the field. One major technical limitation is that no one has been able to isolate intact T4P nanomachines from the bacterial envelope. I will use an advanced imaging technique called cryo-electron tomography (cryo-ET) to reveal the intact architectures of two evolutionarily distinct types of T4P nano-machines directly within the cell envelopes of a significant human pathogen, P. aeruginosa, and the model species Caulobacter crescentus. By comparing structures of the wild-type T4P nanomachines, and those of informative deletion and point mutants, I will visualize the T4P motor complexes in their extension and retraction states at unprecedented resolution. These data will not only reveal the exact positions and roles of individual T4P components but also answer the key question: how does the T4P filament extend and retract? Since mutations in the T4P motor components impair the virulence of bacterial pathogens, results from this work may serve as a guide for the rational design of new therapeutics that can combat infections without encouraging the development of resistance.