Molecular mechanisms underlying Campylobacter jejuni's unusual swimming style

The bacterium Campylobacter jejuni causes food poisoning. In the UK Campylobacter causes more food poisoning than any other bacteria including E. coli or Salmonella (the Food Standards Agency estimates that Campylobacter infections cost us almost one billion pounds per year). Campylobacter is also very similar to other types of 'dangerous' bacteria that cause other stomach problems, including cancers. If we can understand these bacteria better, we'll be better able to develop drugs to fight them. This grant proposes a programme of experiments Campylobacter to understand swimming in this dangerous bacterium; because they need to be able to swim to cause infection, we may be able to develop drugs that "jam" their swimming, in the process halting infection.

Most dangerous bacteria need to be able to swim to cause their disease, but Campylobacter swims in a very unusual manner. Many bacteria 'swim' using a miniature motor that sits in the skin of the bacterium. On the end of the motor's driveshaft is a long tail that spun by the motor; the spinning long tail coils to become a helical propeller, pushing the bacterium through its fluid habitat. Campylobacter uses the same tail, but swims in a very different way to other bacteria, and this may prove to be its Achilles heal: we may in the future develop targeted drugs that only affect Campylobacter and close relatives. Specifically, Campylobacter uses two very powerful motors to swim, one at each end of the cell. Because the two motors are at opposite ends of the cell, they potentially push in opposite directions. Campylobacter has developed a way to combine their thrust productively: one motor "pushes" from behind, while the leading motor coils its tail around Campylobacter's helical cell body to "pull" from in front. But we don't understand how Campylobacter is able to do this. If we can better understand the biology of this curious swimming we may be able to make drugs to prevent it.

To understand bacterial swimming, it is essential to be able to see the motor, see the helical tail, and see the shape of the cell, which are difficult things to do. I believe this is absolutely essential, so to visualize bacteria my "research co-investigator" and I have trained in techniques that enable us to directly see them inside the cell, to see the molecular details of the motor that drives swimming, and to collect videos of bacteria swimming in action. To help us fully understand our data we've recruited a stellar team of international scientists, many of whom we have long-standing collaboratory relationships with.

We're still some way from using these results to fight Campylobacter food poisoning. If we had a detailed understanding of how Campylobacter swims, however, we might start designing drugs that stop it swimming. This MRC proposal requests funds to perform this research.

We propose three aims:

One: A few years ago we published findings that the Campylobacter motor has evolved to be much more powerful than other motors, and this allows it to swim through viscous mucous in our guts to cause food poisoning. Our more recent findings, however, suggest we were naïve: while likely correct, the motor is also probably intimately involved in the unwrapping process. We will test this hypothesis and understand more about its structure in the process.

Two: We will test hypotheses that Campylobacter's long tail has evolved to specifically enable wrapping around the cell body. We will go as far as to deduce the ancestral state, which will provide us with in-depth fascinating insights into precisely which aspects of the tail contribute to wrapping.

Three: Our recent findings suggest that it is not only the motor and tail that contribute to wrapping and unwrapping, but also interactions with the helical cell body of Campylobacter. We will test this hypothesis and characterize factors leading to this.

The aim of this research is to better understand how Campylobacter jejuni swims: although it uses a flagellum, its swimming is considerably different to the flagellar model organisms, E. coli and Salmonella. The work combines electron cryo-tomographic imaging, genetic manipulation, and fluorescence video microscopy to probe hypotheses and mechanisms underlying our recent discovery of a unique "wrapped-mode" swimming by C. jejuni in which the lagging filament pushes the cell while the leading filament-rotating in the same direction with the same handedness-pulls the cell by wrapping around the cell body. Because C. jejuni motility is required for virulence (and a number of other important pathogens from the Helicobacter and Arcobacter genera use the same mechanisms), it presents itself as an excellent target for orthogonal, specific drug development in the era of antimicrobial resistance.

