Understanding how the polar flagella from bacterial pathogens work

Many bacteria need to swim to cause disease, and understanding how they do so is essential for therapeutics development. Bacteria swim using helical propellers called flagella that are rotated by molecular motors embedded in the cell surface. Although understanding flagellar motility has focused on Escherichia coil and Salmonella enterica, whose flagella are dotted over their cell surface, many bacteria polarly-localize their flagella. These polarly-localised flagella are not just differently-positioned versions of those from E. coli and Salmonella: while E. coli and Salmonella rotate all of their flagella counterclockwise to swim forward, polar flagella spin in either direction equally, and—in species with a flagellum at each end—coordinate to rotate in opposite directions. And while E. coli and Salmonella search for new directions by transiently switching to spin clockwise, polar flagella toggle in either direction to reorient their swimming. This proposal seeks to understand the mechanisms behind these differences. We wish to understand how the apparatus that both generates rotation and chooses between clockwise and counterclockwise rotation works—and how it differs from its counterpart in E. coli and Salmonella. We use bacterial genetics to alter characteristics of the motor, and electron cryo-microscopy to visualise molecular structures and understand their mechanisms and the impact of our genetic changes. We focus on polar flagellate Campylobacter jejuni because we can easily genetically manipulate it, and it is well-suited for the structural techniques we use. First, we would like to understand how the polar flagellar motor is not biased to spin counterclockwise. We recently discovered that individual C. jejuni motors spend approximately equal time spinning clockwise and counterclockwise, echoing other studies of polar flagella from Vibrio and Pseudomonas species. We will to determine the structures of purified C. jejuni motors engineered to spin exclusively clockwise or counterclockwise using electron cryo-microscopy. We will compare our structures those already published from Salmonella to understand how polar motors behave differently. Second, we would like to understand how the cell influences the polar flagellar motor’s switching frequency. Recent findings indicate that signalling molecules trigger increased bidirectional switching in polar flagellates, in contrast to E. coli and Salmonella, whose signalling molecules trigger a unidirectional switch to clockwise. To understand how the signalling molecule modulates switching frequency, I will image the signalling molecule bound to flagellar motors in situ using electron cryo-tomography and subtomogram averaging to sufficient resolution to build molecular models of how its binding modulates the switching ability of the motor. Third, we would like to understand how this fits into coordination between two apparently opposed flagella. First we must understand how switching is powered – most likely, this is intrinsic to motor rotation, with forces involved in rotating the motor being harnessed to switch rotation direction. We will first image the motor proteins in action driving rotation. We will build on this knowledge by subsequently assessing how two opposed motors coordinate; we suspect that there is explicit signal to coordinate; rather, we hypothesise that the excessive resistance felt by a motor if it opposes the other motor inherently trigger it to switch rotation. We will test this with mutants and my observing motor switching using light microscopic techniques. This work is relevant to the MRC as basic research into a common feature of many pathogens including Campylobacter jejuni, Helicobacter pylori, Vibrio cholerae, and Pseudomonas aeruginosa.