This proposal describes three independent aims using electron cryo-tomography, bacterial genetics, and video microscopy:

Aim 1: Test hypotheses about how the flagellar motor is used for more than just motility through viscous media: rather, it is also intimately involved in unwrapping wrapped filaments. We will also identify previously unidentified components of the motor with genes deleted or tagged will enable location of proteins.

Aim 2: To test hypotheses about the role of the extracellular flagellar hook and filament on ease of wrapping of the filament around the cell body, essential for effective motility.

Aim 3: Understand the contribution of cell shape to the unwrapping process, and work toward understanding how C. jejuni makes itself into a helical shape, essential for proper swimming.

All electron cryo-tomographic data will be collected using established protocols that have already been published, and video microscopy will be conducted with long-term collaborator Takayuki Nishizaka.

This project has considerable relevance to the MRC. Outcomes will be basic discoveries in a prime area for drug design to tackle antimicrobial resistance, conforming to the MRC's "Antimicrobial resistance" health focus theme: there is increasing antibiotic resistance occurring in Campylobacter, and inhibiting motility would be an under-explored approach to a bacteriostatic therapeutic that disables virulence. This would specifically target Campylobacter and relatives, as their virulence relies upon their unique swimming mode, minimally affecting native microbiota.

This project addresses all four foundations to the MRC's vision:

DISCOVERY SCIENCE: We propose a programme of fundamental research into a neglected topic of cell biology, and have already made substantial findings and preliminary data.

INVESTING IN PEOPLE: I am listing PDRA Dr Eli Cohen as Research Co-Investigator. As discussed in my letter of support, Eli is the best thing that has happened to me as a PI, and I am keen to contribute to his development toward starting his own lab. This project would be critical training, capitalizing upon the infrastructure and preliminary results that he has developed in his first years in lab. If funded, Eli will be able to devote six years to an in-depth project, gain necessary expertise, and be ready to "hit the ground running". Should he wish to take any of his projects with him I am happy for him to do that: there are more than enough question to address in this world for all of us.

NEW TECHNOLOGIES AND INFRASTRUCTURE: Electron cryo-tomography is still rapidly developing and is the "final frontier" in structural biology. By developing methods with Peter Rosenthal, and publishing high-impact work featuring electron cryo-tomography as a mature tool applicable to real-world research (and not just ideal test cases) we will further contribute to its development.

FOSTERING COLLABORATION: I build long-lasting collaboratory partnerships. Here I continue my decade-long collaboration with Campylobacter collaborator David R. Hendrixson (UT Southwestern, USA), decade-long interactions with Daisuke Nakane and five-year-long collaboration with Nakane and Takayuki Nishizaka (Gakushuin University, Japan), and four-year-long Satellite Lab in the lab of Peter Rosental (Francis Crick Institute); I have papers published or available as preprints with all three. I also add a new collaboration with Georg Hochberg (MPI Marburg, Germany), with whom I have interacted for five years, and Cynthia Sharma (University of Würzburg, Germany), with whom we have publishable preliminary data.

Others may also benefit from this research:

Industry. Results may culminate in industrial collaborations, patents, or founding a start-up company to develop novel antimicrobials. This will be realized through Imperial Innovations, Imperial's technology commercialization company.

Basic life scientists. The microbiology and C. jejuni communities will learn about the structure and mechanisms of molecular machinery and cell biology. This information will be communicated by myself and Eli through publications and talks.

Evolutionary biologists. A question in biology lies in how complexity develops, and how proteins have accreted over the course of evolution to form large macromolecular machines.

The public. I publicize my work to the public in a number of ways. I exhibit yearly at the Imperial Festival to present my work, and engage with the Natural History Museum. I also publicize results with press releases through Imperial and journals.

Students. Students are excited to learn about the molecular underpinnings of pathogenesis, particularly when the methods used for study incorporate new, exciting methods such as electron cryo-tomography and fluorescence video microscopy. I already incorporate this research into lectures to inspire students. My PhD students will also benefit.

All work will feature the Imperial and MRC brands.